CN111019913B - A kind of P450SCC mutant and nucleotide, expression vector, host cell - Google Patents

Abstract

The invention relates to the technical field of cholesterol side chain hydrolase, in particular to a P450SCC mutant, nucleotide, an expression vector and a host cell. The P450SCC mutant comprises L109H, N249S, N249Y, L250 based on wild P450SCC amino acid sequence shown in SEQ ID NO.1 mutations at one or several amino acid positions in M, L250I, D253E, F255Y, L257R, F258Y, F258L, F258V, R304C, G307K, N446D. The invention reforms the humanized P450SCC by a site-directed mutagenesis technology, has higher enzyme activity, and can effectively improve the conversion rate of sterol to pregnenolone.

Description

A P450SCC mutant, nucleotide, expression vector, and host cell

Technical Field

The present invention relates to the technical field of cholesterol side chain hydrolase, in particular to a P450SCC mutant and nucleotides, an expression vector and a host cell.

Background Art

Pregnenolone is the precursor for the synthesis of all steroid hormone drugs. Mitochondrial cholesterol side chain hydrolase (P450SCC) can catalyze the synthesis of pregnenolone from cholesterol, which is the first and rate-limiting step in cholesterol degradation. The reaction takes place in the adrenal cortex mitochondria, and is catalyzed by cytochrome P450SCC (also called CYP11A1) and redox electron transfer to adrenal cortex ferredoxin (ADX) and adrenal cortex ferredoxin reductase (ADR) to catalyze the cholesterol side chain through three steps to finally form pregnenolone.

Steroid drugs are the second largest class of drugs in the chemical industry after antibiotics. The initial raw material was bile acid extracted from animal viscera, followed by diosgenin, and now mainly phytosterols. The first step in preparing steroid drugs from phytosterols is the hydrolysis of the sterol side chain, which requires the participation of P450SCC to catalyze the conversion of sterols into pregnenolone. As a result, researchers have done a lot of research on P450SCC.

Al Kandari H found that mutation A359V caused enzyme activity to decrease by 89%. Parajes S et al. found that R360W mutation reduced enzyme activity by 62% with cholesterol as substrate and by 70% with 22R-hydroxycholesterol as substrate, and R405X mutation caused complete loss of activity. Irina A.Pikuleva compared P450 amino acid sequences from different sources, selected site-directed mutation sites, and mutated V206, L208, N210, V211, L218, and F219 of bovine P450SCC. The double mutation of N210S/V211M and L218R/F219Y increased the cytoplasmic expression of P450SCC by 0.5-0.7 times, and the Kcat of L218R/F219Y (Kcat is the transformation number, the larger the Kcat, the higher the enzyme activity) increased by 0.8 times. Irina A. Pikuleva et al. made four single mutations, Y93A, Y93S, Y94A, and Y94S, and one Y93S/Y94S double mutation on bovine P450SCC. The Ks of all mutants increased (Ks is the dissociation constant), and the enzyme activity of all mutations with cholesterol as the substrate decreased. However, the Y93S mutation increased the enzyme activity with 25R-hydroxycholesterol as the substrate by 36%.

The present invention adopts site-directed mutagenesis to mutate amino acids at sites such as L109, N249, L250, D253, F255, L257, F258, R304, G307, and N446 of human P450SCC to obtain mutants with improved enzyme activity, so as to achieve the purpose of using the mutants to increase the conversion rate of sterols to pregnenolone.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of the present invention is to provide a cholesterol side chain hydrolase P450SCC mutant and its application, which is modified by site-directed mutagenesis technology on human P450SCC, has higher enzyme activity, and can effectively improve the conversion rate of sterols to pregnenolone.

The above-mentioned object of the present invention is achieved through the following technical solutions:

A cholesterol side chain hydrolase P450SCC mutant, wherein the mutant comprises a mutation in one or more amino acid sites among L109H, N249S, N249Y, L250M, L250I, D253E, F255Y, L257R, F258Y, F258L, F258V, R304C, G307K, and N446D based on the wild-type P450SCC amino acid sequence as shown in SEQ ID NO.1.

In a preferred example, the present invention can be further configured as follows: the nucleotide sequence of the wild-type P450SCC is as shown in SEQ ID NO.2.

In a preferred example, the present invention can be further configured as follows: the amplification primer pair for the nucleotide sequence of the wild-type P450SCC is as follows:

hcyp-F-BspHI:

AA TCATGA TGCTGGCCAAGGGTCTTCC

hcyp-R-BamHI:

CCGGGATCCTTATTTTTCGAACTGCGGTGGCTCCAAGCGGATCCACCTCCACCCTGCTGGGTTGCTTCCTGGTT .

In a preferred example, the present invention can be further configured as follows: the catalytic target of the P450SCC mutant is the phytosterol side chain.

A nucleotide sequence suitable for encoding the P450SCC mutant.

An expression vector comprising the nucleotide and/or expressing the P450SCC mutant.

In a preferred example, the present invention can be further configured as follows: the expression vector is a PET32a vector.

A host cell comprising the expression vector and/or secreting the P450SCC mutant.

In a preferred example, the present invention can be further configured as follows: the host cell is an Escherichia coli cell.

In a preferred example, the present invention can be further configured as follows: the Escherichia coli cells are Rosetta (DE3) cells.

In summary, the present invention includes at least one of the following beneficial technical effects:

1. The present invention clones the nucleotide sequence of human P450SCC as shown in SEQ ID NO.2 into the PET32a vector, obtains a P450SCC mutant by site-directed mutagenesis, and obtains a mutant with improved enzyme activity by enzyme activity assay screening;

2. The present invention provides a P450SCC mutant, which contains one or more amino acid mutations at sites such as L109, N249, L250, D253, F255, L257, F258, R304, G307, and N446;

3. Compared with the wild-type P450SCC, the activities of the P450SCC mutants L250I, F255Y, L257R, F258V/Y/L, R304C, G307K and mutants containing double mutations in L257 and F258 of the present invention are improved, providing a basis for further utilizing the mutants to catalyze the production of pregnenolone from sterols.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an SDS-PAGE detection diagram of the purified sample of P450SCC.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with the accompanying drawings.

The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

1. Construction of wild-type P450SCC plasmid

(1) Design and synthesis of primers for amplification of the nucleotide sequence of wild-type P450 SCC

Use the following primer online design website for design:

https://www.agilent.com/store/primerDesignProgram.jsp

hcyp-F-BspHI:

AA TCATGA TGCTGGCCAAGGGTCTTCC

(The underline is the BspHI restriction endonuclease cleavage site)

hcyp-R-BamHI:

CCGGGATCCTTATTTTTCGAACTGCGGTGGCTCCAAGCGGATCCACCTCCACCCTGCTGGGTTGCTTCCTGGTT

(BamHI restriction endonuclease cleavage site is underlined)

(2) PCR amplification of the nucleotide sequence of wild-type P450 SCC

(3) Electrophoresis recovery

1% agarose gel electrophoresis was performed and the target gene fragment was recovered using an agarose gel recovery kit from Axygen. The electrophoresis diagram is shown in FIG1 .

According to the method described in the product manual of thermo company, after double digestion with NcoI and BamHI, the PET32a vector (Amp resistance) digested with NcoI (same tail enzyme as BspHI) and BamHI was ligated. The reaction conditions are: the vector and the fragment are mixed in a molar ratio of 1:3, 0.5μL Takara T4 DNA ligase is added, 16℃ ligation for 1h, the ligation system is transformed into E.coli DH5a, and cultured in a 37℃ incubator overnight. The positive clones are screened by colony PCR and sent to the sequencing company for sequencing.

2. Site-directed mutagenesis

Site-directed mutagenesis primers were designed using the following primer online design website:

https://www.agilent.com/store/primerDesignProgram.jsp

Open or copy the nucleotide sequence that needs to be site-directed mutagenesis, select the base to be mutated and the pre-mutation base, submit the primer design to output the mutation primer. If there are multiple bases to be mutated, select the pre-mutation base in order. See Table 1 below for the changes in the bases corresponding to the amino acid mutation.

XL Site-Directed Mutagenesis Kit(定点突变试剂盒)，以PET32a-P450SCC为模板扩增(见SEQ ID NO.2)突变体基因，扩增产物转化E.coli DH5a，37℃培养箱过夜培养，挑单克隆送测序公司测序。多个不相近也不相邻氨基酸突变可在已突变氨基酸的基础上进行，相邻或相近氨基酸的多个氨基酸突变可在突变引物设计时设计成同时突变。use

XL Site-Directed Mutagenesis Kit (site-directed mutagenesis kit), using PET32a-P450SCC as a template to amplify the mutant gene (see SEQ ID NO.2), the amplified product was transformed into E.coli DH5a, cultured in a 37°C incubator overnight, and a single clone was sent to a sequencing company for sequencing. Multiple non-similar and non-adjacent amino acid mutations can be made on the basis of the mutated amino acids, and multiple amino acid mutations of adjacent or similar amino acids can be designed to mutate simultaneously when designing mutation primers.

Table 1 Amino acid mutations corresponding to base changes

Mutation location Original amino acid Original coding base Mutated amino acid Mutated base 109 L CTT H CAT 249 N AAC S AGC 249 N AAC Y TAC 250 L CTT M ATG 250 L CTT I ATT 253 D GAC E GAG 255 F TTC Y TAC 257 L CTG R CGG 258 F TTC Y TAC 258 F TTC L CTC 258 F TTC V GTC 304 R AGA C TGT 307 G GGA K AAA 446 N AAC D GAC

3. P450SCC induced expression

The plasmid with the correct sequencing was transformed into E. coli Rosetta (DE3), and a single clone was selected and inoculated into a small shaking flask. The culture was cultured at 37°C and 180rpm overnight, and then transferred to a shaking flask containing 1L LB at 1% to OD600 = 0.6, and IPTG with a final concentration of 0.2mM was added for induction for 20h. The cells were collected by centrifugation, resuspended in 1×PBS, broken by high-pressure homogenization, and then centrifuged at 10000r/min for 60min. The supernatant was purified by nickel column affinity to obtain purified P450SCC and its mutants.

4. Establishment of the reaction system for determination of P450SCC enzyme activity The following reaction was established with phytosterol as substrate in a reaction volume of 250uL, including P450SCC, Adx, AdR, D-glucose, glucose dehydrogenase, NADPH and phytosterol. The P450SCC enzyme activity was determined by the amount of pregnenolone generated by the reaction system as shown in Table 2 below.

Table 2 P450SCC enzyme activity assay reaction system

Reaction system components Final concentration P450SCC or each mutant 1.8uM Adx 0.1mM AdxR (before adding glycerol) 0.18mM D-Glucose 1.5M Glucose dehydrogenase 10U/mL Phytosterols (dissolved in 2-hydroxypropyl-β-cyclodextrin) 20uM NADPH 0.2M Potassium phosphate buffer 500mM Total volume 250uL

The reaction system was incubated at 37°C for 1 hour.

Parallel reactions were established using the wild-type P450SCC and each of the P450SCC mutants under the above reaction conditions and repeated three times.

The amount of enzyme required to generate 1 nmol of product pregnenolone using phytosterol as substrate within 1 hour at 37°C under specific reaction conditions (reaction system shown in Table 2 above) is defined as one enzyme activity unit (1U).

5. Detection of the product pregnenolone and determination of the enzyme activity of the wild-type P450SCC and each mutant of P450SCC The product pregnenolone was detected using an ELISA kit.

The detection steps are as follows:

(1) Diluting the reaction product in the above reaction system by 10 ¹ , 10 ² , and 10 ³ times respectively with Assay buffer;

(2) Pipette 50 μL of standard samples (A, B, C, D, E, F, with concentrations of pregnenolone of 0 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 2 ng/mL, 8 ng/mL, and 32 ng/mL, respectively), control samples (control 1 and control 2, with concentrations of pregnenolone of 1.17-1.96 ng/mL and 3.65-6.09 ng/mL, respectively), and test samples, and add them to the corresponding reaction wells;

(3) Add 100 μL of enzyme label working solution (provided in the ELISA kit) to each of the above reaction wells;

(4) Incubate on a shaker at 200 rpm for 1 hour at room temperature;

(5) Add 300 μL of 1× Washing buffer to each well and wash the plate three times. Remove all the residual liquid in the reaction wells after the last wash.

(6) Add 150 μL TMB substrate to each well;

(7) Incubate on a shaker at room temperature for 10-15 minutes;

(8) Add 50 μL of reaction stop solution (1 M H2SO4) to each reaction well;

(9) After the reaction is completed, the absorbance at 450 nm is read on an ELISA reader to calculate the relative yield of pregnenolone (abbreviated as pre) generated by the wild type and mutant reactions. The test results are shown in Table 3 below.

Table 3 Comparison of the production of pregnenolone (pre) (μg, nmol) by the wild type (WT) and each mutant of P450SCC

According to the definition of P450SCC enzyme activity (the amount of enzyme required to generate 1 nmol of product pregnenolone using phytosterol as a substrate within 1 hour at 37°C and specific reaction conditions is defined as one enzyme activity unit), the specific activities (U/mg) of the wild-type and mutant enzymes of P450SCC were calculated, as shown in Table 4 below.

Table 4 Comparison of specific activities of wild type (WT) and P450SCC mutant enzymes

For a more intuitive representation, the specific activity of the wild-type P450SCC enzyme is taken as 1, and the multiple relationship of the specific activity of each P450SCC mutant relative to the wild-type enzyme is shown in Table 5 below.

Table 5 Comparison of relative proportions of enzyme activities of wild type (WT) and P450SCC mutants

Referring to Table 5 above, according to the test results of the wild type (WT) and the P450SCC mutants in vitro catalyzing the content of pregnenolone produced by plant sterols, the relative values of the enzyme specific activities of the wild type (WT) and each of the P450SCC mutants were calculated. It can be seen that the content of plant sterols produced by the P450SCC mutants of the present application is generally improved, especially the mutant: L250M257RF258YG307KN446D, compared with the wild type, the enzyme activity is more than 2 times. It can be proved that the P450SCC mutants of the present application have higher enzyme activity and can effectively improve the conversion rate of sterols to pregnenolone.

The present invention is illustrated by the above examples, however, it should be understood that the present invention is not limited to the specific examples and embodiments described herein. The purpose of including these specific examples and embodiments here is to help those skilled in the art to practice the present invention. Any person skilled in the art can easily make further improvements and perfections without departing from the spirit and scope of the present invention, so the present invention is only limited by the content and scope of the claims of the present invention, which is intended to cover all alternatives and equivalents included in the spirit and scope of the present invention defined by the appended claims.

Sequence Listing

Sequence Listing

<110> Suzhou Huizhen Biotechnology Co., Ltd.

<120> A P450SCC mutant, nucleotide, expression vector, and host cell

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<170> PatentIn version 3.5

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Met Leu Ala Lys Gly Leu Pro Pro Arg Ser Val Leu Val Lys Gly Cys

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Gln Thr Phe Leu Ser Ala Pro Arg Glu Gly Leu Gly Arg Leu Arg Val

20 25 30

Pro Thr Gly Glu Gly Ala Gly Ile Ser Thr Arg Ser Pro Arg Pro Phe

35 40 45

Asn Glu Ile Pro Ser Pro Gly Asp Asn Gly Trp Leu Asn Leu Tyr His

50 55 60

Phe Trp Arg Glu Thr Gly Thr His Lys Val His Leu His His Val Gln

65 70 75 80

Asn Phe Gln Lys Tyr Gly Pro Ile Tyr Arg Glu Lys Leu Gly Asn Val

85 90 95

Glu Ser Val Tyr Val Ile Asp Pro Glu Asp Val Ala Leu Leu Phe Lys

100 105 110

Ser Glu Gly Pro Asn Pro Glu Arg Phe Leu Ile Pro Pro Trp Val Ala

115 120 125

Tyr His Gln Tyr Tyr Gln Arg Pro Ile Gly Val Leu Leu Lys Lys Ser

130 135 140

Ala Ala Trp Lys Lys Asp Arg Val Ala Leu Asn Gln Glu Val Met Ala

145 150 155 160

Pro Glu Ala Thr Lys Asn Phe Leu Pro Leu Leu Asp Ala Val Ser Arg

165 170 175

Asp Phe Val Ser Val Leu His Arg Arg Ile Lys Lys Ala Gly Ser Gly

180 185 190

Asn Tyr Ser Gly Asp Ile Ser Asp Asp Leu Phe Arg Phe Ala Phe Glu

195 200 205

Ser Ile Thr Asn Val Ile Phe Gly Glu Arg Gln Gly Met Leu Glu Glu

210 215 220

Val Val Asn Pro Glu Ala Gln Arg Phe Ile Asp Ala Ile Tyr Gln Met

225 230 235 240

Phe His Thr Ser Val Pro Met Leu Asn Leu Pro Pro Asp Leu Phe Arg

245 250 255

Leu Phe Arg Thr Lys Thr Trp Lys Asp His Val Ala Ala Trp Asp Val

260 265 270

Ile Phe Ser Lys Ala Asp Ile Tyr Thr Gln Asn Phe Tyr Trp Glu Leu

275 280 285

Arg Gln Lys Gly Ser Val His His Asp Tyr Arg Gly Ile Leu Tyr Arg

290 295 300

Leu Leu Gly Asp Ser Lys Met Ser Phe Glu Asp Ile Lys Ala Asn Val

305 310 315 320

Thr Glu Met Leu Ala Gly Gly Val Asp Thr Thr Ser Met Thr Leu Gln

325 330 335

Trp His Leu Tyr Glu Met Ala Arg Asn Leu Lys Val Gln Asp Met Leu

340 345 350

Arg Ala Glu Val Leu Ala Ala Arg His Gln Ala Gln Gly Asp Met Ala

355 360 365

Thr Met Leu Gln Leu Val Pro Leu Leu Lys Ala Ser Ile Lys Glu Thr

370 375 380

Leu Arg Leu His Pro Ile Ser Val Thr Leu Gln Arg Tyr Leu Val Asn

385 390 395 400

Asp Leu Val Leu Arg Asp Tyr Met Ile Pro Ala Lys Thr Leu Val Gln

405 410 415

Val Ala Ile Tyr Ala Leu Gly Arg Glu Pro Thr Phe Phe Phe Asp Pro

420 425 430

Glu Asn Phe Asp Pro Thr Arg Trp Leu Ser Lys Asp Lys Asn Ile Thr

435 440 445

Tyr Phe Arg Asn Leu Gly Phe Gly Trp Gly Val Arg Gln Cys Leu Gly

450 455 460

Arg Arg Ile Ala Glu Leu Glu Met Thr Ile Phe Leu Ile Asn Met Leu

465 470 475 480

Glu Asn Phe Arg Val Glu Ile Gln His Leu Ser Asp Val Gly Thr Thr

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Phe Asn Leu Ile Leu Met Pro Glu Lys Pro Ile Ser Phe Thr Phe Trp

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Pro Phe Asn Gln Glu Ala Thr Gln Gln

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atgctggcca agggtcttcc cccacgctca gtcctggtca aaggctgcca gacctttctg 60

agtgccccca gggaggggct ggggcgtctc agggtgccca ctggcgagggg agctggcatc 120

tccacccgca gtcctcgccc cttcaatgag atcccctctc ctggtgacaa tggctggcta 180

aacctgtacc atttctggag ggagacgggc acacacaaag tccaccttca ccatgtccag 240

aatttccaga agtatggccc gatttacagg gagaagctcg gcaacgtgga gtcggtttat 300

gtcatcgacc ctgaagatgt ggcccttctc tttaagtccg agggccccaa cccagaacga 360

ttcctcatcc cgccctgggt cgcctatcac cagtattacc agagacccat aggagtcctg 420

ttgaagaagt cggcagcctg gaagaaagac cgggtggccc tgaaccagga ggtgatggct 480

ccagaggcca ccaagaactttttgcccctg ttggatgcag tgtctcggga cttcgtcagt 540

gtcctgcaca ggcgcatcaa gaaggcgggc tccggaaatt actcggggga catcagtgat 600

gacctgttcc gctttgcctt tgagtccatc actaacgtca tttttgggga gcgccagggg 660

atgctggagg aagtagtgaa ccccgaggcc cagcgattca ttgatgccat ctaccagatg 720

ttccacacca gcgtccccat gctcaacctt cccccagacc tgttccgtct gttcaggacc 780

aagacctgga aggaccatgt ggctgcatgg gacgtgattt tcagtaaagc tgacatatac 840

acccagaact tctactggga attgagacag aaaggaagtg ttcaccacga ttaccgtggc 900

atcctctaca gactcctggg agacagcaag atgtccttcg aggacatcaa ggccaacgtc 960

acagagatgc tggcaggagg ggtggacacg acgtccatga ccctgcagtg gcacttgtat 1020

gagatggcac gcaacctgaa ggtgcaggat atgctgcggg cagaggtctt ggctgcgcgg 1080

caccaggccc agggacat ggccacgatg ctacagctgg tccccctcct caaagccagc 1140

atcaaggaga cactaagact tcaccccatc tccgtgaccc tgcagagata tcttgtaaat 1200

gacttggttc ttcgagatta catgattcct gccaagacac tggtgcaagt ggccatctat 1260

gctctgggcc gagagcccac cttcttcttc gacccggaaa attttgaccc aacccgatgg 1320

ctgagcaaag acaagaacat cacctacttc cggaacttgg gctttggctg gggtgtgcgg 1380

cagtgtctgg gacggcggat cgctgagcta gagatgacca tcttcctcat caatatgctg 1440

gagaacttca gagttgaaat ccaacacctc agcgatgtgg gcaccacatt caacctcatt 1500

ctgatgcctg aaaagcccat ctccttcacc ttctggccct ttaaccagga agcaacccag 1560

cagtga 1566

Claims (10)

1. A P450SCC mutant is characterized in that the mutant comprises one of the following mutations on the basis of the wild P450SCC amino acid sequence shown in SEQ ID NO. 1: L109H; N249Y; L250M; L250I; F255Y; L257R; F258Y; F258L; F258V; R304C; G307K; L250M L257R F Y; L257R F V; L257R F L258; L109H R304C N446D; L250I L257R F Y; L257R F258L R C N446D; L109H N249Y L257R F L; d253E L257R F Y G307K N446D; N249Y L250M L M L257R F258L R304C; L109H N249Y L250M L M L257R F258L R304C N446D.

2. The P450SCC mutant according to claim 1,nucleosides of the wild-type P450SCC The acid sequence is shown as SEQ ID NO.2。

3. The P450SCC mutant according to claim 1, characterized in that the amplification primer pair of the nucleotide sequence of the wild type P450SCC is as follows:

hcyp-F-BspHI：

AATCATGATGCTGGCCAAGGGTCTTCC

hcyp-R-BamHI：

CCGGGATCCTTATTTTTCGAACTGCGGGTGGCTCCAAGCGGATCCACCTCCACCCTGCTGGGTTGCTTCCTGGTT。

4. the P450SCC mutant according to claim 1, characterized in that the catalytic object of the P450SCC mutant is a phytosterol side chain.

5. A nucleotide suitable for encoding the P450SCC mutant according to any one of claims 1 to 4.

6. An expression vector comprising the nucleotide of claim 5 and/or expressing the P450SCC mutant.

7. An expression vector according to claim 6 which is a PET32a vector.

8. A host cell comprising the expression vector of claim 6 and/or secreted with the P450SCC mutant.

9. A host cell according to claim 8, which is an e.

10. A host cell according to claim 9, wherein the e.coli cell is a Rosetta (DE 3) cell.

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