CN116761614A - Engineered uridine phosphorylase variant enzymes - Google Patents

Abstract

The invention provides engineered uridine phosphorylase (UP), polypeptides having UP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. Methods for producing UP enzymes are also provided. The invention also provides compositions comprising UP enzymes, and methods of using engineered UP enzymes. The invention is particularly useful in the production of pharmaceutical compounds.

Description

Engineered uridine phosphorylase variant enzymes

This application claims priority to U.S. Provisional Patent Application Serial No. 63/127,431 filed on December 18, 2020 and U.S. Provisional Patent Application Serial No. 63/148,324 filed on February 11, 2021, both of which are incorporated by reference in their entirety for all purposes.

Field of the Invention

The present invention provides engineered uridine phosphorylase (UP), polypeptides having UP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing UP enzymes. The present invention also provides compositions comprising UP enzymes, and methods of using engineered UP enzymes. The present invention is particularly useful for the production of pharmaceutical compounds. References to sequence listings, tables, or computer programs

An official copy of the sequence listing is submitted with the specification as a text file in ASCII format via EFS-Web, with the file name "CX2-214WO2_ST25.txt", the creation date of December 14, 2021, and the size of 1.84 megabytes. The sequence listing submitted via EFS-Web is part of the present specification and is incorporated herein by reference in its entirety.

Background of the Invention

Increasingly, unnatural nucleoside analogs are being investigated for the treatment of cancer and viral infections, such as COVID-19. Nucleoside analogs, because of their similarity to natural nucleosides used in DNA synthesis, are often potent inhibitors of viral diseases. Similarly, nucleoside analogs are known to stimulate anti-tumor innate immune responses (which are activated by the presence of foreign nucleic acids).

However, the production of nucleoside analogs by standard chemical synthesis techniques may be challenging due to their chemical complexity. Additionally, standard chemical synthesis techniques typically produce undesirable waste. The substrates and intermediates required to produce nucleoside analogs may be unavailable or unsuitable for industrial process conditions.

There is demand for the method for non-natural nucleoside analogs that are used to treat cancer and viral infection for improved generation. Specifically, the improved method of synthesizing the required substrate and intermediate of nucleoside analogs under industrial process conditions is necessary. A method is to utilize the engineered polypeptide with improved properties to produce substrate and intermediate. This biocatalytic method has the advantages of reducing chemical waste and improving efficiency.

Enzymes belonging to the uridine phosphorylase class of enzymes (EC 2.4.2.3) typically catalyze the conversion of uridine and phosphoric acid into uracil and α-D-ribose 1-phosphate. However, uridine phosphorylase (UP) also catalyzes the reverse reaction and can be used for a variety of conversions to synthesize non-natural nucleoside analogs. Improved uridine phosphorylase biocatalysts are needed to produce the necessary intermediates and substrates for the efficient production of non-natural nucleoside analogs.

SUMMARY OF THE INVENTION

The present invention provides engineered uridine phosphorylase (UP), polypeptides having UP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing UP enzymes. The present invention also provides compositions comprising UP enzymes, and methods for using engineered UP enzymes. The present invention is particularly useful for the production of pharmaceutical compounds.

The present invention provides an engineered uridine phosphorylase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 246, SEQ ID NO: 594, SEQ ID NO: 776 and/or SEQ ID NO: 868, or a functional fragment thereof, wherein the engineered uridine phosphorylase comprises a polypeptide comprising at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 246, SEQ ID NO: 594, SEQ ID NO: 776 and/or SEQ ID NO: 868. In some embodiments, the polypeptide sequence is identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 246, SEQ ID NO: 594, SEQ ID NO: 776 and/or SEQ ID NO: 868. NO:2 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 6, 7, 9, 14, 14/38/40 /146/147/179/235/236, 14/38/86/146/147/235/236/240, 14/40, 14/40/86/147/193/236/240, 14/40/136/179/236/240, 14/40/146/235/236, 14 /40/147/181/193/235, 14 /40/235、14/86/146、14/146/147/181/240、14/146/236/240、14/147/193/235/236/240、14/179/181/193/235/240、14/235/236、29、31、38/40/8 6/146/147/179/181, 38/40 /86/147/236/240, 40, 40/43/86/146/240, 40/43/86/147/235, 40/43/146/147, 40/43/147/179/236/240, 40/43/147/179/240, 40/43/147/236/24 0, 40/86/146/235/236, 40 /86/147/235/236/240、40/86/179/235/240、40/86/235/236/240、40/146/147/240、40/147/240、40/235、40/235/236/240、40/236、40/236/240、 42/235/236, 43/86/147/18 1/240, 43/146/147/235/236/240, 43/146/179/240, 43/147, 43/147/179/181, 47, 47/88, 64, 73, 80, 86, 86/136/146/147/179/181, 86/136/146/ 147/179/235/236, 86/147/1 1 46/236/240, 146/240, 147/1 79/181, 147/235/240/249, 157, 167, 179, 179/181, 179/181/193/240, 181, 184, 216, 226, 228, 231, 233, 235, 235/236, 236, 236/240, 237, 239, 240 and 245, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:2. In some embodiments, the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of: 6F, 6G, 6R, 6S, 6W, 6Y, 7T, 9M, 14A, 14A/38L/40S/146E/147L/179R/235R/236A, 14A/38L/86V/146E/147L/235R/236A/240R at one or more positions in the polypeptide sequence. , 14A/40D/86V/147M/193L/236A/240R, 14A/40D/235R, 14A/40N, 14A/40N/146E/235R/236A, 14A/40N/147L/181Q/193L/235R, 14A/40S, 14A/40S/1 36V/179R/236A/240R, 14A/86V/146E, 14A/146E/147L/181Q/240R, 14A/146E/236A/240R , 14A/147L/193L/235R/236A/240R, 14A/179R/181Q/193L/235R/240R, 14A/235R/236A, 29S, 31T, 38L/40D/86V/146E/147L/179R/181Q, 38L/40S/8 6 V/147L/236A/240R, 40D, 40D/86V/147L/235R/236A/240R, 40D/86V/235R/236A/240R, 40D/235R, 40N/43F/86V/146E/240R, 40N/43F/86V/147L/23 5R , 40N/43F/146E/147L, 40N/43F/147L/179R/236A/240R, 40N/86V/179R/235R/240R, 40S/43F/147L/179R/240R, 40S/43F/147L/236A/240R, 40S/86V/146E/235R/236A, 40S/86V/147L/235R/236A/240R, 40S/146E/147L/240R, 40S/147L/240R, 40S/235R/236A/240R, 40S/236A, 40S/236A/240R, 42I/235R/236A, 43F/8 6V/147L/181Q/240R, 43F/146E/147L/235R/236A/240R, 43F/146E/179R/240R, 43F/ 147L, 43F/147L/179R/181Q, 47M, 47R, 47V, 47V/88A, 64C, 73G, 80G, 80I, 80K, 80M, 80S, 80T, 86V, 86V/136V/146E/147L/179R/181Q, 86V/136V/146 E/147L/179R/235R/236A, 86V/147L/179R/181Q, 86V/235R, 86V/235R/236A/240R, 86V 1 46E/236A/240R, 146E/240R, 147L/179R/181Q, 147L/235R/240R/249V, 157S, 167S, 1 79R, 179R/181Q, 179R/181Q/193L/240R, 181Q, 184R, 184S, 216M, 226R, 228A, 228G, 228K, 228L, 228R, 231I, 231P, 231T, 231W, 231Y, 233S, 235R, 235R/236A, 236A, 236A/240R, 237V, 239A, 239K, 239R, 240R and 245S, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:2. In some embodiments, the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of V6F, V6G, V6R, V6S, V6W, V6Y, F7T, L9M, N14A, N14A/M38L/K40S/S146E/I147L/H179R/K235R/Q236A, N14A/M38L/I86V/S146E/I147L/K235R/Q236A/H240R at one or more positions in the polypeptide sequence. , N14A/K40D/I86V/I147M/M193L/Q236A/H240R, N14A/K40D/K235R, N14A/K40N, N14A/K40N/S146E/K235R/Q236A , N14A/K40N/I147L/K181Q/M193L/K235R, N14A/K40S, N14A/K40S/C136V/H179R/Q236A/H240R, N14A/I86V/S146E, N14A/S146E/I147L/K181Q/H24 0R, N14A/S146E/Q236A/H240R, N14A/I147L/M193L/K235R/Q236A/H240R, N14A/H179R/K181Q/M193L/K235R/H240R, N14A/K235R/Q236A, D29S, V31T , M38L/K40D/I86V/S146E/I147L/H179R/K181Q, M38L/K40S/I86V/I147L/Q236A/H240R, K40D, K40D/I86V/I147L/K235R/Q236A/H240R, K40D/I86V /K235R/Q236A/H240R, K40D/K235R, K40N/K43F/I86V/S146E/H240R, K40N/K43F/I86V/I147L/K235R, K40N/K43F/S146E/I147L, K40N/K43F/I147L/H179R/Q236A/H240R , K40N/I86V/H179R/K235R/H240R, K40S/K43F/I147L/H179R/H240R, K40S/K43F/I147L/Q236A/H240R, K40S/I86V/S146E/K235R/Q236A, K40S/I86 V/I147L/K235R/Q236A/H240R, K40S/S146E/I147L/H240R, K40S/I147L/H240R, K40S/K235R/Q236A/H240R, K40S/Q236A, K40S/Q236A/H240R, V 42I/K235R/Q236A, K43F/I86V/I147L/K181Q/H240R, K43F/S146E/I147L/K235R/Q236A/H240R, K43F/S146E/H179R/H240R, K43F/I147L, K43F/I14 7L/H179R/K181Q, H47M, H47R, H47V, H47V/T88A, V64C, S73G, E80G, E80I, E80K, E80M, E80S, E80T, I86V, I86V/C136V/S146E/I147L/H179R/K181Q , I86V/C136V/S146E/I147L/H179R/K235R/Q236A, I86V/I147L/H179R/K181Q, I86V/K235R, I86V/K235R/Q236A/H240R, I86V/Q236A/H240R, I86 V/H240R, I92V, A97T, Q99E, N103G/A249V, N103S, V104T, G105M , D106E, T110A, T110C, T110S, S146E/I147L, S146E/I147L/K235R/Q236A, S146E/K235R/H240R, S146E/Q236A/H240R, S146E/H240R, I147L/H179R/K 181Q、I147 L/K235R/H240R/A249V, A157S, E167S, H179R, H179R/K181Q, H179R/K181Q/M193L/H240R, K181Q, M184R, M184S, V216M, Q226R, I228A, I228G, I228K, I228L, I2 28R, A231I, A231P, A231T, A231W, A231Y, T233S, K235R, K235R/Q236A, Q236A, Q236A/H240R, T237V, S239A, S239K, S239R, H240R, and V245S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2.

In some embodiments, the present invention provides an engineered uridine phosphorylase having a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 3, 3/9/2 16. 3/9/216/236, 3/9/235/237, 3/31/47/179/181/216, 3/31/47/179/181/237, 3/31/47/179/216, 3/31/47/179/216/237, 3/31/179, 3/31/179/1 81.3/31/179/181/237, 3/31/179/216, 3/31/181/216, 3/31/181/237, 3/47/179/181/216, 3/47/179/216/237, 3/47/181, 3/179, 3/179/181, 3/179/181/216, 3/179/181 /237、3/179/216、3/179/ 216/237, 3/179/237, 3/181/216, 3/181/216/237, 3/216/236/240, 9/216/236/237, 9/237, 13, 24, 31, 31/47, 31/47/179/181/237, 31/47/179/216 ,31/47/181,31/47/216 , 31/179, 31/179/181, 31/179/216, 31/181, 31/181/216, 31/181/216/237, 31/181/237, 31/216, 31/216/237, 31/236/237/240, 31/237, 33, 46, 4 7. 47/147/181/231, 47/1 10 0, 101, 105, 106, 108, 111 ,137,151,152,155,159,160,170,173,177,179,179/181,179/181/216,179/181/216/237,179/181/231,179/181/241,179/216,179/228/231, 179/237, 181, 181/216, 1 81/216/237, 181/237, 183, 185, 188, 189, 191, 201, 216, 216/237, 218, 222, 228, 231, 233, 235, 235/237, 236, 236/237/240, 237, 238, 240, 241 and 248, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:4. In some embodiments, the present invention provides an engineered uridine phosphorylase having a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of: 3E, 3E/9M/216M, 3E/9M/216M/236A, 3E/9M/235R/237V, 3E/31T/47V/179R/181Q/216M, 3E/31T/47V/179R/181Q/237V, 3E/31T/47V/179R/216M, 3E/31T/47V/179R/216M/237V, 3E/31T/179R, 3E/31T/179R/181Q, 3E/31T/179R/181Q/237 V, 3E/31T/179R/216M, 3E/31T/181Q/216M, 3E/31T/181Q/237V, 3E/47V/1 79R/181Q/216M, 3E/47V/179R/216M/237V, 3E/47V/181Q, 3E/179R, 3E/179R/181Q, 3E/179R/181Q/216M, 3E/179R/181Q/237V, 3E/179R/216M, 3E/ 179R/216M/237V, 3E/179R/237V, 3E/181Q/216M, 3E/181Q/216M/237V, 3E/2 16M/236A/240R, 9M/216M/236A/237V, 9M/237V, 13L, 24L, 24M, 24Q, 31T, 31T/47V, 31T/47V/179R/181Q/237V, 31T/47V/179R/216M, 31T/47V/181Q , 31T/47V/216M, 31T/179R, 31T/179R/181Q, 31T/179R/216M, 31T/181Q, 31T /181Q/216M, 31T/181Q/216M/237V, 31T/181Q/237V, 31T/216M, 31T/216M/237V, 31T/236A/237V/240R, 31T/237V, 33L, 33N, 33R, 33V, 33Y, 46Q, 47V , 47V/147L/181Q/231I, 47V/179R/181Q, 47V/179R/181Q/216M, 47V/179R/ 184R, 47V/179R/216M, 47V/181Q/216M, 47V/181Q/216M/237V, 47V/181Q/231W, 47V/216M, 47W, 52L, 52W, 63M, 67G, 83G, 87H/160C, 92M, 95S, 97S, 9 9L, 99R, 99V, 100D, 100E, 100G, 100R, 100T, 101A, 101T, 101V, 101W, 105S, 10 179 R/181Q/216M/237V, 179R/181Q/231W, 179R/181Q/241V, 179R/216M, 179R/2 28A/231I, 179R/237V, 181Q, 181Q/216M, 181Q/216M/237V, 181Q/237V, 183L, 183T, 183W, 185A, 185G, 185H, 185K, 185Q, 185R, 185S, 185V, 185W, 188 A. 188L, 189L, 189R, 189V, 191F, 191R, 201L, 216M, 216M/237V, 218A, 218V, 222L, 222R, 228H, 228L, 228T, 231G, 231V, 233A, 233G, 233N, 233S, 235H, 235R/237V, 235S, 236A, 236A/237V/240R, 236P, 236S, 236T, 237V, 238G, 238P, 238S, 240G, 241G, 241L, 241M, 241P, 241W and 248T, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 4. In some embodiments, the present invention provides an engineered uridine phosphorylase having the same amino acid position as SEQ ID NO: 4. NO:4 has a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of K3E, K3E/L9M/V216M, K3E/ L9M/V216M/Q236A, K3E/L9M/K235R/T237V, K3E/V31T/H47V/H179R/K181Q/V216M, K3E/V31T/H47V/H179R/K181Q/T237V, K3E/V31T/H47V/H179R/V2 16M, K3E/V31T/H47V/H179R/V216M/T237V, K3E/V31T/H179R, K3E/V31T/H179R/K181Q, K3E/V31T/H179R/K181Q/T237V, K3E/V31T/H179R/V216M, K3E/V31T/K181Q/V216M, K3E/V31T/K181Q/T 237V, K3E/H47V/H179R/K181Q/V216M, K3E/H47V/H179R/V216M/T237V, K3E/H47V/K181Q, K3E/H179R, K3E/H179R/K181Q, K3E/H179R/K181Q/V216M , K3E/H179R/K181Q/T237V, K3E/H179R/V216M, K3E/H179R/V216M/T237V, K3E/H179R/T237V, K3E/K181Q/V216M, K3E/K181Q/V216M/T237V, K3E/ V216M/Q236A/H240R, L9M/V216M/Q236A/T237V, L9M/T237V, K13L, V24L, V24M, V24Q, V31T, V31T/H4 7V, V31T/H47V/H179R/K181Q/T237V, V31T/H47V/H179R/V216M, V31T/H47V/K181Q, V31T/H47V/V216M, V31T/H179R, V31T/H179R/K181Q, V31T/H179 R/V216M, V31T/K181Q, V31T/K181Q/V216M, V31T/K181Q/V216M/T237 V, V31T/K181Q/T237V, V31T/V216M, V31T/V216M/T237V, V31T/Q236A/T237V/H240R, V31T/T237V, K33L, K33N, K33R, K33V, K33Y, S46Q, H47V, H47V/I1 47L/K181Q/A231I, H47V/H179R/K181Q, H47V/H179R/K181Q/V216M, H47V/H179R/M184R, H47V/H179R/V216M, H47V/K181Q/V216M, H47V/K181Q/V216M/T237V, H47V/K181Q/A231W, H47V/V216M, H47W, T52L, T52W, I63M, T 67G, Q83G, R87H/D160C, I92M, T95S, A97S, Q99L, Q99R, Q99V, P100D, P100E, P100G, P100R, P100T, H101A, H101T, H101V, H101W, G105S, D106E, L108A, L108M, L108V, T111K, T111R, T137G, T151S, H152L, H152S, H152V, V1 55R, S159A, S159T, D160G, D170A, S173R, V177R, H179R, H179R/K181 Q. H179R/K181Q/V216M, H179R/K181Q/V216M/T237V, H179R/K181Q/A231W, H179R/K181Q/A241V, H179R/V216M, H179R/I228A/A231I, H179R/T237V, K181Q, K181Q/V216M, K181Q/V216M/T237V, K181Q/T237V, S183L, S18 3T, S183W, E185A, E185G, E185H, E185K, E185Q, E185R, E185S, E185V, E185W, Q188A, Q188L, A189L, A189R, A189V, G191F, G191R, T201L, V216M, V216 M/T237V, G218A, G218V, N222L, N222R, I228H, I228L, I228T, A231G, A2 31V, T233A, T233G, T233N, T233S, K235H, K235R/T237V, K235S, Q236A, Q236A/T237V/H240R, Q236P, Q236S, Q236T, T237V, E238G, E238P, E238S, H240G, A241G, A241L, A241M, A241P, A241W and A248T, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 4. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 4. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 4.

In some embodiments, the invention provides an engineered uridine phosphorylase having a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 246, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 3/24/33/47/100/183/185, 3/24/33/47/100/216/228/233, 3/24/33/47/100/217/229/230, 3/24/33/47/100/228/231. 3/100/183/185/228, 3/24/33/108, 3/24/47/100, 3/24/47/100/108/111/160/185/233/241, 3/24/47/108/160/241, 3/24/47/160/189, 3/24/47 /189/228/233, 3/24/47/228, 3/24/47/228/233, 3/24/95/100, 3/24/95/100/160/189/ 228/241, 3/24/100, 3/24/100/160/218/241, 3/24/111, 3/24/111/183/228/233/241, 3/24/111/228/233, 3/24/183/185/216, 3/24/189/233, 3/3 3/47/95/100/241, 3/33/47/100/108/189/216/228/233, 3/33/47/100/111/228, 3/33 /47/100/111/233/241、3/33/47/100/216、3/33/47/108/111/233、3/33/47/108/189/233/241、3/33/160/233、3/47、3/47/95/100/108/189/233、 3/47/95/100/111/241, 3/47/95/160/189, 3/47/100/108/183/185/189/241, 3/47/10 0/160/185, 3/47/100/185/189/228, 3/47/108/111, 3/47/183/189/228/233, 3/47/189, 3/47/228/233, 3/95/100/160/228/233, 3/95/100/183/ 216/228/233, 3/95/100/183/233, 3/95/185/189/216, 3/95/189, 3/95/233, 3/160, 3/ 183/185/189/228/233, 3/183/189/228/233, 3/185, 3/185/189, 3/189, 24, 24/33/47, 24/33/47/228/241, 24/33/100/108/241, 24/47/95/100, 2 4/47/95/100/160/228/233/241, 24/47/185/216/218, 24/47/216, 24/95/183, 24/100/ 160/233, 24/160/183/185, 24/189/228/233, 33/47/95/100/233, 33/47/95/100/233/241, 33/47/160, 33/47/233, 33/100/183/185, 33/100/185 /233, 47, 47/100/111/233, 47/100/189, 47/100/189/233, 47/108/160/228/241, 47/11 1, 47/160/185/189/233, 47/228/233, 95/100/183, 95/100/189, 95/100/228, 95/100/228/233, 95/100/233, 100/160/185, 100/228/233, 108, 108/183/189/233, 108/185/216/228/233, 160/233, 228 and 228/233, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 246. In some embodiments, the invention provides an engineered uridine phosphorylase having the same amino acid position as SEQ ID NO: 246. NO:246 has a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of: 3E/24 L/33Y/47V/100E/216M/228L/233S, 3E/24L/33Y/47W/100E/183L/185A, 3E/24L/33Y/100E/183L/185A/228L, 3E/24L/33Y/108V, 3E/24L/47V/228L ,3E/24L/47V/228L/233S , 3E/24L/47W/100E, 3E/24L/47W/100E/108V/111K/160G/185A/233S/241A, 3E/24L/47W/108V/160G/241A, 3E/24L/47W/160G/189R, 3E/24L /47W/189R/228L/233S, 3E/24L/95S/100E, 3E/24L/95S/100E/160G/189R/228L/241A, 3E/24L/100E, 3E/24L/100E/160G/218V/241A, 3E/24L/111K, 3E/24L/111K/183L/228L/233S/241A, 3E/24L/111K/228L/233S, 3E/24L/183L/185A/216M, 3E/24L/189R/233S, 3E/33Y/47V/100E /111K/228L, 3E/33Y/47V/100E/111K/233S/241A, 3E/33Y/47V/100E/216M, 3E/33Y/47W/95S/100E/241A , 3E/33Y/47W/100E/108V/189R/216M/228L/233S, 3E/33Y/47W/108V/111K/233S, 3E/33Y/47W/108V/189R/233S/241A, 3E/33Y/160G/233S, 3E/4 7V/95S/100E/111K/241A, 3E/47V/95S/160G/189R, 3E/47V/100E/160G/185A, 3E/47W, 3E/47W/9 5S/100E/108V/189R/233S, 3E/47W/100E/108V/183L/185A/189R/241A, 3E/47W/100E/185A/189R/228L, 3E/47W/108V/111K, 3E/47W/183L/189R/ 228L/233S, 3E/47W/189R, 3E/47W/228L/233S, 3E/95S/100E/160G/228L/233S , 3E/95S/100E/183L/216M/228L/233S, 3E/95S/100E/183L/233S, 3E/95S/185A/189R/216M, 3E/95S/189R, 3E/95S/233S, 3E/160G, 3E/183L/185A /189R/228L/233S, 3E/183L/189R/228L/233S, 3E/185A, 3E/185A/189R, 3E/189R, 24L, 24L/33Y/47W, 24L/33Y /47W/228L/241A, 24L/33Y/100E/108V/241A, 24L/47V/95S/100E, 24L/47V/95S/100E/160G/228L/233S/241A, 24L/47V/185A/216M/218V, 24L/47V /216M、24L/95S/183L、24L/100E/160G/233S、24L/160G/183L/185A、24L/189R/228L/233S、33Y/47V/95S/10 0E/233S, 33Y/47W/95S/100E/233S/241A, 33Y/47W/160G, 33Y/47W/233S, 33Y/100E/183L/185A, 33Y/100E/185A/233S, 47V, 47V/100E/111K/233S, 47V/100E/189R, 47V/108V/160G/228L/241A, 47V/111K, 47V/160G/185A/189R/233S, 47V/228L/233S, 47W/1 : 00E/189R/233S, 95S/100E/183L, 95S/100E/189R, 95S/100E/228L, 95S/100E/228L/233S, 95S/100E/233S, 100E/160G/185A, 100E/228L/233S, 108V, 108V/183L/189R/233S, 108V/185A/216M/228L/233S, 160G/233S, 228L and 228L/233S, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:246. In some embodiments, the present invention provides an engineered uridine phosphorylase having a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 246, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the following at one or more positions in the polypeptide sequence: K3E/V24L/K33Y/H47V/P100E/V216M/I228L/T233S, K3E/V24L/K33Y/H47W/P100E/S183L/E185A , K3E/V24L/K33Y/P100E/S183L/E185A/I228L, K3E/V24L/K33Y/L108V, K3E/V24L/H47V/I228L, K3E/V24L/H47V/I228L/T233S, K3E/V24L/H47W/P 100E , K3E/V24L/H47W/P100E/L108V/T111K/D160G/E185A/T233S/M241A, K3E/V24L/H47W/L108V/D160G/M241A, K3E/V24L/H47W/D160G/A189R, K3E/V24 L/H47W/ A189R/I228L/T233S, K3E/V24L/T95S/P100E, K3E/V24L/T95S/P100E/D160G/A189R/I228L/M241A, K3E/V24L/P100E, K3E/V24L/P100E/D160G/G218 V/M241A, K3E/V24L/T111K, K3E/V24L/T111K/S183L/I228L/T233S/M241A , K3E/V24L/T111K/I228L/T233S, K3E/V24L/S183L/E185A/V216M, K3E/V24L/A189R/T233S, K3E/K33Y/H47V/P100E/T111K/I228L, K3E/K33Y/H47V/ P100 E/T111K/T233S/M241A, K3E/K33Y/H47V/P100E/V216M, K3E/K33Y/H47W/T95S/P100E/M241A, K3E/K33Y/H47W/P100E/L108V/A189R/V216M/I228L/T2 33S . 41A, K3E/H47V/T95S/D160G/A189R, K3E/H47V/P100E/D160G/E185A, K3E/H47W, K3E/H47W/T95S/P100E/L108V/A189R/T233S ,K3E/H47W/P100E/L108V/S183L/E185A/A189R/M241A , K3E/H47W/P100E/E185A/A189R/I228L, K3E/H47W/L108V/T111K, K3E/H47W/S183L/A189R/I228L/T233S, K3E/H47W/A189R, K3E/H47W/I228L/T23 3S, K3E/T95S/P100E/D160G/I228L/T233S, K3E/T95S/P100E/S183L/V216M/I228L/T233S, K3E/T95S/P100E/S183L/T233S, K3E/T95S/E185A/A18 9R/V216M, K3E/T95S/A189R, K3E/T95S/T233S, K3E/D160G, K3E/S183L/E185A/A189R/I228L/T233S, K3E/S183L/A189R/I228L/T233S, K3E/E185A, K3E/E185A/A189R, K3E/A189R, V24L, V24L/K33Y/H47W, V24L/K33Y/H47W/I228L/M241A, V24L/K33Y/P100E/L108V/M241A, V24L/H47V/T95S/P100E . G/T233S, V24L/D160G/S183L/E185A, V24L/A189R/I228L/T233S, K33Y/H47V/T95S/P100E/T233S, K33Y/H47W/T95S/P100E/T233S/M241A, K33Y/H47W/D160G, K33Y/H47W/T233S, K33Y/P100E/S183L/E185A, K33Y/P1 00E/E185A/ T233S, H47V, H47V/P100E/T111K/T233S, H47V/P100E/A189R, H47V/L108V/D160G/I228L/M241A, H47V/T111K, H47V/D160G/E185A/A189R/T233S, H47 V/I228L/T 233S, H47W/P100E/A189R/T233S, T95S/P100E/S183L, T95S/P100E/A189R, T95S/P100E/I228L, T95S/P100E/I228L/T233S, T95S/P100E/T233S, P10 0E/D160G/E 185A, P100E/I228L/T233S, L108V, L108V/S183L/A189R/T233S, L108V/E185A/V216M/I228L/T233S, D160G/T233S, I228L, and I228L/T233S, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 246. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 246. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 246. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 246.

In some embodiments, the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:594, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 6, 6/9/29/40/100/121/126/179/181/189/237, 6/9/121/179/181, 6/46/52/63/97/121/126/179, 6/52/180/181, 6/63/126/179/24 2, 9, 9/40/46/97/100/106/135/179/181/207/231, 9/52/126/189/242, 9/97/100/106/126/180/207/231, 9/181/242, 29, 40, 46, 52, 52/63/126, 52/179/189, 61, 63, 97, 100, 106, 121, 126, 126/180/189, 135, 142, 179, 180, 181, 189, 201, 207, 230, 231, 236, 237 and 242, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:594. In some embodiments, the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:594, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of 6S, 6S/9M/29A/40N/100G/121G/126M/179R/181Q/189E/237V, 6S/9M/121G/179R/181Q, 6S/46Q/52S/63V/97T/121G/126M/179R, 6S/52S/180L/181Q, 6S/63V/126M/179R/242I, 9M, 9M/40N/46Q/97T/100G/106E/135D/179R/181Q/207S/231E , 9M/52S/126M/189E/242I, 9M/97T/100G/106E/126M/180L/207S/231E, 9M/181Q/242I, 29A, 40N, 46Q, 52S, 52S/63V/126M, 52S/179R/189E, 61A, 63V, 97T, 100G, 106E, 121G, 126M, 126M/180L/189E, 135D, 142A, 179R, 180L, 181Q, 189D, 189E, 201L, 207S, 230D, 231E, 236E, 237V and 242I, wherein the amino acid positions of the polypeptide sequences are referenced to SEQ ID NO: 594. In some embodiments, the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 594, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of V6S, V6S/L9M/D29A/K40N/P100G/L121G/L126M/H179R/K181Q/R189E/T237V, V6S/L9M/L121G/H179R/K181Q ,V6S/S46Q/T52S/I63V/A97T/L121G/L126M/H179R , V6S/T52S/F180L/K181Q, V6S/I63V/L126M/H179R/V242I, L9M, L9M/K40N/S46Q/A97T/P100G/D106E/E135D/H179R/K181Q/A207S/A231E, L9M/T52 S/L126M/R189E/V242I, L9M/A97T/P100G/D106E/L126M/F180L/A207S/A231E, L9M/K181Q/V242I, D29A, [0147] The invention relates to polypeptides of the invention wherein the amino acid positions of the polypeptides are numbered with reference to SEQ ID NO: 594. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 594. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 594. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 594.

In some embodiments, the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 776, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 3, 3/8/22/36/181/235/250, 6, 6/45/51/81 /126/226/233、6/45/51/144、6/45/51/188/189、6/45/51/189/208、6/45/51/189/228/236、6/45/51/208/233、6/45/149/188/189/208/233/236、 6/51/126/144/208, 6/51/126/189/208/233/236, 6/ 51/126/189/231/236, 6/51/126/189/233, 6/51/188/189/208/226/228, 6/51/188/189/236, 6/51/189/208/231/233, 6/51/208/226/233, 6/51/ 208/231, 6/126/188/231/233, 6/144/208, 6/188/189 /208/228/233, 8, 8/36/143/147/235, 8/36/181/235, 8/142/147, 8/147/181/250, 8/147/235, 9, 19, 20, 22, 22/147/181/235/250, 24, 36, 36/143/ 147/181, 36/143/147/235, 40, 41, 43, 45, 45/51, 45/5 1/126/144/208/226/228, 45/51/144, 45/51/144/208/226/231/233, 45/51/188/189, 45/51/189/233, 45/51/208/226/231, 45/51/208/233, 45/ 51/226, 45/126/189, 45/126/189/208/226, 45/144/1 89/228, 45/144/226/231/233, 45/188/189, 45/188/189/208/228, 45/188/189/226/228, 45/188/189/231/233, 45/189, 45/189/208, 46, 51, 51/ 126/144/208, 51/126/144/226/231/233/236, 51/126/ 208, 51/144, 51/144/226, 51/188/189/228, 51/189, 51/189/208/226, 51/208, 51/233, 57, 58, 80, 80/135/147, 81, 81/126/144/188/208/228, 86 ,103,126,126/144/188/189/226,134,135,141,142, 143, 143/147/235, 144, 144/188/228, 146, 147, 149, 181, 188, 188/189/233, 189, 207, 208, 208/226/233, 208/228, 208/231/233, 226, 228, 230, 231, 232, 233, 235, 236, 240 and 250, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 776. In some embodiments, the polypeptide sequence of the engineered uridine phosphorylase is the same as SEQ ID NO: 776. NO:776 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of 3N, 3N/8A/22G/36I/181R/235G/250A, 6T, 6T/45G/51I/81W/126Y/226G/233D, 6T/45G/51I/144G, 6T /45G/51I/188R/189Y, 6T/45G/51I/189V/228M/236W, 6T/45G/51I/189Y/208V, 6T/45G/51I/208V/233D, 6T/45G/149T/188R/189Y/208V/233D/236G , 6T/51I/126Y/144G/208V, 6T/51I/126Y/189V/233D, 6T/51I/126Y/189Y/208V/233D/236G, 6T/51I/126Y/18 9Y/231V/236W, 6T/51I/188R/189Y/208V/226G/228M, 6T/51I/188R/189Y/236G, 6T/51I/189Y/208V/231V/233D, 6T/51I/208V/226G/233D, 6T/51 I/208V/231V, 6T/126Y/188R/231V/233D, 6T/144G/208V, 6T/188R/189Y/208V/228M/233D, 8A, 8A/36I/143G/14 7M/235G, 8A/36I/181R/235G, 8A/142L/147C, 8A/147C/181R/250A, 8A/147M/235G, 9V, 19V, 20L, 22G, 22G/147C/181R/235G/250A, 24V, 36I, 36I/1 43G/147C/181R, 36I/143G/147C/235G, 40L, 40V, 41G, 43P, 45G, 45G/51I, 45G/51I/126Y/144G/208V/226G/228M , 45G/51I/144G, 45G/51I/144G/208V/226G/231V/233D, 45G/51I/188R/189V, 45G/51I/189V/233D, 45G/51I/208V/226G/231V, 45G/51I/208V/233D, 45G/51I/226G, 45G/126Y/189Y, 45G/126Y/189Y/208 V/226G, 45G/144G/189V/228M, 45G/144G/226G/231V/233D, 45G/188R/189Y, 45G/188R/189Y/208V/228M, 45 G/188R/189Y/226G/228M, 45G/188R/189Y/231V/233D, 45G/189V, 45G/189V/208V, 45G/189Y, 45T, 46Q, 51I, 51I/126Y/144G/208V, 51I/126Y/144G /226G/231V/233D/236W, 51I/126Y/208V, 51I/144G, 51I/144G/226G, 51I/188R/189Y/228M, 51I/189V/208V /226G, 51I/189Y, 51I/208V, 51I/233D, 57S, 57T, 58T, 80M, 80M/135V/147C, 81W, 81W/126Y/144G/188R/208V/228M, 86L, 103G, 126L, 126Q, 126V, 12 6Y, 126Y/144G/188R/189V/226G, 134L, 135V, 141L, 142I, 142L, 143G, 143G/147C/235G, 144G, 144G/188R/22 8M, 146V, 147C, 147M, 149F, 181R, 188R, 188R/189V/233D, 189T, 189V, 189Y, 207C, 207G, 208V, 208V/226G/233D, 208V/228M, 208V/231V/233D, 226G, 228M, 230E, 231V, 232S, 233D, 235G, 235P, 236A, 236G, 236I, 236W, 240F, 240W, and 250A, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 776. In some embodiments, the polypeptide sequence of the engineered uridine phosphorylase is the same as SEQ ID NO: 776. NO:776 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of E3N, E3N/H8A/A22G/A36I/Q181R/K235G/R250A, S6T, S6T/A45G/T51I/L81W/M126Y/Q226G/S233D, S6T/A45G/T51I/A144G, S6T/A45G /T51I/Q188R/E189Y, S6T/A45G/T51I/E189V/L228M/Q236W, S6T/A45G/T51I/E189Y/S208V, S6T/A45G/T51I/S208V/S233D, S6T/A45G/A149T/Q188R /E189Y/S208V/S233D/Q236G, S6T/T51I/M126Y/A144G/S208V, S6T/T51I/M126Y/E189V/S233D, S6T/T51I/M126Y/E189Y/S208V/S233D/Q236G . V/A231V/S233D, S6T/T51I/S208V/Q226G/S233D, S6T/T51I/S208V/A231V, S6T/M126Y/Q188R/A231V/S233D, S6T/A144G/S208V . 250A, H8A/I147M/K235G, M9V, A19V, T20L, A22G, A22G/I147C/Q181R/K235G/R250A, L24V, A36I, A36I/A143G/I147C/Q181R, A36I/A143 G/I147C/K235G, N40L, N40V, P41G, K43P, A45G, A45G/T51I, A45G/T51I/M126Y/A144G/S208V/Q226G/L228M, A45G/T51I/A144G, A45G/T51I/A144G/S2 08V/Q226G/A231V/S233D, A45G/T51I/Q188R/E189V , A45G/T51I/E189V/S233D, A45G/T51I/S208V/Q226G/A231V, A45G/T51I/S208V/S233D, A45G/T51I/Q226G, A45G/M126Y/E189Y , A45G/M126Y/E189Y/S208V/Q226G, A45G/A144G/E189V/L228M, A45G/A144G/Q226G/A231V/S233D, A45G/Q188R/E189Y, A45G/Q188R/E189Y/S208V /L228M、A45G/Q188R/E189Y/Q226G/L228M、A45G/Q188R/E189Y/A231V/S233D、A45G/E189V、A45G/E189V/S208V、A45G/E189 Y, A45T, S46Q, T51I, T51I/M126Y/A144G/S208V, T51I/M126Y/A144G/Q226G/A231V/S233D/Q236W, T51I/M126Y/S208V, T51I/A144G, T51I/A144G/Q22 6G, T51I/Q188R/E189Y/L228M, T51I/E189V/S208V/Q226G, T51I/E189Y, T51I/S208V, T51I/S233D, L57S, L57T, D58T, I80M , I80M/E135V/I147C, L81W, L81W/M126Y/A144G/Q188R/S208V/L228M, I86L, N103G, M126L, M126Q, M126V, M126Y, M126Y/A144G/Q188R/E189V/Q226 G, F134L, E135V, V141L, E142I, E142L, A143G, A143G/I147C/K235G, A144G, A144G/Q188R/L228M, S146V, I147C, I147M, A149F , Q181R, Q188R, Q188R/E189V/S233D, E189T, E189V, E189Y, A207C, A207G, S208V, S208V/Q226G/S233D, S208V/L228M, S208V/A231V/S233D, Q226G, L228M, N230E, A231V, E232S, S233D, K235G, K235P, Q236A, Q236G, Q236I, Q236W, H240F, H240W and R250A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:776. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 776. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 776. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 776.

In some embodiments, the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 6, 6/8/9/126/147, 6/9/24/181/189, 6/9/181/189/235, 6/9/208/233/235, 6/24/24/181/189/235. /43/46/181/189, 6/24/43/126/147/189, 6/24/46/103/181/208, 6/24/46/147/240, 6/24/103/189, 6/24/126/189, 6/24/147, 6/46/126/147/181/ 235/240, 6/46/147/189/240, 6/103, 6/103/147/230/233, 6/103/189/235, 6/126/181/189, 6/1 26/181/189/235, 6/126/233/235, 6/181/230/233/235, 9/43/46/103/189/233/240, 9/46/126/147/181, 9/46/147/233, 24/43/46/147/230/235 ,24/46/126, 24/46/147/189, 24/46/208/230/233/235, 24/103/126/147, 24/103/126/147/181/ 189/208/233, 24/147, 24/147/189/230/233, 24/181/189/230/233/235, 24/189/230, 24/208, 43/46/126/147/189/240, 43/103/189/208/233, 103/126/189/233/235, 103/147/181, 147/181/233, 189, 189/235 and 208/233, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 868. In some embodiments, the polypeptide sequence of the engineered uridine phosphorylase is the same as SEQ ID NO: 868. NO:868 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of: 6T, 6T/8A/9V/126Y/147C, 6T/9V/24V/181R/189E, 6T/9V/181R/189E/235P, 6T/9V/208V/233D/235P, 6T/24V, 6T/24V/43P/46Q/181R/189E, 6T/24V/43P/46Q/181R/189E, 6T/24V/43P/46Q/181R/189E, 6T/24V/43P/46Q/181R/189E 24V/43P/126Y/147C/189E, 6T/24V/46Q/103G/181R/208V, 6T/24V/46Q/147C/240F, 6T/24V/103G/189E, 6T/24V/126Y/189E, 6T/24V/147C, 6T/46 Q/12 6Y/147C/181R/235P/240F, 6T/46Q/147C/189E/240F, 6T/103G, 6T/103G/147C/230E/233D, 6T/103G/189E/235P, 6T/126Y/181R/189E, 6T/126Y/1 81R/ 189E/235P, 6T/126Y/233D/235P, 6T/181R/230E/233D/235P, 9V/43P/46Q/103G/189E/233D/240W, 9V/46Q/126Y/147C/181R, 9V/46Q/147C/233D, 24V/ 43P/46Q/147C/230E/235P, 24V/46Q/126Y, 24V/46Q/147C/189E, 24V/46Q/208V/230E/233D/235P, 24V/103G/126Y/147C, 24V/103G/126Y/147C/1 81R/ 189E/208V/233D, 24V/147C, 24V/147C/189E/230E/233D, 24V/181R/189E/230E/233D/235P, 24V/189E/230E, 24V/208V, 43P/46Q/126Y/147C/189E/240F, 43P/103G/189E/208V/233D, 103G/126Y/189E/233D/235P, 103G/147C/181R, 147C/181R/233D, 189E, 189E/235P and 208V/233D, wherein the amino acid positions of the polypeptide sequences refer to SEQ ID NO: 868. In some embodiments, the polypeptide sequence of the engineered uridine phosphorylase is the same as SEQ ID NO:868 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions selected from the group consisting of S6T, S6T/H8A/M9V/M126Y/I147C, S6T/M9V/L24V/Q181R/V189E, S6T/M9V/Q181R/V189E/K235P, S6T/M9V/S208V/S233D/K235P, S6T/L24V, S6T/L24V/K43P/S46Q/Q181R/V189E , S6T/L24V/K43P/M126Y/I147C/V189E, S6T/L24V/S46Q/N103G/Q181R/S208V, S6T/L24V/S46Q/I147C/H240F, S6T/L24V/N103G/V189E, S6T/L24 V/M126Y/V189E, S6T/L24V/I147C, S6T/S46Q/M126Y/I147C/Q181R/K235P/H240F, S6T/S46Q/I147C/V189E/H240F, S6T/N103G , S6T/N103G/I147C/N230E/S233D, S6T/N103G/V189E/K235P, S6T/M126Y/Q181R/V189E, S6T/M126Y/Q181R/V189E/K235P, , S6T/Q181R/N230E/S233D/K235P, M9V/K43P/S46Q/N103G/V189E/S233D/H240W, M9V/S46Q/M126Y/I147C/Q181R, M9V/S46Q/I147C/S233D, L24V/K43P/S46Q/I147C/N230E/K235P, L24V/S46Q/M126Y, L24V/S46Q/I147C/V189E, L24V/S46Q/S208V/N230E/S233D/K235P, L24V/N103G/M126Y/I147C, L24V/N103G/M126Y/I147C/Q181R /V189E/S208V/S233D、L24V/I147C、L24V/I147C/V189E/N230E/S233D、L24V/Q181R/V189E/N230E/S233D/ K235P, L24V/V189E/N230E, L24V/S208V, K43P/S46Q/M126Y/I147C/V189E/H240F, K43P/N103G/V189E/S208V/S233D, N103G/M126Y/V189E/S233D/K235P, N103G/I147C/Q181R, I147C/Q181R/S233D, V189E, V189E/K235P and S208V/S233D, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:868. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868. In some embodiments, the engineered uridine phosphorylase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868.

In some additional embodiments, the present invention provides engineered uridine phosphorylases, wherein the engineered uridine phosphorylase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered uridine phosphorylase variant listed in Table 1.2, Table 2.2, Table 3.1, Table 4.1, Table 5.2 and/or Table 6.1.

In some additional embodiments, the invention provides engineered uridine phosphorylases, wherein the engineered uridine phosphorylase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 246, SEQ ID NO: 594, SEQ ID NO: 776 and/or SEQ ID NO: 868. In some embodiments, the engineered uridine phosphorylase comprises a variant engineered uridine phosphorylase listed in SEQ ID NO: 4, SEQ ID NO: 246, SEQ ID NO: 594, SEQ ID NO: 776 and/or SEQ ID NO: 868.

The present invention also provides engineered uridine phosphorylases, wherein the engineered uridine phosphorylase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered uridine phosphorylase variant listed in the even-numbered sequences of SEQ ID NOs: 4-1196.

The present invention also provides engineered uridine phosphorylases, wherein the engineered uridine phosphorylases comprise at least one improved property compared to wild-type Escherichia coli (Escherichiacoli) uridine phosphorylases. In some embodiments, the improved properties include improved activity on substrates. In some other embodiments, the substrates include 5'-isobutyryl ribose-1-phosphate (compound (2)) and uracil (compound (3)). In some other embodiments, the improved properties include improved production of compound (1) from compound (2) and compound (3). In some other embodiments, the engineered uridine phosphorylase is purified. The present invention also provides compositions comprising at least one engineered uridine phosphorylase provided herein.

The present invention also provides polynucleotide sequences encoding at least one engineered uridine phosphorylase provided herein. In some embodiments, the polynucleotide sequence encoding at least one engineered uridine phosphorylase comprises a polynucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 245, SEQ ID NO: 593, SEQ ID NO: 775 and/or SEQ ID NO: 867. In some embodiments, the polynucleotide sequence encoding at least one engineered uridine phosphorylase comprises a polynucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 245, SEQ ID NO: 593, SEQ ID NO: 775, and/or SEQ ID NO: 867, wherein the polynucleotide sequence of the engineered uridine phosphorylase comprises at least one substitution at one or more positions. In some other embodiments, the polynucleotide sequence encoding at least one engineered uridine phosphorylase or its functional fragment comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:245, SEQ ID NO:593, SEQ ID NO:775 and/or SEQ ID NO:867. In some other embodiments, the polynucleotide sequence is operably linked to a control sequence. In some other embodiments, the polynucleotide sequence is codon optimized. In some other embodiments, the polynucleotide sequence comprises a polynucleotide sequence listed in an odd-numbered sequence in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:245, SEQ ID NO:593, SEQ ID NO:775 and/or SEQ ID NO:867.

The present invention also provides an expression vector comprising at least one polynucleotide sequence provided herein. The present invention also provides a host cell comprising at least one expression vector provided herein. In some embodiments, the present invention provides a host cell comprising at least one polynucleotide sequence provided herein.

The present invention also provides a method for producing an engineered uridine phosphorylase in a host cell, the method comprising culturing a host cell provided herein under suitable conditions, thereby producing at least one engineered uridine phosphorylase. In some embodiments, the method further comprises recovering at least one engineered uridine phosphorylase from the culture and/or host cell. In some other embodiments, the method further comprises the step of purifying the at least one engineered uridine phosphorylase.

Description of the invention

The present invention provides engineered uridine phosphorylase (UP), polypeptides having UP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing UP enzymes. The present invention also provides compositions comprising UP enzymes, and methods for using engineered UP enzymes. The present invention is particularly useful for the production of pharmaceutical compounds.

Unless otherwise defined, all technical terms and scientific terms used herein generally have the same meanings as those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the experimental procedures in cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well known in the art and generally adopted. Such technology is well known and described in many textbooks and reference works well known to those skilled in the art. Standard techniques or their modified forms are used for chemical synthesis and chemical analysis. All patents, patent applications, articles and publications mentioned herein (both above and below) are hereby expressly incorporated herein by reference.

Although any suitable methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, some methods and materials are also described herein. It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described, as these may be varied according to the circumstances in which they are used by those skilled in the art. Therefore, the terms defined immediately below are more fully described by reference to the present invention as a whole.

It should be understood that the general description above and the detailed description below are only exemplary and illustrative, rather than limiting the present invention. The section titles used herein are only for organizational purposes and are not to be construed as limiting the subject matter described. Numerical ranges include numbers that limit the range. Therefore, each numerical range disclosed herein is intended to cover each narrower numerical range that falls within such a wider numerical range, as such a narrower numerical range is all explicitly written herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as such a lower (or higher) numerical limit is explicitly written herein.

Abbreviations and definitions

The abbreviations for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

When three-letter abbreviations are used, unless specifically preceded by "L" or "D", or it is clear from the context in which the abbreviation is used, an amino acid can be in the L-configuration or the D-configuration about the α-carbon (Cα). For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented as an amino (N) to carboxyl (C) direction according to conventional practice.

Abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, abbreviated nucleosides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated individually or collectively as ribonucleosides or 2'-deoxyribonucleosides. When a nucleic acid sequence is represented by a string of single-letter abbreviations, the sequence is presented in a 5' to 3' direction according to conventional convention, and the phosphate is not shown.

With reference to the present invention, technical and scientific terms used in the description herein shall have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes more than one polypeptide.

Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. Thus, as used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

It should also be understood that where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific cases, the embodiments may be alternatively described using the language of "consisting essentially of" or "consisting of."

As used herein, the term "about" means an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

As used herein, "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions that they catalyze.

As used herein, "ATCC" refers to the American Type Culture Collection, whose biological deposit collections include genes and strains.

As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

As used herein, "uridine phosphorylase" ("UP") is an enzyme that catalyzes the reversible conversion of 5'-isobutyrylribose-1-phosphate (compound (2)) and uracil (compound (3)) and/or related compounds to 5'-isobutyryluridine (compound (1)) and/or related compounds. The UP enzyme may be naturally occurring, including wild-type E. coli UP enzyme or other uridine phosphorylases or nucleoside transferases present in humans, bacteria, fungi, plants or other species, or the UP enzyme may be an engineered polypeptide produced by human manipulation.

"Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). Included in this definition are D- and L-amino acids, as well as mixtures of D- and L-amino acids, and polymers comprising mixtures of D- and L-amino acids and D- and L-amino acids.

"Amino acids" are referred to herein by either their commonly known three letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

As used herein, "hydrophilic amino acids or residues" refer to amino acids or residues having side chains that exhibit a hydrophobicity less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179: 125-142 [1984]). Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K), and L-Arg (R).

As used herein, "acidic amino acids or residues" refer to hydrophilic amino acids or residues having side chains that exhibit a pKa value of less than about 6 when the amino acid is contained in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of hydrogen ions. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, "basic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that exhibits a pKa value greater than about 6 when the amino acid is contained in a peptide or polypeptide. Basic amino acids generally have positively charged side chains at physiological pH due to association with hydronium ions. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

As used herein, "polar amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH but has at least one bond in which a pair of electrons shared by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T).

As used herein, "hydrophobic amino acids or residues" refer to amino acids or residues having side chains that exhibit a hydrophobicity greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179: 125-142 [1984]). Genetically encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A), and L-Tyr (Y).

As used herein, "aromatic amino acid or residue" refers to a hydrophilic or hydrophobic amino acid or residue having a side chain comprising at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), and L-Trp (W). Although L-His (H) is sometimes classified as a basic residue due to the pKa of its heteroaromatic nitrogen atom, or as an aromatic residue because its side chain includes a heteroaromatic ring, histidine is classified as a hydrophilic residue or as a "constrained residue" (see below) herein.

As used herein, "constrained amino acid or residue" refers to an amino acid or residue with constrained geometry. Herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline has a constrained geometry because it also has a five-membered ring.

As used herein, "non-polar amino acid or residue" refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and has a bond in which an electron pair shared by two atoms is usually equally held by each of the two atoms (i.e., the side chain is not polar). Genetically encoded non-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

As used herein, "aliphatic amino acid or residue" refers to a hydrophobic amino acid or residue with an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L), and L-Ile (I). It is noteworthy that cysteine (or "L-Cys" or "[C]") is unusual because it can form disulfide bridges with other L-Cys (C) amino acids or other sulfonyl or sulfhydryl-containing amino acids. "Cysteine-like residues" include cysteine and other amino acids containing sulfhydryl moieties that can be used to form disulfide bridges. The ability of L-Cys (C) (and other amino acids with -SH side chains) to be present in a peptide in a reduced free-SH or oxidized disulfide-bridged form affects whether L-Cys (C) contributes a net hydrophobic or hydrophilic character to the peptide. Although L-Cys(C) exhibits a hydrophobicity of 0.29 according to Eisenberg's normalized consensus scale (Eisenberg et al., 1984, supra), it is understood that for the purposes of the present disclosure, L-Cys(C) is classified into its own unique group.

As used herein, "small amino acid or residue" refers to an amino acid or residue having a side chain comprising three or fewer carbon atoms and/or heteroatoms (excluding α-carbon and hydrogen). According to the above definition, small amino acids or residues can be further classified as aliphatic, non-polar, polar or acidic small amino acids or residues. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D).

As used herein, "hydroxyl-containing amino acid or residue" refers to an amino acid containing a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides covalently linked together. A polynucleotide may be completely comprised of ribonucleotides (i.e., RNA), completely comprised of 2' deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2' deoxyribonucleotides. Although nucleosides are usually linked together via standard phosphodiester bonds, polynucleotides may include one or more non-standard bonds. A polynucleotide may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions. In addition, although a polynucleotide may generally include naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may also include one or more modified and/or synthesized nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthesized nucleobases are nucleobases encoding amino acid sequences.

As used herein, "nucleoside" refers to a glycosylamine comprising a nucleoside base (i.e., a nitrogenous base) and a 5-carbon sugar (e.g., ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In contrast, the term "nucleotide" refers to a glycosylamine comprising a nucleoside base, a 5-carbon sugar, and one or more phosphate groups. In some embodiments, nucleosides can be phosphorylated by kinases to produce nucleotides.

As used herein, "nucleoside diphosphate" refers to a glycosylamine comprising a nucleoside base (i.e., a nitrogenous base), a 5-carbon sugar (e.g., ribose or deoxyribose) and a diphosphate (i.e., pyrophosphate) portion. In some embodiments herein, "nucleoside diphosphate" is abbreviated as "NDP". Non-limiting examples of nucleoside diphosphates include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP) and inosine diphosphate (IDP). In some cases, the terms "nucleoside" and "nucleotide" are used interchangeably.

As used herein, "coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

As used herein, the terms "biocatalysis," "biocatalytic," "bioconversion," and "biosynthesis" refer to the use of enzymes to perform chemical reactions on organic compounds.

As used herein, "wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

As used herein, "recombinant," "engineered," "variant," and "non-naturally occurring" when used with respect to cells, nucleic acids, or polypeptides refer to materials that have been modified in a manner that does not otherwise exist in nature or to materials that correspond to the native or native form of the material. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived from synthetic materials and/or through manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not found in the natural (non-recombinant) form of the cell or that express natural genes that are otherwise expressed at different levels.

The term "percentage (%) of sequence identity" is used herein to refer to the comparison between polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences. The percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences or where the nucleic acid base or amino acid residue is aligned with a gap to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used to align two sequences. Optimal alignment of sequences for comparison can be performed by any suitable method, including, but not limited to, the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482 [1981]), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443 [1970]), by the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection, as is known in the art. Examples of algorithms suitable for determining percentages of sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms, described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 215: 403-410 [1990]; and Altschul et al., Nucl. Acids Res., 3389-3402 [1977]). Software for performing BLAST analysis is available to the public through the website of the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (see, Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence to the extent that the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extensions in each direction are stopped when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score reaches 0 or less due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as defaults: word length (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989]). Exemplary determinations of sequence alignments and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

As used herein, "reference sequence" refers to a defined sequence used as a basis for sequence and/or activity comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the total length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) be included in a sequence similar to the two sequences (i.e., a part of a complete sequence), and (2) can also be included in a divergent sequence between the two sequences, the sequence comparison between two (or more) polynucleotides or polypeptides is usually performed by comparing the sequences of the two polynucleotides or polypeptides on a "comparison window" to identify and compare the sequence similarity of a local region. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more variations in a primary sequence.

As used herein, "comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window may include 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100 or more.

As used herein, "corresponding to," "reference," and "relative to," when used in the context of numbering a given amino acid or polynucleotide sequence, refer to numbering the residues of a given reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to a reference sequence, rather than being specified by the actual numerical positions of the residues within the given amino acid or polynucleotide sequence. For example, given an amino acid sequence, such as an engineered uridine phosphorylase, the residue matching between the two sequences can be optimized by introducing gaps to align with the reference sequence. In these cases, the residues in a given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned, despite the presence of gaps.

As used herein, "substantially identical" refers to a polynucleotide or polypeptide sequence having at least 80% sequence identity, at least 85% identity, at least 89% to 95% sequence identity, or more generally at least 99% sequence identity in a comparison window of at least 20 residue positions, usually in a window of at least 30-50 residues, compared to a reference sequence, wherein the percentage of sequence identity is calculated by comparing the reference sequence and a sequence comprising a deletion or addition totaling 20% or less of the reference sequence over the comparison window. In some specific embodiments applied to polypeptides, the term "substantially identical" means that when the best alignment is performed using the default gap weights such as by the program GAP or BESTFIT, two polypeptide sequences share at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or higher (e.g., 99% sequence identity). In some embodiments, the residue positions that are not identical in the compared sequences differ due to conservative amino acid substitutions.

As used herein, "amino acid difference" and "residue difference" refer to the difference of the amino acid residue at one position of the polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. In some cases, the reference sequence has a histidine tag, but the numbering remains unchanged relative to the equivalent reference sequence without a histidine tag. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X93 compared to SEQ ID NO:4" refers to the difference of the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:4. Therefore, if the reference polypeptide SEQ ID NO:4 has serine at position 93, "the residue difference at position X93 compared to SEQ ID NO:4" refers to the amino acid substitution of any residue except serine at the polypeptide position corresponding to position 93 of SEQ ID NO:4. In most examples herein, the specific amino acid residue difference at one position is indicated as "XnY", where "Xn" specifies the corresponding position as described above, and "Y" is a single letter identifier of the amino acid found in the engineered polypeptide (i.e., a residue different from the reference polypeptide). In some examples (e.g., in the tables presented in the Examples), the present invention also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a single-letter identifier for a residue in a reference sequence, "n" is the number of a residue position in a reference sequence, and B is a single-letter identifier for a residue substitution in the sequence of an engineered polypeptide. In some examples, a polypeptide of the present invention may comprise one or more amino acid residue differences relative to a reference sequence, indicated by a column of designated positions where residue differences exist relative to a reference sequence. In some embodiments, when more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by "/" (e.g., X307H/X307P or X307H/P). Slashes can also be used to indicate more than one substitution within a given variant (i.e., there is more than one substitution in a given sequence such as in a combinatorial variant). In some embodiments, the present invention includes engineered polypeptide sequences containing one or more amino acid differences, the amino acid differences comprising conservative amino acid substitutions or non-conservative amino acid substitutions. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative amino acid substitutions and non-conservative amino acid substitutions.

As used herein, "conservative amino acid substitutions" refer to the replacement of a residue with a different residue having a similar side chain, and thus generally include the replacement of an amino acid in a polypeptide with an amino acid in the same or similar amino acid defined class. For example, but not limited to, in some embodiments, an amino acid with an aliphatic side chain is replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and/or a hydrophobic amino acid or a hydrophilic amino acid is replaced by another hydrophobic amino acid or a hydrophilic amino acid, respectively.

As used herein, "non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups, and affect (a) the structure of the peptide backbone in the region of the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

As used herein, "deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include the removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids that make up the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and/or retaining the improved properties of the engineered uridine phosphorylase. Deletion can involve internal portions and/or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous. Deletions in amino acid sequences are generally represented by "-".

As used herein, "insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. The insertion may be in an internal portion of the polypeptide or may be an insertion into the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions may be continuous stretches of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

The term "amino acid substitution set" or "substitution set" refers to a set of amino acid substitutions in a polypeptide sequence compared to a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, a substitution set refers to a collection of amino acid substitutions present in any of the variant uridine phosphorylases listed in the tables provided in the Examples.

"Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to polypeptides having an amino-terminal deletion and/or a carboxyl-terminal deletion and/or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., a full-length engineered uridine phosphorylase of the invention) and retains substantially all of the activity of the full-length polypeptide.

As used herein, "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that are naturally associated with it. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or via in vitro synthesis). Recombinant uridine phosphorylase polypeptides can be present in cells, in cell culture media, or prepared in various forms (such as lysates or isolated preparations). Therefore, in some embodiments, the recombinant uridine phosphorylase polypeptide can be an isolated polypeptide.

As used herein, "substantially pure polypeptide" or "purified protein" refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition), and when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight, it is generally a substantially purified composition. However, in some embodiments, the composition comprising uridine phosphorylase comprises less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40% or about 50%) uridine phosphorylase. Typically, a substantially pure uridine phosphorylase composition constitutes about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition by mole or % weight. In some embodiments, the target species is purified to substantial homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species.In some embodiments, the isolated recombinant uridine phosphorylase polypeptide is a substantially pure polypeptide composition.

As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the invention provides an engineered uridine phosphorylase polypeptide that shows an improvement in any enzyme property compared to a reference uridine phosphorylase polypeptide and/or a wild-type uridine phosphorylase polypeptide and/or another engineered uridine phosphorylase polypeptide. Therefore, the level of "improvement" can be determined and compared between various uridine phosphorylase polypeptides, including wild-type and engineered uridine phosphorylases. Improved properties include, but are not limited to, properties such as: increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzyme activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and changed temperature profiles. In other embodiments, the term is used to refer to at least one improved property of uridine phosphorylase. In some embodiments, the invention provides engineered uridine phosphorylase polypeptides that demonstrate improvements in any enzyme property compared to a reference uridine phosphorylase polypeptide and/or a wild-type uridine phosphorylase polypeptide and/or another engineered uridine phosphorylase polypeptide. Therefore, the level of "improvement" can be determined and compared between various uridine phosphorylase polypeptides, including wild-type and engineered uridine phosphorylases.

As used herein, "increased enzymatic activity" and "enhanced catalytic activity" refer to the improved properties of engineered polypeptides, which can be expressed as an increase in specific activity (e.g., product produced/time/weight protein) or an increase in the percentage of conversion of substrate to product (e.g., using a specified amount of enzyme, the percentage of conversion of the starting amount of substrate to product within a specified time period) compared to a reference enzyme. In some embodiments, these terms refer to the improved properties of engineered uridine phosphorylase polypeptides provided herein, which can be expressed as an increase in specific activity (e.g., product produced/time/weight protein) or an increase in the percentage of conversion of substrate to product (e.g., using a specified amount of uridine phosphorylase, the percentage of conversion of the starting amount of substrate to product within a specified time period) compared to a reference uridine phosphorylase. In some embodiments, these terms are used to refer to the improved uridine phosphorylase provided herein. Exemplary methods for determining the enzymatic activity of the engineered uridine phosphorylase of the present invention are provided in the Examples. Any property associated with enzymatic activity can be affected, including typical enzyme properties K _m , V _max or k _cat , changes of which can result in an increase in enzymatic activity. For example, the improvement in enzyme activity can be from about 1.1 times the enzyme activity of the corresponding wild-type enzyme to up to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more enzyme activity compared to a naturally occurring uridine phosphorylase or another engineered uridine phosphorylase from which the uridine phosphorylase polypeptide is derived.

As used herein, "conversion" refers to the enzymatic conversion (or bioconversion) of one or more substrates into one or more corresponding products. "Conversion percentage" refers to the percentage of substrate converted to product under specified conditions within a certain period of time. Therefore, the "enzyme activity" or "activity" of a uridine phosphorylase polypeptide can be expressed as the "conversion percentage" of substrate to product within a specific period of time.

An enzyme with "generalist properties" (or "generalist enzymes") refers to an enzyme that exhibits improved activity on a wide range of substrates compared to the parent sequence. A generalist enzyme need not exhibit improved activity for every possible substrate. In some embodiments, the invention provides uridine phosphorylase variants with generalist properties because they exhibit similar or improved activity on a wide range of spatially and electronically different substrates relative to the parent gene. In addition, the generalist enzymes provided herein are engineered to be improved across a wide range of differentiated molecules to increase the production of metabolites/products.

The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, the stability of the hybrid is reflected in the melting temperature ( _Tm ) of the hybrid. Typically, the stability of the hybrid varies with ionic strength, temperature, G/C content, and the presence of a chaotropic agent. The _Tm value of a polynucleotide can be calculated using known methods for predicting melting temperatures (see, e.g., Baldino et al., Meth. Enzymol., 168:761-777 [1989]; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390 [1962]; Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897 [1986]; Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377 [1986]; Kierzek et al., Biochem., 25:7840-7846 [1986]; Rychlik et al., Nucl. Acids Res., 18:6409-6412 [1990] (erratum, Nucl. Acids Res., 19:698 [1991]); Sambrook et al., supra); Suggs et al., 1981, in Developmental Biology Using Purified Genes , Brown et al., [eds.], pp. 683-693, Academic Press, Cambridge, MA [1981]; and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-259 [1991]). In some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered uridine phosphorylase of the invention.

As used herein, "hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washings of different but higher stringency. The term "moderate stringency hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids having about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity with target polynucleotides. Exemplary moderate stringency conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart solution, 5×SSPE, 0.2%SDS, followed by washing at 42°C in 0.2×SSPE, 0.2%SDS. "High stringency hybridization" generally refers to conditions that differ by about 10°C or less from the thermal melting temperature _Tm as determined under solution conditions for a defined polynucleotide sequence. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65° C. (i.e., if the hybrid is unstable in 0.018 M NaCl at 65° C., it will be unstable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridizing under conditions equivalent to 50% formamide, 5× Denhart solution, 5× SSPE, 0.2% SDS at 42° C., followed by washing in 0.1× SSPE and 0.1% SDS at 65° C. Another high stringency condition is hybridization under conditions equivalent to hybridization in 5× SSC containing 0.1% (w/v) SDS at 65° C. and washing in 0.1× SSC containing 0.1% SDS at 65° C. Other high stringency hybridization conditions, as well as moderate stringency conditions, are described in the references cited above.

As used herein, "codon optimized" refers to that the codons of the polynucleotides encoding proteins are changed to those codons preferentially used in a particular organism so that the encoded protein is effectively expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that the codon usage of a particular organism is non-random and biased for specific codon triplets. With respect to the aggregated protein coding region of a given gene, a gene with a common function or ancestral origin, a high-expression protein contrast low copy number protein, and an organism's genome, this codon usage bias may be higher. In some embodiments, codon optimization can be performed on the polynucleotides encoding uridine phosphorylase for optimized production in a host organism selected for expression.

As used herein, "preferred,""optimal," and "high codon usage bias" codons, when used alone or in combination, refer interchangeably to codons in a protein coding region that are used at a higher frequency than other codons encoding the same amino acid. Preferred codons can be determined based on codon usage in a single gene, a group of genes with a common function or origin, highly expressed genes, codon frequency in aggregating protein coding regions of an entire organism, codon frequency in aggregating protein coding regions of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are generally the optimal codons for expression. Various methods for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in a particular organism are known, including multivariate analysis, such as using cluster analysis or correlation analysis, and the effective number of codons used in a gene (see, e.g., GCG Codon Preference, Genetics Computer Group Wisconsin Package; Codon W, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73 [1998]; Stenico et al., Nucl. Acids Res., 222437-46 [1994]; and Wright, Gene 87:23-29 [1990]). Codon usage tables for many different organisms are available (see, e.g., Wada et al., Nucl. Acids Res., 20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000]; Duret et al., supra; Henaut and Danchin, in Escherichiacoli and Salmonella , Neidhardt et al. (Eds.), ASM Press, Washington D.C., pp. 2047-2066 [1996]). The data source for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein. These data sets include nucleic acid sequences that are actually known to encode expressed proteins (e.g., complete protein coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see, e.g., Mount, Bioinformatics: Sequence and Genome Analysis , Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [2001]; Uberbacher, Meth. Enzymol., 266:259-281 [1996]; and Tiwari et al., Comput. Appl. Biosci., 13:263-270 [1997]).

As used herein, "control sequences" include all components that are necessary or advantageous for the expression of the polynucleotides and/or polypeptides of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided with a linker for the purpose of introducing specific restriction sites that facilitate the connection of the control sequence to the coding region of the nucleic acid sequence encoding the polypeptide.

"Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed (ie, in a functional relationship) relative to a polynucleotide of interest such that the control sequence directs or regulates expression of the polynucleotide and/or polypeptide of interest.

"Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence includes transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that shows transcriptional activity in a selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

The phrase "suitable reaction conditions" refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvents, etc.) under which the uridine phosphorylase polypeptide of the invention is able to convert the substrate into a desired product compound. Some exemplary "suitable reaction conditions" are provided herein.

As used herein, "loading," such as in "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

As used herein, in the context of an enzymatic conversion reaction process, "substrate" refers to a compound or molecule that is acted upon by an engineered enzyme (eg, an engineered uridine phosphorylase polypeptide) provided herein.

As used herein, an "increased" yield of a product (e.g., a deoxyribose phosphate analog) produced by a reaction occurs when the presence of a particular component (e.g., uridine phosphorylase) during the reaction results in the production of more product compared to a reaction performed under the same conditions with the same substrate and other substituents, but in the absence of the component of interest.

A reaction is said to be "substantially free" of a particular enzyme if the amount of that enzyme is less than about 2%, about 1%, or about 0.1% (wt/wt) compared to other enzymes involved in catalyzing the reaction.

As used herein, "fractionating" a liquid (e.g., culture broth) means applying a separation process (e.g., salt precipitation, column chromatography, size exclusion, and filtration) or a combination of these processes to provide a solution in which the desired protein constitutes a greater percentage of the total protein in the solution than in the initial liquid product.

As used herein, "starting composition" refers to any composition comprising at least one substrate. In some embodiments, the starting composition comprises any suitable substrate.

As used herein, "product" in the context of an enzymatic conversion process refers to a compound or molecule resulting from the action of an enzyme polypeptide on a substrate.

As used herein, "equilibrium" as used herein refers to the process that leads to a steady-state concentration of a chemical species in a chemical or enzymatic reaction (e.g., the interconversion of two species A and B), including the interconversion of stereoisomers, as determined by the forward rate constant and the reverse rate constant of the chemical or enzymatic reaction.

As used herein, "alkyl" refers to a saturated hydrocarbon group having from 1 to 18 carbon atoms (inclusive), linear or branched, more preferably from 1 to 8 carbon atoms (inclusive), and most preferably 1 to 6 carbon atoms (inclusive). Alkyl groups having a specified number of carbon atoms are indicated in brackets (e.g., (C1-C4) alkyl refers to alkyl groups of 1 to 4 carbon atoms).

As used herein, "alkenyl" refers to a group having from 2 to 12 carbon atoms (inclusive), linear or branched, containing at least one double bond but optionally containing more than one double bond.

As used herein, "alkynyl" refers to a group having from 2 to 12 carbon atoms (inclusive), straight or branched, containing at least one triple bond but optionally containing more than one triple bond, and further optionally containing one or more double bonded moieties.

As used herein, "heteroalkyl", "heteroalkenyl" and "heteroalkynyl" refer to alkyl, alkenyl and alkynyl groups as defined herein in which one or more carbon atoms are each independently replaced by the same or different heteroatoms or heteroatom groups. The heteroatoms and/or heteroatom groups that may replace carbon atoms include, but are not limited to, -O-, -S-, -SO-, -NRα-, -PH-, -S(O)-, -S(O) ₂ -, -S(O)NRα-, -S(O) ₂ NRα-, and the like, including combinations thereof, wherein each Rα is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

As used herein, "alkoxy" refers to a group -OR[beta], wherein R[beta] is an alkyl group as defined above, including optionally substituted alkyl groups also as defined herein.

As used herein, "aryl" refers to an unsaturated aromatic carbocyclic group having from 6 to 12 carbon atoms (including endpoints) having a single ring (e.g., phenyl) or more than one condensed ring (e.g., naphthyl or anthracenyl). Exemplary aryl groups include phenyl, pyridyl, naphthyl, etc.

As used herein, "amino" refers to the group -NH2. Substituted amino refers to the groups -NHRδ, NRδRδ and NRδRδRδ, where each Rδ is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, etc. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfonylamino, etc.

As used herein, "oxygen/oxo" refers to =0.

As used herein, "oxy" refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

As used herein, "carboxyl" refers to -COOH.

As used herein, "carbonyl" refers to -C(O)-, which can have various substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

As used herein, "alkyloxycarbonyl" refers to -C(O)ORε, wherein Rε is an alkyl group as defined herein, which may be optionally substituted.

As used herein, "aminocarbonyl" refers to -C(O)NH ₂ . Substituted aminocarbonyl refers to -C(O)NRδRδ, wherein the amino group NRδRδ is as defined herein.

As used herein, "halogen" and "halo" refer to fluorine, chlorine, bromine and iodine.

As used herein, "hydroxy" refers to -OH.

As used herein, "cyano" refers to -CN.

As used herein, "heteroaryl" refers to an aromatic heterocyclic group having 1 to 10 carbon atoms (inclusive) and 1 to 4 heteroatoms (inclusive) selected from oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups may have a single ring (e.g., pyridyl or furanyl) or more than one condensed ring (e.g., indolizinyl or benzothienyl).

As used herein, "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group (i.e., a heteroaryl-alkyl-group), preferably having from 1 to 6 carbon atoms (inclusive) in the alkyl portion and from 5 to 12 ring atoms (inclusive) in the heteroaryl portion. Examples of such heteroarylalkyl groups are pyridylmethyl and the like.

As used herein, "heteroarylalkenyl" refers to an alkenyl group substituted with a heteroaryl group (i.e., a heteroaryl-alkenyl- group), preferably having from 2 to 6 carbon atoms (inclusive) in the alkenyl portion and from 5 to 12 ring atoms (inclusive) in the heteroaryl portion.

As used herein, "heteroarylalkynyl" refers to an alkynyl group substituted with a heteroaryl group (i.e., a heteroaryl-alkynyl-group), preferably having from 2 to 6 carbon atoms (inclusive) in the alkynyl portion and from 5 to 12 ring atoms (inclusive) in the heteroaryl portion.

As used herein, "heterocycle", "heterocyclic" and interchangeably "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or more than one fused ring, having from 2 to 10 carbon ring atoms (inclusive) and from 1 to 4 heterocyclic atoms (inclusive) selected from nitrogen, sulfur or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuranyl) or more than one fused ring (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline, and the like.

As used herein, "membered ring" is intended to encompass any cyclic structure. The number before the term "membered" represents the number of backbone atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyrans, and thiopyrans are 6-membered rings, and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

Unless otherwise specified, the positions occupied by hydrogen in the foregoing groups may be further substituted with substituents such as, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxyl, alkoxycarbonyl, formylamino, substituted formylamino, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonylamino, substituted sulfonylamino, cyano, amino, substituted The preferred heteroatoms are amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy and (heterocycle)alkyl; and preferred heteroatoms are oxygen, nitrogen and sulfur. It is understood that when open valences are present on these substituents, they may be further substituted with alkyl, cycloalkyl, aryl, heteroaryl and/or heterocyclic groups, when these open valences are present on carbon, they may be further substituted with halogens and oxygen-, nitrogen- or sulfur-bonded substituents, and when more than one such open valence is present, these groups may be connected to form a ring by direct bond formation or by bonding to new heteroatoms (preferably, oxygen, nitrogen or sulfur). It is also understood that the above substitutions may be made provided that replacement of hydrogen with a substituent does not introduce unacceptable instability into the molecules of the invention and is otherwise chemically reasonable.

As used herein, the term "culturing" refers to the growth of a population of microbial cells under any suitable conditions (eg, using liquid, gel, or solid culture media).

Recombinant polypeptides can be produced using any suitable method known in the art. The gene encoding the wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host such as Escherichia coli. Variants of recombinant polypeptides can be produced by various methods known in the art. In fact, there are various different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits can also be obtained from many commercial molecular biology suppliers. The method can be used to make specific substitutions at a determined amino acid (site-directed), specific (region-specific) mutations or random mutations in a local region of a gene, or random mutagenesis (e.g., saturation mutagenesis) in the entire gene. Many suitable methods for producing enzyme variants are known to those skilled in the art, including but not limited to, using PCR to single-stranded DNA or double-stranded DNA site-directed mutagenesis, cassette mutagenesis, gene synthesis, error-prone PCR, reorganization and chemical saturation mutagenesis, or any other suitable method known in the art. Mutagenesis and directed evolution methods can be easily applied to polynucleotides encoding enzymes to produce variant libraries that can be expressed, screened and measured. Any suitable mutagenesis and directed evolution methods can be used in the present invention and are well known in the art (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6, No. 132,970, No. 6,165,793, No. 6,180,406, No. 6,251,674, No. 6,265,201, No. 6,277,638, No. 6,287,861, No. 6,287,862, No. 6,291,242, No. 6,297,053, No. 6,303,344, No. 6,309,883 No. 6,319,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, No. 6,358,740, No. 6,358,742, No. 6,36 No. 5,377, No. 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,337,186, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6,391,552, No. 6,391,640, No. 6,395,547, No. 6,406,855, No. 6,406,910, No. 6,413,745, No. 6,413,774, No. 6,420,175, No. 6,423,542, No. 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253, No. 6,479,652, No. 6,482,647, No. 6,483, 011, No. 6,484,105, No. 6,489,146, No. 6,500,617, No. 6,500,639, No. 6,506,602, No. 6,506,603, No. 6,518,065, No. 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,746, No. 6 ,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,605,430, No. 6,613,514, No. 6,653,072, No. 6,686,515, No. 6,703,240, No. 6,716,631, No. 6,825,00 No. 1, No. 6,902,922, No. 6,917,882, No. 6,946,296, No. 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,054, No. 7,220,566, No. 7,288,375, No. 7,3 No. 84,387, No. 7,421,347, No. 7,430,477, No. 7,462,469, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629,170, No. 7,702,464, No. 7,747,391, No. 7,747,393, No. 7,751,986 No. 7,776,598, No. 7,783,428, No. 7,795,030, No. 7,853,410, No. 7,868,138, No. 7,783,428, No. 7,873,477, No. 7,873,499, No. 7,904,249, No. 7,957,912, No. 7,981,614, No. 8,01 No. 4,961, No. 8,029,988, No. 8,048,674, No. 8,058,001, No. 8,076,138, No. 8,108,150, No. 8,170,806, No. 8,224,580, No. 8,377,681, No. 8,383,346, No. 8,457,903, No. 8,504,498, Nos. 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,665,694, 9,684,771, and all related U.S. and PCT and non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale ... et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol. , 17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of which are incorporated herein by reference).

In some embodiments, the enzyme clones obtained after mutagenesis are screened by subjecting the enzyme preparation to a specified temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatment or other suitable assay conditions. Clones containing polynucleotides encoding polypeptides are then isolated from the gene, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in a host cell. Measuring enzyme activity from an expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques, such as HPLC analysis).

Once variants are generated, they can be screened for any desired property (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermal stability and/or acidic pH stability, etc.). In some embodiments, "recombinant uridine phosphorylase polypeptides" (also referred to herein as "engineered uridine phosphorylase polypeptides," "variant uridine phosphorylases," "uridine phosphorylase variants," and "uridine phosphorylase combinatorial variants") can be used.

As used herein, "vector" is a DNA construct for importing a DNA sequence into a cell. In some embodiments, a vector is an expression vector operably linked to a suitable control sequence capable of achieving expression of a polypeptide encoded in a DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgenic) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

As used herein, the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from a cell.

As used herein, the term "production" refers to the production of proteins and/or other compounds by cells. It is intended that the term includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of a polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is heterologous to another sequence to which it is operably linked if the two sequences are not associated in nature. For example, a "heterologous" polynucleotide is any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector comprising a DNA as provided herein (e.g., a polynucleotide encoding a uridine phosphorylase variant). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques as known in the art.

The term "analog" means a polypeptide having more than 70% sequence identity, but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, an analog means a polypeptide comprising one or more non-naturally occurring amino acid residues (including but not limited to homoarginine, ornithine and norvaline) and naturally occurring amino acids. In some embodiments, an analog also includes one or more D-amino acid residues and non-peptide connections between two or more amino acid residues.

The term "effective amount" means an amount sufficient to produce the desired result. An ordinary technician in this field can determine the effective amount by using routine experiments.

The terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components that are separated from at least one other component with which they are naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

As used herein, "stereoselectivity" refers to the preferential formation of one stereoisomer over another stereoisomer in a chemical or enzymatic reaction. Stereoselectivity can be partial, where the formation of one stereoisomer is superior to another, or it can be complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, i.e. the fraction of one enantiomer in the sum of the two (usually reported as a percentage). The art usually optionally reports it as the enantiomeric excess ("e.e.") (usually a percentage) calculated therefrom according to the following formula: [major enantiomer-minor enantiomer]/[major enantiomer+minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity is referred to as diastereoselectivity, i.e. the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), usually optionally reported as diastereomeric excess ("d.e."). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

As used herein, "regioselectivity" and "regioselective reaction" refer to a reaction in which one direction of bond formation or cleavage occurs in preference to all other possible directions. A reaction can be completely (100%) regioselective if the discrimination is complete, substantially regioselective (at least 75%) if the reaction product of one site predominates over the reaction products of other sites, or partially regioselective (x%, where the percentage depends on the reaction setting of interest).

As used herein, "chemoselectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

As used herein, "pH stable" refers to a uridine phosphorylase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hours) compared to the untreated enzyme.

As used herein, "thermostable" refers to a uridine phosphorylase polypeptide that retains similar activity (e.g., more than 60% to 80%) after exposure to an elevated temperature (e.g., 40-80°C) for a certain period of time (e.g., 0.5-24 h) compared to a wild-type enzyme exposed to the same elevated temperature.

As used herein, "solvent stable" refers to a uridine phosphorylase polypeptide that retains similar activity (e.g., more than 60% to 80%) after exposure to varying concentrations (e.g., 5%-99%) of a solvent (ethanol, isopropanol, dimethyl sulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a certain period of time (e.g., 0.5 h to 24 h) compared to a wild-type enzyme exposed to the same solvent at the same concentration.

As used herein, "thermostable and solvent stable" refers to a uridine phosphorylase polypeptide that is both thermostable and solvent stable.

As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances in which the event or circumstance does not occur. One of ordinary skill in the art will understand that for any molecule described as containing one or more optional substituents, only sterically feasible and/or synthetically feasible compounds are intended to be included.

As used herein, "optionally substituted" refers to all subsequent modifiers in a term or a series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" portion and the "aryl" portion of the molecule may or may not be substituted, and for a series of "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl, cycloalkyl, aryl and heteroaryl groups may or may not be substituted independently of each other.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides engineered uridine phosphorylase (UP), polypeptides having UP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing UP enzymes. The present invention also provides compositions comprising UP enzymes, and methods for using engineered UP enzymes. The present invention is particularly useful for the production of pharmaceutical compounds.

An increasing number of unnatural nucleoside analogs are being investigated for the treatment of cancer and viral infections, such as COVID-19. Industrial process conditions generally require the efficient production of unnatural nucleosides using readily available and cost-effective intermediates and substrates. One such intermediate is compound (1), 5'-isobutyryl uridine.

There is a need in the art for methods of synthesizing compound (1) under industrial process conditions, more particularly green chemistry methods. One such method is to use a biocatalyst or an engineered enzyme to produce compound (1). In some embodiments, the present disclosure provides an engineered uridine phosphorylase for producing compound (1). The engineered uridine phosphorylase of the present disclosure produces compound (1) from a 5'-isobutyryl ribose-1-phosphate substrate compound (2) and a uracil compound (3). See Scheme 1 below.

Inorganic phosphate is also produced in the conversion of compounds (2) and (3) to compound (1) (not shown). Addition of sucrose phosphorylase (or any of a number of enzymes that consume inorganic phosphate, such as pyruvate oxidase) is used to drive the equilibrium of the reversible reaction toward the product compound (1).

Engineered uridine phosphorylases with improved properties compared to naturally occurring uridine phosphorylases can be used under relevant process conditions and/or in multi-enzyme systems. These engineered UP enzymes can result in increased production of compound (1) and/or can have other improved properties.

There is a need for engineered UPs that have improved activity and operate under typical industrial conditions and/or as part of a multi-enzyme system. The present invention addresses this need and provides engineered UPs suitable for use in these and other reactions under industrial conditions.

Engineered UP peptides

The present invention provides engineered UP polypeptides, polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and methods for using the polypeptides. When describing a polypeptide, it should be understood that it also describes a polynucleotide encoding the polypeptide. In some embodiments, the present invention provides an engineered, non-naturally occurring UP enzyme with improved properties compared to a wild-type UP enzyme. Any suitable reaction conditions can be used in the present invention. In some embodiments, methods are used to analyze the improved properties of the engineered polypeptide for phosphorylation reactions. In some embodiments, as further described below and in the Examples, the reaction conditions are changed according to the concentration or amount of the engineered UP, one or more substrates, one or more buffers, one or more solvents, pH, conditions including temperature and reaction time, and/or the conditions under which the engineered UP polypeptide is fixed on a solid support.

In some embodiments, additional reaction components or additional techniques are used to supplement reaction conditions. In some embodiments, these include taking measures to stabilize the enzyme or prevent enzyme inactivation, reduce product inhibition, and shift the reaction equilibrium toward the desired product formation.

In some other embodiments, any of the above-described methods for converting a substrate compound into a product compound may also include one or more steps selected from the following: extraction, separation, purification, crystallization, filtration and/or lyophilization of the product compound. Methods, techniques and protocols for extracting, separating, purifying and/or crystallizing products from a biocatalytic reaction mixture produced by the methods provided herein are known to those of ordinary skill and/or are obtainable by routine experimentation. In addition, illustrative methods are provided in the examples hereinafter.

Engineered UP polynucleotides encoding engineered polypeptides, expression vectors and host cells

The present invention provides polynucleotides encoding engineered enzyme polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing a polypeptide. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding an engineered enzyme polypeptide is introduced into an appropriate host cell to express the corresponding enzyme polypeptide.

As will be apparent to those skilled in the art, the availability of protein sequences and the knowledge of codons corresponding to a variety of amino acids provide an explanation of all polynucleotides that can encode subject polypeptides. The degeneracy of genetic coding (wherein the same amino acid is encoded by optional or synonymous codons) allows a very large number of nucleic acids to be prepared, all of which encode engineered enzymes (e.g., UP) polypeptides. Therefore, the invention provides methods and compositions for producing each possible enzyme polynucleotide variation that can be prepared, the enzyme polynucleotides encode enzyme polypeptides described herein by selecting a combination based on possible codon options, and all such variations are considered to be specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the embodiments (e.g., in each table).

In some embodiments, codons are preferably optimized for protein production by the selected host cell. For example, the preferred codons used in bacteria are generally used for expression in bacteria. Therefore, the codon-optimized polynucleotides encoding engineered enzyme polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% of the codon positions in the full-length coding region.

In some embodiments, the enzyme polynucleotide encodes an engineered polypeptide having enzyme activity and properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from a reference sequence of a SEQ ID NO provided herein, or any variant (e.g., those provided in the Examples), and one or more residue differences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions) compared to the amino acid sequence of one or more reference polynucleotides or any variant as disclosed in the Examples. In some embodiments, the reference polypeptide sequence is selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:246, SEQ ID NO:594, SEQ ID NO:776 and/or SEQ ID NO:868.

In some embodiments, polynucleotide can be selected from any polynucleotide sequence provided herein or its complementary sequence or the polynucleotide sequence of encoding any variant enzyme polypeptide provided herein hybridized under high stringency conditions. In some embodiments, the polynucleotide encoding enzyme polypeptide that can hybridize under high stringency conditions comprises an amino acid sequence with one or more residue differences compared to the reference sequence.

In some embodiments, the polynucleotide of any separation in the engineered enzyme polypeptide of encoding this paper is manipulated in various ways to promote the expression of enzyme polypeptide.In some embodiments, the polynucleotide of encoding enzyme polypeptide constitutes expression vector, wherein there is one or more control sequence to regulate the expression of enzyme polynucleotide and/or polypeptide.According to the expression vector used, the manipulation of the polynucleotide of separation before the polynucleotide of separation is inserted into the vector can be desired or necessary.The technology of utilizing recombinant DNA method to modify polynucleotide and nucleotide sequence is well known in the art.In some embodiments, control sequence comprises, among others, promoter, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence and transcription terminator.In some embodiments, suitable promoter is selected based on the selection of host cell. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters obtained from the Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75:3727-3731 [1978]), and the tac promoter (See, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80:21-25 [1983]). Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 99/05455). 96/00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutants, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488 [1992]).

In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the selected host cell can be used in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., supra).

In some embodiments, the control sequence is also a suitable leader sequence (i.e., a non-translated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable leader sequence that is functional in the selected host cell can be used in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the following genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence that is operably linked to the 3' end of the nucleic acid sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990 [1995]).

In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence that is linked to the amino terminus of a polypeptide and directs the encoded polypeptide to the secretory pathway of the cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in translation reading frame to the segment of the coding region encoding the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide to the secretory pathway of the selected host cell can be used for expression of one or more engineered polypeptides. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the following genes: Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993]). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase.

In some embodiments, the control sequence is also a propeptide coding region encoding an amino acid sequence positioned at the amino terminus of a polypeptide. The polypeptide produced is referred to as a "proenzyme", "propolypeptide" or "zymogen". The propolypeptide can be converted into a mature active polypeptide by catalyzing or autocatalyzing the cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source including but not limited to the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease and Myceliophthora thermophila lactase (see, e.g., WO 95/33836). When both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned adjacent to the amino terminus of the polypeptide and the signal peptide region is positioned adjacent to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also utilized. These sequences promote the regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause the expression of genes to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). In prokaryotic host cells, suitable regulatory sequences include but are not limited to lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include but are not limited to ADH2 systems or GAL1 systems. In filamentous fungi, suitable regulatory sequences include but are not limited to TAKA alpha-amylase promoters, Aspergillus niger glucoamylase promoters and Aspergillus oryzae glucoamylase promoters.

On the other hand, the present invention relates to a polynucleotide comprising an engineered enzyme polypeptide, and one or more expression regulatory regions such as promoters and terminators, replication origins, etc., according to the type of host to be introduced. In some embodiments, various nucleic acids and control sequences described herein are linked together to produce a recombinant expression vector comprising one or more convenient restriction sites to allow insertion or replacement of the nucleic acid sequence encoding the enzyme polypeptide at such a site. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into a suitable vector for expression. In some embodiments relating to the generation of an expression vector, the coding sequence is located in a vector so that the coding sequence is operably linked to a suitable control sequence for expression.

The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and cause expression of the enzyme polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid.

In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some selectable embodiments, the vector is a vector that, when introduced into a host cell, is integrated into the genome and replicated with one or more chromosomes into which it is integrated. In addition, in some embodiments, a single vector or plasmid is utilized, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon.

In some embodiments, the expression vector comprises one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., from S. Hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. oryzae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

In another aspect, the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered enzyme polypeptide of the present invention, the polynucleotide being operably connected to one or more control sequences for expressing engineered enzymes in host cells. Host cells suitable for use in expressing polypeptides encoded by the expression vector of the present invention are well known in the art, and include but are not limited to bacterial cells, such as Escherichia coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC deposit accession number 201178)); insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21). Examples of bacterial selectable markers include, but are not limited to, the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, and/or tetracycline resistance.

In some embodiments, the expression vectors of the invention contain elements that allow the vector to be integrated into the host cell genome or to autonomously replicate the vector in the cell independently of the genome. In some embodiments involving integration into the host cell genome, the vector relies on the nucleic acid sequence encoding the polypeptide or any other element of the vector to integrate the vector into the genome by homologous or nonhomologous recombination.

In some optional embodiments, expression vector contains other nucleic acid sequences for guiding integration into the genome of host cell by homologous recombination.Other nucleic acid sequences enable carrier to be integrated into the host cell genome at the precise position in chromosome.In order to increase the possibility of integration at the precise position, integration element preferably comprises a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous to the corresponding target sequence, to improve the possibility of homologous recombination.Integration element can be any sequence homologous to the target sequence in the genome of host cell.In addition, integration element can be non-coding or encoding nucleic acid sequence.On the other hand, carrier can be integrated into the genome of host cell by non-homologous recombination.

For autonomous replication, the vector may also include an origin of replication that enables the vector to autonomously replicate in the host cell in question. Examples of bacterial origins of replication are P15A ori or plasmids pBR322, pUC19, pACYC177 (the plasmid has P15A ori) or pACYC184 that allow replication in Escherichia coli and pUB110, pE194 or pTA1060 that allow replication in bacillus. Examples of origins of replication for yeast host cells are 2 micron origins of replication, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6. The origin of replication may be an origin of replication with a mutation that makes its function in the host cell sensitive to temperature (see, e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of the nucleic acid sequence of the present invention is inserted into the host cell to increase the production of the gene product. The increase in the number of nucleic acid sequence copies can be obtained by integrating at least one other copy of the sequence into the host cell genome or by including an amplifiable selection marker gene with the nucleic acid sequence, wherein the cells containing the amplified copy of the selection marker gene and therefore the other copy of the nucleic acid sequence can be selected by culturing cells in the presence of a suitable selection agent (selectable agent).

Many expression vectors for the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to, the p3xFLAG ^{™ TM} expression vector (Sigma-Aldrich Chemicals), which contains a CMV promoter and an hGH polyadenylation site for expression in mammalian host cells and a pBR322 origin of replication and an ampicillin resistance marker for amplification in Escherichia coli. Other suitable expression vectors include, but are not limited to, pBluescriptII SK(-) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen), or pPoly (see, e.g., Lathe et al., Gene 57:193-201 [1987]).

Therefore, in some embodiments, a vector comprising a sequence encoding at least one variant uridine phosphorylase is transformed into a host cell to allow the propagation of the vector and the expression of the variant uridine phosphorylase. In some embodiments, the variant uridine phosphorylase is post-translationally modified to remove the signal peptide, and in some cases, can be cleaved after secretion. In some embodiments, the host cell of the conversion described above is cultivated in a suitable nutrient medium under conditions that allow the variant uridine phosphorylase to be expressed. Any suitable culture medium for cultivating host cells can be used in the present invention, including but not limited to minimal culture medium or composite culture medium containing suitable supplements. In some embodiments, host cells are grown in HTP culture medium. Suitable culture medium can be obtained from various commercial suppliers or can be prepared according to the formula (for example, in the catalog of the American Type Culture Collection) announced.

On the other hand, the invention provides a host cell comprising a polynucleotide encoding an improved uridine phosphorylase polypeptide provided herein, the polynucleotide being operably connected to one or more control sequences for expressing uridine phosphorylase in a host cell. The host cell for expressing the uridine phosphorylase polypeptide encoded by the expression vector of the present invention is well known in the art, and includes but is not limited to bacterial cells such as Escherichia coli, Bacillus megaterium, Lactobacillus kefir, Streptomyces and Salmonella typhimurium cells; Fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC accession number 201178)); Insect cells such as Drosophila S2 and Spodoptera Sf9 cells; Animal cells such as CHO, COS, BHK, 293 and Bowes melanoma cells; And plant cells. Suitable culture medium and growth conditions for the host cells described above are well known in the art.

Polynucleotides for expressing uridine phosphorylase can be introduced into cells by various methods known in the art. Techniques include electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion, among others. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algae cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, and Fungi imperfecti. In some embodiments, the fungal host cell is a yeast cell and a filamentous fungal cell. The filamentous fungal host cell of the present invention includes all filamentous forms of Eumycotina and Oomycota. Filamentous fungi are characterized in that the vegetative mycelium, the cell wall is composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungal host cell of the present invention is morphologically different from yeast.

In some embodiments of the invention, the filamentous fungal host cell is of any suitable genus and species, including, but not limited to, Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothia, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea ), Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytali The invention further comprises any of the genera Trichoderma, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium and/or Volvariella, and/or their sexual or asexual forms, and their synonyms, baxinies or taxonomic equivalents.

In some embodiments of the invention, the host cell is a yeast cell, including but not limited to a cell of a Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kudamae, Pichia membranaefaciens, Pichia cactus, Pichia thermotolerans, Pichia salictaria, Pichia somnifera, Pichia spp. quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

In some embodiments of the invention, the host cell is an algal cell such as Chlamydomonas (eg, C. reinhardtii) and Phormidium (Phormidium sp. ATCC 29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include but are not limited to Gram-positive, Gram-negative and Gram-variable bacterial cells. Any suitable bacterial organism can be used for the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Campylobacter, Clostridium, Corynebacterium ), Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus , Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces ), Streptococcus, Synechococcus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azotobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments, the bacterial host strain is an industrial strain. Many bacterial industrial strains are known and suitable for the present invention. In some embodiments of the present invention, the bacterial host cell is a species of Agrobacterium (e.g., A. radiobacter, A. rhizogenes and A. rubi). In some embodiments of the invention, the bacterial host cell is a species of the genus Arthrobacter (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfurus, and A. ureafaciens). In some embodiments of the invention, the bacterial host cell is a species of the genus Bacillus (e.g., B. thuringensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens). In some embodiments, the host cell is an industrial bacillus strain, including but not limited to bacillus subtilis, bacillus pumilus, bacillus licheniformis, bacillus megaterium, bacillus clausii, bacillus stearothermophilus or bacillus amyloliquefaciens. In some embodiments, the bacillus host cell is bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus stearothermophilus and/or bacillus amyloliquefaciens. In some embodiments, the bacterial host cell is a species of the genus Clostridium (e.g., acetobutylicum, Clostridium tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens and C. beijerinckii). In some embodiments, the bacterial host cell is a species of the genus Corynebacterium (e.g., Corynebacterium glutamicum (C.glutamicum) and Corynebacterium acetoacetic acid (C.acetoacidophilum)). In some embodiments, the bacterial host cell is a species of the genus Escherichia (e.g., Escherichia coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments, the bacterial host cell is a species of the genus Erwinia (e.g., Erwinia uredovora (E.uredovora), Erwinia carotovora (E.carotovora), Erwinia ananas (E.ananas), Erwinia herbicola (E.herbicola), Erwinia punctata (E.punctata) and Erwinia terreus (E.terreus)). In some embodiments, the bacterial host cell is a species of the genus Pantoea (e.g., Pantoea citrea (P.citrea) and Pantoea agglomerans (P.agglomerans)). In some embodiments, the bacterial host cell is a species of Pseudomonas (e.g., Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas mevalonii, and P.sp. D-0110). In some embodiments, the bacterial host cell is a species of Streptococcus (e.g., Streptococcus equisimiles, Streptococcus pyogenes, and Streptococcus uberis). In some embodiments, the bacterial host cell is a species of the genus Streptomyces (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, and S. lividans). In some embodiments, the bacterial host cell is a species of the genus Zymomonas (e.g., Zymomonas mobilis and Zymomonas lipolytica).

Many prokaryotic and eukaryotic strains that can be used in the present invention are readily available to the public from a number of culture collections, such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms and Fungi (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSM), the Netherlands Central Agricultural Research Center (Centraalbureau Voor Schimmelcultures, CBS), and the U.S. Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to have improved protein secretion, protein stability and/or other properties desired for protein expression and/or secretion.Genetic modification can be achieved by genetic engineering techniques and/or traditional microbiological techniques (for example, chemical or UV mutagenesis and subsequent selection). In fact, in some embodiments, the combination of recombinant modification and classical selection techniques is used to produce host cells.Utilizing recombinant technology, nucleic acid molecules can be introduced, lacked, suppressed or modified in a manner that causes an increase in uridine phosphorylase variant output in the host cell and/or in the culture medium.For example, the knockout of Alp1 function causes a cell with protease defect, and the knockout of pyr5 function causes a cell with a pyrimidine defect phenotype.In a genetic engineering method, homologous recombination is used to induce targeted gene modification to suppress the expression of encoded protein by specific targeting genes in vivo.In an optional method, siRNA, antisense and/or ribozyme technology can be used to inhibit gene expression.It is known in the art to include but not limited to the expression of all or part of the genes encoding the protein and site-specific mutagenesis to destroy the expression or active various methods of the gene product for reducing the expression of the protein in the cell. (See, e.g., Chaveroche et al., Nucl. Acids Res., 28:22e97 [2000]; Cho et al., Molec. Plant Microbe Interact., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett., 30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272:344-352 [2004]; and You et al., Arch. Microbiol., 191:615-622 [2009], all of which are incorporated herein by reference). Random mutagenesis followed by screening for desired mutations is also useful (See, e.g., Combier et al., FEMS Microbiol. Lett., 220: 141-8 [2003]; and Firon et al., Eukary. Cell 2:247-55 [2003], both of which are incorporated by reference).

The introduction of a vector or DNA construct into a host cell can be accomplished using any suitable method known in the art, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, PEG-mediated transformation, electroporation, or other conventional techniques known in the art. In some embodiments, the E. coli expression vector pCK100900i (see U.S. Pat. No. 9,714,437, which is hereby incorporated by reference) can be used.

In some embodiments, the engineered host cells of the invention (i.e., "recombinant host cells") are cultured in conventional nutrient media modified appropriately to activate promoters, select transformants, or amplify uridine phosphorylase polynucleotides. Culture conditions, such as temperature, pH, etc., are those previously used for the host cell selected for expression and are well known to those skilled in the art. As described, many standard references and textbooks are available for the culture and production of many cells, including cells of bacterial, plant, animal (particularly mammalian) and archaeal origin.

In some embodiments, cells expressing variant uridine phosphorylase polypeptides of the present invention are grown under batch fermentation or continuous fermentation conditions. Classical "batch fermentation" is a closed system in which the composition of the culture medium is set at the beginning of the fermentation and is not subject to artificial changes during the fermentation. A variation of the batch system is "fed-batch fermentation", which can also be used in the present invention. In this variation, the substrate is added incrementally as the fermentation proceeds. Fed-batch systems are useful when catabolite inhibition may inhibit the metabolism of the cell and it is desirable to have a limited amount of substrate in the culture medium. Batch fermentation and fed-batch fermentation are conventional and well known in the art. "Continuous fermentation" is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is removed for processing. Continuous fermentation generally maintains the culture at a constant high density, in which the cells are primarily in the logarithmic growth phase. Continuous fermentation systems strive to maintain steady-state growth conditions. Methods for regulating nutrients and growth factors in continuous fermentation processes and techniques for maximizing product formation rates are well known in the field of industrial microbiology.

In some embodiments of the invention, a cell-free transcription/translation system can be used to produce one or more variant uridine phosphorylases. Several systems are commercially available and methods are well known to those skilled in the art.

The present invention provides a method for preparing a variant uridine phosphorylase polypeptide or a biologically active fragment thereof. In some embodiments, the method comprises: providing a host cell transformed with a polynucleotide encoding the following amino acid sequence, the amino acid sequence comprising at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 246, SEQ ID NO: 594, SEQ ID NO: 776 and/or SEQ ID No: 868, and comprising at least one mutation provided herein; culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the encoded variant uridine phosphorylase polypeptide; and optionally recovering or isolating the expressed variant uridine nucleoside phosphorylase polypeptide, and/or recovering or isolating the culture medium containing the expressed variant uridine nucleoside phosphorylase polypeptide. In some embodiments, the method also provides optionally lysing the transformed host cell after expressing the encoded uridine phosphorylase polypeptide, and optionally recovering and/or isolating the expressed variant uridine phosphorylase polypeptide from the cell lysate. The present invention also provides a method for preparing a variant uridine phosphorylase polypeptide, the method comprising culturing a host cell transformed with a variant uridine phosphorylase polypeptide under conditions suitable for producing a variant uridine phosphorylase polypeptide, and recovering the variant uridine phosphorylase polypeptide. Typically, the uridine phosphorylase polypeptide is recovered or isolated from the host cell culture medium, the host cell, or both using protein recovery techniques well known in the art, including those described herein. In some embodiments, the host cells are harvested by centrifugation, broken by physical or chemical means, and the resulting crude extract is retained for further purification. The microbial cells used in the expression of the protein can be broken by any conventional method, including but not limited to freeze-thaw cycles, sonication, mechanical disruption, and/or the use of cell lysis agents, as well as many other suitable methods well known to those skilled in the art.

The engineered uridine phosphorylase expressed in the host cell can be recovered from cells and/or culture medium using any one or more of the techniques known in the art for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation and chromatography. Suitable for cracking and efficiently extracting proteins from bacteria (such as Escherichia coli) is commercially available under the trade name CelLytic B ^TM (Sigma-Aldrich). Therefore, in some embodiments, the resulting polypeptide is recovered/separated, and optionally purified by any of many methods known in the art. For example, in some embodiments, polypeptide is separated from a nutrient medium by conventional methods, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing and size exclusion) or precipitation. In some embodiments, as desired, a protein refolding step is used in the configuration of the mature protein. In addition, in some embodiments, high performance liquid chromatography (HPLC) is adopted in the final purification step. For example, in some embodiments, methods known in the art can be used in the present invention (see, e.g., Parry et al., Biochem. J., 353: 117 [2001]; and Hong et al., Appl. Microbiol. Biotechnol., 73: 1331 [2007], both of which are incorporated herein by reference). In fact, any suitable purification method known in the art can be used in the present invention.

Chromatographic techniques used to separate uridine phosphorylase polypeptides include, but are not limited to, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. The conditions used to purify a particular enzyme will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., which are known to those skilled in the art.

In some embodiments, affinity techniques can be used to isolate improved uridine phosphorylase. For affinity chromatography purification, any antibody that specifically binds to uridine phosphorylase polypeptides can be used. In order to produce antibodies, various host animals can be immunized by injection of uridine phosphorylase, including but not limited to rabbits, mice, rats, etc. The uridine phosphorylase polypeptide can be attached to a suitable carrier such as BSA by means of side chain functional groups or linkers attached to side chain functional groups. Depending on the host species, various adjuvants can be used to enhance the immune response, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpets. keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacillus Calmette-Guérin) and Corynebacterium parvum.

In some embodiments, the uridine phosphorylase variant is prepared and used in the form of cells expressing the enzyme, as a crude extract, or as an isolated or purified product. In some embodiments, the uridine phosphorylase variant is prepared as a lyophilized agent, a powder form (e.g., acetone powder), or as an enzyme solution. In some embodiments, the uridine phosphorylase variant is in the form of a substantially pure product.

In some embodiments, the uridine phosphorylase polypeptide is connected to any suitable solid substrate. Solid substrates include but are not limited to solid phases, surfaces and/or membranes. Solid supports include but are not limited to organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy and polyacrylamide and their copolymers and grafts. Solid supports can also be inorganic, such as glass, silica, controlled pore glass (CPG), reversed silica or metals such as gold or platinum. The configuration of the substrate can be in the form of beads, balls, microparticles, particles, gels, membranes or surfaces. The surface can be planar, substantially planar or non-planar. The solid support can be porous or non-porous and can have swelling or non-swelling characteristics. The solid support can be configured in the form of a hole, a depression or other container, a vessel, a feature or a position. More than one support can be configured at multiple positions on the array, and the multiple positions are automatic delivery of reagents or addressable by detection methods and/or instruments.

In some embodiments, immunological methods are used to purify uridine phosphorylase variants. In one method, antibodies generated using conventional methods against wild-type or variant uridine phosphorylase polypeptides (e.g., against polypeptides comprising any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:246, SEQ ID NO:594, SEQ ID NO:776 and/or SEQ ID NO:868 and/or variants thereof and/or immunogenic fragments thereof) are fixed to beads, mixed with cell culture medium under conditions where variant uridine phosphorylase is bound, and precipitated. In a related method, immunochromatography can be used.

In some embodiments, variant uridine phosphorylase is expressed as a fusion protein comprising a non-enzyme portion. In some embodiments, the variant uridine phosphorylase sequence is fused to a purification facilitating domain. As used herein, the term "purification facilitating domain" refers to a domain that mediates the purification of a polypeptide fused thereto. Suitable purification domains include, but are not limited to, metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, sequences that bind glutathione (e.g., GST), hemagglutinin (HA) tags (corresponding to epitopes derived from influenza hemagglutinin proteins; see, e.g., Wilson et al., Cell 37: 767 [1984]), maltose binding protein sequences, FLAG epitopes utilized in FLAGS extension/affinity purification systems (e.g., systems available from Immunex Corp), etc. It is contemplated that an expression vector for the compositions and methods described herein provides expression of a fusion protein comprising a polypeptide of the present invention fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography; e.g., see Porath et al., Prot. Exp. Purif., 3:263-281 [1992]), while the enterokinase cleavage site provides a means for separating the variant uridine phosphorylase polypeptide from the fusion protein. The pGEX vector (Promega) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions), followed by elution in the presence of free ligand.

Therefore, in another aspect, the invention provides a method for producing an engineered enzyme polypeptide, wherein the method includes culturing a host cell capable of expressing a polynucleotide encoding an engineered enzyme polypeptide under conditions suitable for polypeptide expression. In some embodiments, the method also includes the step of separating and/or purifying the enzyme polypeptide as described herein.

Suitable culture media and growth conditions for host cells are well known in the art. It is contemplated that any suitable method for introducing a polynucleotide for expressing an enzyme polypeptide into a cell can be used in the present invention. Suitable techniques include, but are not limited to, electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

Various features and embodiments of the invention are illustrated in the following representative examples, which are intended to be illustrative rather than limiting.

experiment

The following examples are provided, including the results of experiments and acquisitions, for illustrative purposes only and should not be construed as limiting the present invention. In fact, many reagents and equipment described below have various suitable sources. The present invention is not intended to be limited to any particular source of any reagent or equipment item.

In the following experimental disclosure, the following abbreviations apply: M (mole/liter); mM (millimol/liter), uM and μΜ (micromol/liter); nM (nanomoles/liter); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min (minute); h and hr (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); psi and PSI (pounds per square inch) number); °C (degrees Celsius); RT and rt (room temperature); CV (coefficient of variation); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-L-thiogalactopyranoside); LB (lysogenic broth); TB (terrific broth); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); Escherichia coli W3110 (a common laboratory Escherichia coli strain, obtained from Coli =The results are as follows: (a) DNA sequencing (DNA sequencing (DNA sequencing) (available at Genetic Stock Center [CGSC], New Haven, CT); (b) HTP (high throughput); (c) HPLC (high pressure liquid chromatography); (d) HPLC-UV (HPLC-ultraviolet visible detector); (e) HNMR (proton nuclear magnetic resonance spectroscopy); (e) FIOPC (improvement factor over positive control); (f) Sigma and Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); (f) Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); (f) Microfluidics (Microfluidics, Westwood, MA); (f) Life Technologies (Life Technologies, part of Fisher Scientific, Waltham, MA); (f) Amresco (Amresco, LLC, Solon, OH); (f) Carbosynth (Carbosynth, Ltd., Berkshire, UK); (f) Varian (Varian Medical Systems, Palo Alto, CA); (f) Agilent (Agilent Technologies, Inc., Santa Clara, CA); (f) Infors (Infors USA Inc., Annapolis Junction, MD); and Thermotron (Thermotron, Inc., Holland, MI).

Example 1

Escherichia coli expression host containing recombinant uridine phosphorylase gene

The initial uridine phosphorylase (UP) for producing variants of the present invention is obtained from the Escherichia coli genome and cloned into the expression vector pCK110900 (see Fig. 3 of U.S. Patent Application Publication No. 2006/0195947), which is operably connected to the lac promoter under the control of the lacI repressor. The expression vector also comprises a P15a origin of replication and a chloramphenicol resistance gene. The resulting plasmid is transformed into Escherichia coli W3110 using standard methods known in the art. As known in the art, transformants are isolated by making cells undergo chloramphenicol selection (see, for example, U.S. Patent No. 8,383,346 and WO2010/144103).

Example 2

HTP preparation of wet cell pellets and lysates containing UP

The E. coli cells containing recombinant UP coding genes from monoclonal colonies are inoculated into 180 μl LB containing 1% glucose and 30 μg/mL chloramphenicol in the holes of 96-well shallow-well microtiter plates. The plate is sealed with O ₂ permeable seals (seal), and the culture is grown overnight at 30 ℃, 200rpm and 85% humidity. Then, 10 μL of each of the cell cultures is transferred to the holes of the 96-well deep-well plates containing 390mL TB and 30 μg/mL CAM. The deep-well plates are sealed with O ₂ permeable seals, and cultivated at 30 ℃, 250rpm and 85% humidity until OD ₆₀₀ 0.6-0.8 is reached. Then the cell culture is induced with the IPTG reaching the ultimate concentration of 1mM, and incubated overnight at 30 ℃. Then 4,000rpm, 10 minutes of centrifugal precipitation cells are used. The supernatant is discarded, and the precipitate is frozen at -80 ℃ before lysis.

For lysis, 400 μL lysis buffer containing 100 mM triethanolamine (TEoA) buffer, pH 7.5, 1 g/L lysozyme and 0.5 g/L polymyxin b sulfate (PMBS) was added to the cell paste in each well. The cells were lysed at room temperature for 2 hours with shaking on a bench top shaker. The plate was then centrifuged at 4000 rpm and 4 ° C for 15 min. The clarified supernatant was then used for biocatalytic reactions to determine its activity level.

Example 3

Preparation of lyophilized lysates from shake flask (SF) cultures

Selected HTP cultures grown as described above were plated onto LB agar plates containing 1% glucose and 30 μg/ml CAM and grown overnight at 37°C. A single colony from each culture was transferred to 6 ml LB containing 1% glucose and 30 μg/ml CAM. The cultures were grown at 30°C, 250 rpm for 18 hours and subcultured to 250 ml TB containing 30 μg/ml CAM at approximately 1:50 to a final OD ₆₀₀ of 0.05. The cultures were grown at 30°C, 250 rpm for approximately 195 minutes to an OD ₆₀₀ between 0.6-0.8 and induced with 1 mM IPTG. The cultures were then grown at 30°C, 250 rpm for 20 hours. The cultures were centrifuged at 4,000 rpm for 20 min. The supernatant was discarded and the pellet was resuspended in 30 ml of 20 mM triethanolamine, pH 7.5 and spun using Processor system (Microfluidics) lysed at 18,000 psi. The lysate was pelleted (10,000 rpm for 60 min) and the supernatant was frozen and lyophilized to produce shake flask (SF) enzyme powder.

Example 4

Improvement in 5'-isobutyryl uridine phosphorylation compared to SEQ ID NO: 2

Based on the results of screening variants for phosphorylation of 5&apos;-isobutyryluridine, SEQ ID NO:2 was selected as the parent enzyme (Scheme II, below).

The reaction of Scheme II is reversible, and in the initial stages of research, the reverse reaction was successfully used as an alternative to the desired forward reaction depicted in Scheme I above.

The library of engineered genes was generated using established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated with HTP as described in Example 2. For all variants, the cell pellet was lysed by adding 400 μL lysis buffer (comprising 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme and 0.5 g/L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. The plate was centrifuged at 4000 rpm for 15 minutes at 4 ° C to remove cell debris.

Reactions were performed in a 96-well format in a 2 mL deep well plate with a total volume of 100 μL. Reactants included 11.1 g/L (3.5 mM) 5'-isobutyryl uridine, 10 mM potassium chloride, 50 mM sodium phosphate, pH 7.4, and 10× diluted UP lysate. The reactions were set up as follows: (i) all reaction components except UP were premixed in a single solution, and 87.5 μL of this solution was then aliquoted into each well of the 96-well plate (ii) 12.5 μL of 10× diluted UP lysate was then added to the wells to start the reaction. The reaction plates were heat sealed with foil seals and incubated at 35°C with shaking at 600 rpm for 18-20 hours. The reactions were quenched with 100 μL of a 1:1 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The quenched reactions were shaken on a benchtop shaker for 3 min, followed by centrifugation at 4,000 rpm at 4°C for 10 min to pellet any precipitate. The supernatant (50 μL) was then transferred to a 96-well round-bottom plate pre-filled with 100 μL of a 1:1 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The samples were analyzed according to the HILIC analysis method summarized in Table 1.1.

Activity relative to SEQ ID NO: 2 (Active FIOP) was calculated as the % conversion of the variant to uracil (uracil peak area/[uracil peak area + 5'-isobutyryl uridine peak area]) compared to the % conversion of uracil produced by the reaction with SEQ ID NO: 2. The results are shown in Table 1.2.

Example 5

Improvements compared to SEQ ID NO: 4 regarding the synthesis of 5'-isobutyryl uridine

Based on the results of a screen of variants for the synthesis of 5'-isobutyryluridine from 5'-isobutyrylribose-1-phosphate and uracil, SEQ ID NO:4 was selected as the parent enzyme (Scheme I, above).

Libraries of engineered genes were generated using established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were generated with HTP as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (comprising 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme and 0.5 g/L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. The plate was centrifuged at 4,000 rpm for 15 minutes at 4°C to remove cell debris.

Reactions were performed in a 96-well format in a 2 mL deep well plate in a total volume of 100 μL. Reactants included 67 mM 5'-isobutyryl ribose-1-phosphate solution, 7.5 g/L (67 mM) uracil, 91 g/L (267 mM, 4 equivalents) sucrose, 0.019 g/L (0.25 wt% wrt uracil) SUP-101 (SEQ ID NO: 852), 50 mM triethanolamine, pH 7.5, and 100× diluted UP lysate. The reactions were set up as follows: (i) all reaction components except UP were premixed in a single solution, and 90 μL of this solution was then aliquoted into each well of a 96-well plate; (ii) 10 μL of 100× diluted UP lysate was then added to the well to start the reaction. The reaction plates were heat sealed with foil seals and incubated at 30°C with shaking at 600 rpm for 18-20 hours. The reaction was quenched by 400 μL of a 1:1 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The quenched reaction was shaken on a benchtop shaker for 5 min, followed by centrifugation at 4,000 rpm at 4°C for 10 min to pellet any precipitate. The supernatant (30 μL) was then transferred to a 96-well round-bottom plate pre-filled with 120 μL of a 1:9 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The samples were analyzed according to the reverse phase analytical method summarized in Table 2.1.

Activity relative to SEQ ID NO: 4 (Active FIOP) was calculated as the peak area of the 5'-isobutyryl uridine product formed by the reaction of the variant compared to the peak area of the 5'-isobutyryl uridine product produced by the reaction of SEQ ID NO: 4. The results are shown in Table 2.2.

Example 6

Improvement over SEQ ID NO: 246 for the synthesis of 5'-isobutyryluridine Based on the results of screening variants for the synthesis of 5'-isobutyryluridine (compound (1)) from 5'-isobutyrylribose-1-phosphate (compound (2)) and uracil (compound (3)), SEQ ID NO: 246 was selected as the parent enzyme (Scheme I).

Libraries of engineered genes were generated using established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were generated with HTP as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (comprising 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme and 0.5 g/L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. The plate was centrifuged at 4,000 rpm for 15 minutes at 4°C to remove cell debris.

Reactions were performed in a 96-well format in a 2 mL deep well plate in a total volume of 100 μL. Reactants included 67 mM 5'-isobutyryl ribose-1-phosphate solution, 7.5 g/L (67 mM) uracil, 91 g/L (267 mM, 4 equivalents) sucrose, 0.019 g/L (0.25 wt% wrt uracil) SUP-101 (SEQ ID NO: 852), 50 mM triethanolamine, pH 7.5, and 100× diluted UP lysate. The reactions were set up as follows: (i) all reaction components except UP were premixed in a single solution, and 90 μL of this solution was then aliquoted into each well of a 96-well plate; (ii) 10 μL of 100× diluted UP lysate was then added to the well to start the reaction. The reaction plates were heat sealed with foil seals and incubated at 30°C with shaking at 600 rpm for 18-20 hours. The reaction was quenched by 400 μL of a 1:1 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The quenched reaction was shaken on a benchtop shaker for 5 min, followed by centrifugation at 4,000 rpm at 4°C for 10 min to pellet any precipitate. The supernatant (30 μL) was then transferred to a 96-well round-bottom plate pre-filled with 120 μL of a 1:9 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The samples were analyzed according to the reverse phase analytical method summarized in Table 2.1.

Activity relative to SEQ ID NO: 246 (Active FIOP) was calculated as the peak area of the 5'-isobutyryl uridine product formed by the reaction of the variant compared to the peak area of the 5'-isobutyryl uridine product produced by the reaction of SEQ ID NO: 246. The results are shown in Table 3.1.

Example 7

Improvements compared to SEQ ID NO:594 regarding the synthesis of 5'-isobutyryl uridine

Based on the results of screening variants for the synthesis of 5'-isobutyryluridine (compound (1)) from 5'-isobutyrylribose-1-phosphate (compound (2)) and uracil (compound (3)), SEQ ID NO:594 was selected as the parent enzyme (Scheme I).

Libraries of engineered genes were generated using established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were generated with HTP as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (containing 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme and 0.5 g/L PMBS) and shaking on a benchtop shaker for 2 hours at room temperature. The plate was centrifuged at 4,000 rpm for 15 minutes at 4°C to remove cell debris.

Reactions were performed in a 96-well format in a 2 mL deep well plate in a total volume of 100 μL. Reactants included 67 mM 5'-isobutyryl ribose-1-phosphate solution, 7.5 g/L (67 mM) uracil, 91 g/L (267 mM, 4 equivalents) sucrose, 0.019 g/L (0.25 wt% wrt uracil) SUP-101 (SEQ ID NO: 852), 50 mM triethanolamine, pH 7.5, and 100× diluted UP lysate. The reactions were set up as follows: (i) all reaction components except UP were premixed in a single solution, and 90 μL of this solution was then aliquoted into each well of a 96-well plate; (ii) 10 μL of 100× diluted UP lysate was then added to the well to start the reaction. The reaction plates were heat sealed with foil seals and incubated at 30°C with shaking at 600 rpm for 18-20 hours. The reaction was quenched by 400 μL of a 1:1 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The quenched reaction was shaken on a benchtop shaker for 5 min, followed by centrifugation at 4,000 rpm at 4°C for 10 min to pellet any precipitate. The supernatant (30 μL) was then transferred to a 96-well round-bottom plate pre-filled with 120 μL of a 1:9 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The samples were analyzed according to the reverse phase analytical method summarized in Table 2.1.

Activity relative to SEQ ID NO: 594 (Active FIOP) was calculated as the peak area of the 5'-isobutyryl uridine product formed by the reaction of the variant compared to the peak area of the 5'-isobutyryl uridine product produced by the reaction of SEQ ID NO: 594. The results are shown in Table 4.1.

Example 8

Improvements compared to SEQ ID NO: 776 regarding the synthesis of 5'-isobutyryl uridine

Based on the results of screening variants for the synthesis of 5'-isobutyryluridine (compound (1)) from 5'-isobutyrylribose (compound (4)) and uracil (compound (3)), SEQ ID NO: 776 was selected as the parent enzyme (Scheme III).

The uridine phosphorylase reaction is reversible. Initial experiments examined the "reverse" reaction or the phosphorylation of compound (1) via the UP enzyme reaction (Scheme II). Subsequent experiments studied the "forward" reaction or the production of compound (1) from compounds (2) and (3) (as shown in Scheme I). In addition, UP enzyme variants were also studied in the "forward" direction in a reaction produced in situ with compound (2). In this embodiment, compound (2) is produced in the reaction via an evolved 5-methylthioglycan (MTR) kinase, which catalyzes the phosphorylation of the C-1 hydroxyl group of the ribose portion of compound (4). The ability of the UP enzyme variants in the same reaction to convert compound (2) produced by the MTR kinase into the form of compound (1) can be analyzed. Scheme III above summarizes the entire cascade reaction. The cascade described below also includes auxiliary enzymes that support the kinase reaction: (1) pyruvate oxidase (with TPP, FAD, and pyruvate) both consumes phosphate (driving the equilibrium toward the product) and produces acetyl phosphate, (2) catalase consumes the peroxide byproduct formed by pyruvate oxidase, and (3) acetate kinase produces ATP (from ADP + acetyl phosphate), which is then used by the evolved MTR kinase. UP enzyme variants were screened in this cascade because it is an example of a possible scalable process. Other similar enzyme cascades are also available.

Libraries of engineered genes were generated using established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were generated with HTP as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (comprising 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme and 0.5 g/L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. The plate was centrifuged at 4,000 rpm for 15 minutes at 4°C to remove cell debris.

Reactions were performed in a 96-well format in 2 mL deep well plates in a total volume of 100 μL. The reactants included 72.6 g/L (330 mM) 5'-isobutyryl ribose solution (19.5 wt%), 37 g/L (330 mM) uracil, 0.76 g/L (0.5 mol%) TPP, 0.94 g/L (0.5 mol%) ATP, 0.14 g/L (0.05 mol%) FAD, 46.3 g/L (412 mM) pyruvate, 3.7 g _{/ L} (33 mM) K2HPO4, 0.5 wt% acetate kinase (ACK-101 Codexis, Inc.), 1.9 wt% evolved MTR-kinase (SEQ ID NO: 1198), 0.5 wt% catalase, 0.8 wt% pyruvate oxidase (SEQ ID NO: 1200), and 10 mM _MgCl2 . The reaction was set up as follows: (i) An aqueous solution of pyruvate and potassium dihydrogen phosphate was cooled to 0°C and then 5'-isobutyryl ribose-1-phosphate solution (in portions) was added. The pH of the resulting solution was adjusted to 7.2 using 8N KOH while maintaining at 0°C. To this solution were added: _MgCl2 , TPP, FAD and ATP, followed by AcK, MTR kinase, catalase and pyruvate oxidase. The resulting solution (90 μL) was then aliquoted into each well of a 96-well plate. (ii) 10 μL of 100× diluted UP lysate was then added to the wells to start the reaction. The reaction plate was sealed with breathable tape and incubated at 25°C for 18-20 h at 85% humidity with shaking at 250 rpm. The reaction was quenched by 1000 μL of a 1:1 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The quenched reactions were shaken vigorously on a benchtop shaker for 15 min, followed by centrifugation at 4,000 rpm for 10 min at room temperature to pellet any precipitate. The supernatant (15 μL) was then transferred to a 96-well round-bottom plate pre-filled with 120 μL of a 1:9 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The samples were analyzed according to the reverse phase analytical method summarized in Table 5.1 below.

Activity relative to SEQ ID NO: 776 (Active FIOP) was calculated as the peak area of the 5'-isobutyryl uridine product formed by the reaction of the variant compared to the peak area of the 5'-isobutyryl uridine product produced by the reaction of SEQ ID NO: 776. The results are shown in Table 5.2.

Example 9

Improvements compared to SEQ ID NO: 868 regarding the synthesis of 5'-isobutyryl uridine

Based on the results of screening variants for the synthesis of 5'-isobutyryluridine (compound (1)) from 5'-isobutyrylribose (compound (4)) and uracil (compound (3)), SEQ ID NO: 868 was selected as the parent enzyme (Scheme III).

Libraries of engineered genes were generated using established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were generated with HTP as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (comprising 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme and 0.5 g/L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. The plate was centrifuged at 4,000 rpm for 15 minutes at 4°C to remove cell debris.

Reactions were performed in a 96-well format in 2 mL deep well plates in a total volume of 100 μL. The reactants included 72.6 g/L (330 mM) 5'-isobutyryl ribose solution (19.5 wt%), 37 g/L (330 mM) uracil, 0.76 g/L (0.5 mol%) TPP, 0.94 g/L (0.5 mol%) ATP, 0.14 g/L (0.05 mol%) FAD, 46.3 g/L (412 mM) pyruvate, 3.7 g _{/ L} (33 mM) K2HPO4, 0.5 wt% acetate kinase (ACK-101 Codexis, Inc.), 1.9 wt% evolved MTR-kinase (SEQ ID NO: 1198), 0.5 wt% catalase, 0.8 wt% pyruvate oxidase (SEQ ID NO: 1200), and 10 mM _MgCl2 . The reaction was set up as follows: (i) An aqueous solution of pyruvate and potassium dihydrogen phosphate was cooled to 0°C and then 5'-isobutyryl ribose-1-phosphate solution (in portions) was added. The pH of the resulting solution was adjusted to 7.2 using 8N KOH while maintaining at 0°C. To this solution were added: _MgCl2 , TPP, FAD and ATP, followed by AcK, MTR kinase, catalase and pyruvate oxidase. The resulting solution (90 μL) was then aliquoted into each well of a 96-well plate. (ii) 10 μL of 100× diluted UP lysate was then added to the wells to start the reaction. The reaction plate was sealed with breathable tape and incubated at 25°C for 18-20 h at 85% humidity with shaking at 250 rpm. The reaction was quenched by 1000 μL of a 1:1 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The quenched reactions were shaken vigorously on a benchtop shaker for 15 min, followed by centrifugation at 4,000 rpm for 10 min at room temperature to pellet any precipitate. The supernatant (15 μL) was then transferred to a 96-well round-bottom plate pre-filled with 120 μL of a 1:9 mixture of acetonitrile in 20 mM TEoA pH 7.5 buffer. The samples were analyzed according to the reverse phase analytical method summarized in Table 5.1.

Activity relative to SEQ ID NO: 868 (Active FIOP) was calculated as the peak area of the 5'-isobutyryl uridine product formed by the reaction of the variant compared to the peak area of the 5'-isobutyryl uridine product produced by the reaction of SEQ ID NO: 868. The results are shown in Table 6.1.

All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims (31)

1. An engineered uridine phosphorylase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 246, SEQ ID No. 594, SEQ ID No. 776 and/or SEQ ID No. 868 or a functional fragment thereof, wherein the polypeptide sequence of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions, and wherein the amino acid position of the polypeptide sequence is numbered with reference to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 246, SEQ ID No. 594, SEQ ID No. 776 and/or SEQ ID No. 868.

2. The engineered uridine phosphorylase according to claim 1, wherein the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 2, and wherein the polypeptide sequence of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 45/51/80/188/189/241, 16, 7, 9, 14, 14/38/40/146/147/179/235/236, 14/38/86/146/147/235/236/240, 14/40, 14/40/86/147/193/236/240, 14/40/136/179/236/240, 14/40/146/235/236, 14/40/147/181/193/235, 14/40/235, 14/86/146, 14/146/147/181/240, 14/146/236/240, 14/147/193/235/236/240, 14/179/181/193/235/240, 14/235/236, 29, 31, 38/40/86/146/147/179/181, 38/40/86/147/236/240, 40, 40/43/86/146/240, 40/43/86/147/235, 40/43/146/147, 40/43/147/179/236/240, 40/43/147/179/240, 40/43/147/236/240, 40/86/146/235/236, 40/86/147/235/236/240, 40/86/179/235/240, 40/86/235/236/240, and combinations thereof, 40/146/147/240, 40/147/240, 40/235, 40/235/236/240, 40/236/240, 42/235/236, 43/86/147/181/240, 43/146/147/235/236/240, 43/146/179/240, 43/147, 43/147/179/181, 47/88, 64, 73, 80, 86, 86/136/146/147/179/181, 86/136/146/147/179/235/236, 86/147/179/181, 86/235, 86/235/236/240, 86/236/240, 86/240, 92, 97, 99, 103/249, 104, 105, 106, 110, 146/147, 146/147/235/236, 146/235/240, 146/236/240, 146/240, 147/179/181, 147/235/240/249, 157, 167, 179/181, 179/181/193/240, 181, 184, 216, 226, 228, 231, 233, 235/236, 236/240, 237, 239, 240 and 245, wherein the amino acid position of the polypeptide sequence is referenced to SEQ ID NO: 2.

3. The engineered uridine phosphorylase according to claim 1, wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID No. 4, and wherein the polypeptide sequence of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 3. 3/9/216, 3/9/216/236, 3/9/235/237, 3/31/47/179/181/216, 3/31/47/179/181/237, 3/31/47/179/216, 3/31/47/179/216/237, 3/31/179, 3/31/179/181, 3/31/179/181/237, 3/31/179/216, 3/31/181/216, 3/31/181/237, 3/47/179/181/216, 3/47/179/216/237, 3/47/181, 3/179/181, 3/179/181/216, 3/179/181/237, 3/179/216, 3/179/216/237, 3/179/237, 3/181/216, 3/181/216/237, 3/216/236/240, 9/216/236/237, 9/237, 13, 24, 31/47, 31/47/179/181/237, 31/47/179/216, 31/47/181, 31/47/216, 31/179/181/216, 31/181/216, 31/181/216/237, 31/181/237, 31/216, 31/216/237, 31/236/237/240, 31/237, 33, 46, 47, 47/147/181/231, 47/179/181, 47/179/181/216, 47/179/184, 47/179/216, 47/181/216, 47/181/216/237, 47/181/231, 47/216, 52, 63, 67, 83, 87/160, 92, 95, 97, 99, 100, 101, 105, 106, 108, 111, 137, 151, 152, 155, 159, 160, 170, 173, 177, 179/181/216, 179/181/216/237, 179/181/231, 179/181/241, 179/216, 179/228/231, 179/237, 181/216, 181/237, 183, 185, 188, 189, 191, 201, 216/237, 218, 222, 228, 231, 233, 235/236, 236/237, 240, 241 and 248, wherein the amino acid position of the polypeptide sequence is referenced to SEQ ID NO: 4.

4. The engineered uridine phosphorylase according to claim 1, wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 246, and wherein the polypeptide sequence of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 3/24/33/47/100/183/185, 3/24/33/47/100/216/228/233, 3/24/33/100/183/185/228, 3/24/33/108, 3/24/47/100, 3/24/47/100/108/111/160/185/233/241, 3/24/47/108/160/241, 3/24/47/160/189, 3/24/47/189/228/233, 3/24/47/228, 3/24/47/228/233, 3/24/95/100, 3/24/95/100/160/189/228/241, 3/24/100, 3/24/100/160/218/241, 3/24/111, 3/24/111/183/228/233/241, 3/24/111/228/233, 3/24/183/185/216, 3/24/189/233, 3/33/47/95/100/241, 3/33/47/100/108/189/216/228/233, 3/33/47/100/111/228, 3/33/47/100/111/233/241, 3/33/47/100/216, 3/33/47/108/111/233, 3/33/47/108/189/233/241, 3/33/160/233, 3/47, 3/47/95/100/108/189/233, 3/47/95/100/111/241, 3/47/95/160/189, 3/47/100/108/183/185/189/241, 3/47/100/160/185, 3/47/100/185/189/228, 3/47/108/111, 3/47/183/189/228/233, 3/47/189, 3/47/228/233, 3/95/100/160/228/233, 3/95/100/183/216/228/233, 3/95/100/183/233, 3/95/185/189/216, 3/95/189, 3/95/233, 3/160, 3/183/185/189/228/233, 3/183/189/228/233, 3/185/189, 3/189, 24/33/47, 24/33/47/228/241, 24/33/100/108/241, 24/47/95/100, 24/47/95/100/160/228/233/241, 24/47/185/216/218, 24/47/216, 24/95/183, 24/100/160/233, 24/160/183/185, 24/189/228/233, 33/47/95/100/233, 33/47/95/100/233/241, 33/47/160, 33/47/233, 33/100/183/185, 33/100/185/233, 47, 47/100/111/233, 47/100/189, 47/100/189/233, 47/108/160/228/241, 47/111, 47/160/185/189/233, 47/228/233, 95/100/183, 95/100/189, 95/100/228, 95/100/228/233, 95/100/233, 100/160/185, 100/228/233, 108, 108/183/189/233, 108/185/216/228/233, 160/233, 228 and 228/233, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID No. 246.

5. The engineered uridine phosphorylase according to claim 1, wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID No. 594, and wherein the polypeptide sequence of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 6. 6/9/29/40/100/121/126/179/181/189/237, 6/9/121/179/181, 6/46/52/63/97/121/126/179, 6/52/180/181, 6/63/126/179/242, 9, 9/40/46/97/100/106/135/179/181/207/231, 9/52/126/189/242, 9/97/100/106/126/180/207/231, 9/181/242, 29, 40, 46, 52/63/126, 52/179/189, 61, 63, 97, 100, 106, 121, 126/180/189, 135, 142, 179, 180, 181, 189, 201, 207, 230, 231, 236, 237 and 242, wherein the amino acid position of the polypeptide sequence is numbered with reference to SEQ ID NO 594.

6. The engineered uridine phosphorylase according to claim 1, wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID No. 776, and wherein the polypeptide sequence of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 3. 3/8/22/36/181/235/250, 6, 6/45/51/81/126/226/233, 6/45/51/144, 6/45/51/188/189, 6/45/51/189/208, 6/45/51/189/228/236, 6/45/51/208/233, 6/45/149/188/189/208/233/236, 6/51/126/144/208, 6/51/126/189/208/233/236, 6/51/126/189/231/236, 6/51/126/189/233, 6/51/188/189/208/226/228, 6/51/188/189/236, 6/51/189/208/231/233, 6/51/208/226/233, 6/51/208/231, 6/126/188/231/233, 6/144/208, 6/188/189/208/228/233, 8, 8/36/143/147/235, 8/36/181/235, 8/142/147, 8/147/181/250, 8/147/235, 9, 19, 20, 22, 22/147/181/235/250, 24, 36, 36/143/147/181, 36/143/147/235, 40, 41, 43, 45/51, 45/51/126/144/208/226/228, 45/51/144, 45/51/144/208/226/231/233, 45/51/188/189, 45/51/189/233, 45/51/208/226/231, 45/51/208/233, 45/51/226, 45/126/189 45/126/189/208/226, 45/144/189/228, 45/144/226/231/233, 45/188/189, 45/188/189/208/228, 45/188/189/226/228, 45/188/189/231/233, 45/189/208, 46, 51, 51/126/144/208, 51/126/144/226/231/233/236 51/126/208, 51/144/226, 51/188/189/228, 51/189, 51/189/208/226, 51/208, 51/233, 57, 58, 80/135/147, 81, 81/126/144/188/208/228, 86, 103, 126, 126/144/188/189/226, 134, 135, 141, 142, 143/147/235, 144/188/228, 146, 147. 149, 181, 188/189/233, 189, 207, 208/226/233, 208/228, 208/231/233, 226, 228, 230, 231, 232, 233, 235, 236, 240 and 250, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID No. 776.

7. The engineered uridine phosphorylase according to claim 1, wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 868, and wherein the polypeptide sequence of the engineered uridine phosphorylase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 6. 6/8/9/126/147, 6/9/24/181/189, 6/9/181/189/235, 6/9/208/233/235, 6/24, 6/24/43/46/181/189, 6/24/43/126/147/189, 6/24/46/103/181/208, 6/24/46/147/240, 6/24/103/189, 6/24/126/189, 6/24/147, 6/46/126/147/181/235/240, 6/46/147/189/240, 6/103, 6/103/147/230/233, 6/103/189/235, 6/126/181/189, 6/126/181/189/235, 6/126/233/235, 6/181/230/233/235, 9/43/46/103/189/233/240, 9/46/126/147/181, 9/46/147/233, 24/43/46/147/230/235, 24/46/126, 24/46/147/189, 24/46/208/230/233/235, 24/103/126/147, 24/103/126/147/181/189/208/233, 24/147, 24/147/189/230/233, 24/181/189/230/233/235, 24/189/230, 24/208, 43/46/126/147/189/240, 43/103/189/208/233, 103/126/189/233/235, 103/147/181, 147/181/233, 189/235 and 208/233, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 868.

8. The engineered uridine phosphorylase according to claim 1, wherein said engineered uridine phosphorylase comprises a polypeptide sequence which is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered uridine phosphorylase variant listed in table 1.2, table 2.2, table 3.1, table 4.1, table 5.2 and/or table 6.1.

9. The engineered uridine phosphorylase according to claim 1, wherein the engineered uridine phosphorylase comprises a polypeptide sequence which is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 246, SEQ ID No. 594, SEQ ID No. 776 and/or SEQ ID No. 868.

10. The engineered uridine phosphorylase according to claim 1, wherein said engineered uridine phosphorylase comprises the variant engineered uridine phosphorylase listed in SEQ ID No. 4, SEQ ID No. 246, SEQ ID No. 594, SEQ ID No. 776 and/or SEQ ID No. 868.

11. The engineered uridine phosphorylase according to claim 1, wherein the engineered uridine phosphorylase comprises a polypeptide sequence which is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered uridine phosphorylase variant listed in the even numbered sequence in SEQ ID No. 4-1196.

12. The engineered uridine phosphorylase according to claim 1, wherein the engineered uridine phosphorylase comprises a polypeptide sequence listed in at least one of the even numbered sequences in SEQ ID NOs 4-1196.

13. The engineered uridine phosphorylase according to any of claims 1-12, wherein said engineered uridine phosphorylase comprises at least one improved property compared to a wild-type Escherichia coli (Escherichia coli) uridine phosphorylase.

14. The engineered uridine phosphorylase according to claim 13, wherein the improved property comprises improved activity towards one or more substrates.

15. The engineered uridine phosphorylase according to claim 14, wherein the one or more substrates comprise 5' -isobutyrylribose-1-phosphate (compound (2)) and/or uracil (compound (3)).

16. The engineered uridine phosphorylase according to any of claims 13-15, wherein said improved property comprises improved production of 5' -isobutyryl uridine compound (1).

17. The engineered uridine phosphorylase according to any of claims 1-16, wherein said engineered uridine phosphorylase is purified.

18. The engineered uridine phosphorylase according to any of claims 1-17, wherein the engineered uridine phosphorylase is part of a multi-enzyme system for producing nucleoside analogues.

19. A composition comprising at least one engineered uridine phosphorylase according to any of claims 1-16.

20. A polynucleotide sequence encoding at least one engineered uridine phosphorylase according to any of claims 1-18.

21. A polynucleotide sequence encoding at least one engineered uridine phosphorylase, said polynucleotide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 245, SEQ ID No. 593, SEQ ID No. 775 and/or SEQ ID No. 867, wherein the polynucleotide sequence of said engineered uridine phosphorylase comprises at least one substitution at one or more positions.

22. A polynucleotide sequence encoding at least one engineered uridine phosphorylase, or a functional fragment thereof, said polynucleotide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 245, SEQ ID No. 593, SEQ ID No. 775 and/or SEQ ID No. 867.

23. The polynucleotide sequence of any one of claims 20-22, wherein the polynucleotide sequence is operably linked to a control sequence.

24. The polynucleotide sequence of any one of claims 20-23, wherein the polynucleotide sequence is codon optimized.

25. The polynucleotide sequence of any one of claims 20-24, wherein the polynucleotide sequence comprises the polynucleotide sequence set forth in the odd-numbered sequences of SEQ ID NOs 3-1195.

26. An expression vector comprising at least one polynucleotide sequence according to any one of claims 20-25.

27. A host cell comprising at least one expression vector according to claim 26.

28. A host cell comprising at least one polynucleotide sequence according to any one of claims 20-25.

29. A method of producing an engineered uridine phosphorylase in a host cell, the method comprising culturing the host cell according to claim 27 and/or 28 under suitable conditions such that at least one engineered uridine phosphorylase is produced.

30. The method of claim 29, further comprising recovering at least one engineered uridine phosphorylase from the culture and/or host cell.

31. The method of claim 29 and/or 30, further comprising the step of purifying the at least one engineered uridine phosphorylase.