JP2001510048A - Transformed yeast strain and its use for producing mono- and di-terminal aliphatic carboxylate - Google Patents

Abstract

(57) [Abstract] The present invention relates to a method for producing an aliphatic compound represented by the chemical formula CH ₃ (CH ₂ ) _n CH ₃ (where n = 4 to 20) using a living organism subjected to genetic engineering technology. It consists of a bioprocess that converts the terminal carboxylate. The present invention relates to a method of expressing alkane hydroxylation activity in genetically engineered yeasts Pichia pastoris and CH ₃ (CH ₂ ) _n CH ₃ Candida maltosa. In addition, the present invention describes a method for producing a genetically transformed Candida maltosa strain with increased cytochrome P450 activity and / or gene disruption of the β-oxidation pathway.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to a method for producing an aliphatic compound having a chemical formula of CH ₃ (CH ₂ ) _n CH ₃ (where n = 4 to 20) by a genetically engineered organism. Bioprocess for conversion to mono and di terminal carboxylate. The present invention further relates to a yeast strain with enhanced alkane hydroxylation activity and / or gene disruption of the β-oxidation pathway that produces carboxylate.

BACKGROUND OF THE INVENTION Diterminal carboxylate of an aliphatic compound represented by the chemical formula HO (O) C (CH ₂ ) _n C (O) OH (where n = 7 to 16) is used as a polymer intermediate. It is useful (US 4,767,828) and is also useful as an anticorrosion compound (JP
081133771). Monoterminal carboxylate of an aliphatic compound represented by the chemical formula CH ₃ (CH ₂ ) _n C (O) OH (where n = 7 to 16) is useful as an intermediate of a surfactant (U.S. Pat. , 863, 619). These compounds can be produced from natural plant sources (Dale et al., J. Sci. Fo).
od Agr. , 6: 162, (1955)), and generally purification of the compound produces large amounts of by-products. Means for reducing waste by-products and synthesizing such compounds in an enriched form are commercially useful.

[0003] Many yeasts are known to grow the formula _{CH 3 (CH 2) n CH} 3 ( provided that, n = 4 to 20) by metabolize aliphatic compound represented by. See, for example, Klug et al. (Adv. In Microbiological Physilog).
y, 5: 1-43, (1971)). In addition, Mauersber
ger et al. (Non-conventional Yeasts in Biot)
technology. A Handbook. Klause Wolf (ed.
), Springer-Verlag, the Berlin (1996)), it is described that Candida Marutosa (Candida maltosa) can grow in chain length of the aliphatic compounds of C ₆ -C _40. In all cases examined to date, growth on aliphatic compounds relies on a unique enzymatic step that converts one or both terminal methyl groups to the carboxylate form. The first step in such a transformation is the hydroxylation of the terminal methyl group by the yeast cytochrome P450 hydroxylation system: 2CH ₃ (CH ₂ ) _n CH ₃ + O ₂ + NADPH → 2CH ₃ (CH ₂ ) _n CH ₂ OH + NAD ⁺ (However, n = 7 to 16).

[0004] Such hydroxylation systems include at least three biological components: cytochrome P450 monooxygenase, cytochrome P450-NADP
H reductase and NADPH are included. Cytochrome P450-NA
DPH reductase converts NADPH (or NADPH) to cytochrome P4.
Transfer electrons to 50 monooxygenase and activate it. In the presence of oxygen and an aliphatic substrate, activated cytochrome P450 catalyzes the reaction between oxygen and the aliphatic substrate to form the corresponding alcohol. Electron transfer between reductase and cytochrome P450 monooxygenase requires proper structural orientation of the two components. In addition, the stoichiometric requirement of NADPH means that the hydroxylation activity requires a continuous supply of NADPH. Generally, this supply of NADPH is obtained from the central metabolic pool of living cells.

In general, hydroxylated compounds are further metabolized to the corresponding mono- or di-terminal carboxylate, which can supply energy and carbon for yeast growth (Klug et al., Adv. In Micr.
obial Physiology, 5: 1-43, (1971)). Candida maltosa, a diploid yeast, can grow on alkanes as a single carbon source by extracting carbon and energy via the β-oxidation pathway. Because this pathway is so efficient, usually wild-type strains do not produce dicarboxylic acids via ω-oxidation while growing on alkanes. However, the carboxylate production rate can exceed the growth requirements of the organism. Under appropriate conditions, this excess carboxylate product is released into the growth medium. In order to industrially produce carboxylate-rich solutions from which the desired carboxylate compound can be easily separated, a net production of carboxylate derived from aliphatic starting materials was developed.

[0006] The use of yeast to produce mono- and di-terminal carboxylate is known in the art. Various natural ("wild-type") strains have been developed for producing diterminal acid. US4,275,158
Includes the Debaryomyces vanriji
IAE) using ATCC 20588, has been described to generate the di terminal carboxylate of C ₁₀ -C ₁₈ aliphatic hydrocarbon or a fatty acid. US
Nos. 4,220,720 include Debaryomyces puffy for similar purposes.
omyces phaffi) It has been reported to use ATCC 20499. Such carboxylates also include the production of diterminal carboxylate from C ₉ -C ₁₉ aliphatic hydrocarbons by Pichia polymorpha (JP 700002492) and the production of carboxylate by Candida cloacae (JP 76600750). It has been reported to use other natural strains for the production of.

The diterminal carboxylate produced by fermentation by most yeasts, including Candida maltosa, is in most cases shorter than the original aliphatic substrate by one or more pairs of carbon atoms, and mixtures are common. (Ogino et al., Agr. Biol.
. Chem. 29: 1009-1015 (1965); Shiio et al., Arg.
. Biol. Chem. 35: 2033-2012 (1971); Hill et al., Appl. Microbiol. Biotechnol. 24: 168-17
4 (1986)). The shorter chains are due to the degradation of the substrates and products by the peroxisomal β-oxidation pathway after activation to the corresponding acyl-CoA ester. The first step in the β-oxidation (fatty acid) pathway involves acyl CoA
Oxidation of the ester to its enoyl-CoA is involved and is catalyzed by acyl-CoA oxidase. In addition, enoyl-CoA is activated by the action of enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase.
-Metabolized to ketoacyl-CoA. The fourth and final step in the β-oxidation pathway involves acyl-CoA acetyltransferase (more generally, acyl-CoA
(Referred to as thiolase), which facilitates the reaction of β-ketoacyl-CoA with free coenzyme A molecules and hydrolyzes the two carboxy-terminal carbon fragments of the starting fatty acid, such as acetyl-CoA.

[0008] Gene mutations that cause partial interference in these late reactions produce unsaturated or hydroxylated by-products (Meussdo).
erffer et al., Proc. -World Conf. Biotechnol
. Fats Oils Ind. 142-147 (1988)). These unwanted by-products are often associated with the biological production of diterminal carboxylate. Mutants generated by typical mutagenesis or genetic engineering that increase carboxylate production in excess of growth requirements have been reported in the prior art. Candida lipolytica (Candida lipolytica)
(EP 229252, DE 3929337, DE 4019166), Candida tropicalis (DE 392)
9337, DE 4019166, EP 296506, EP 316072,
US 5,254,466), Pichia carb
ofelas) (JP 57129694), Torulopsis candida (Torr)
ulopsis Candida (JP 52018885) and Torulopsis bombicola (US 3,
796,630) have been described. If the yeast's ability to consume the desired carboxylate as part of its normal metabolism was reduced, the production of excess carboxylate could be increased. Mutants lacking in part the ability to grow on alkanes, fatty acids or dicarboxylic acid substrates often show increased dicarboxylic acid yields. However, most mutants have not been characterized, except for their reduced ability to use these compounds as carbon sources for growth. Presumably, the ability to produce diterminal carboxylate is increased over partial inhibition of the β-oxidation pathway. In addition, compounds known to inhibit β-oxidation (ie, acrylates) also increase diterminal carboxylate yield.

With respect to biocatalysts for producing carboxylate, it is desirable to effectively inhibit the β-oxidation pathway in a first reaction catalyzed by acyl-CoA oxidase. Complete inhibition in this step prevents recycling of the diterminal carboxylate product by the β-oxidation pathway, while increasing the yield of diterminal carboxylate by resubmitting the substrate to the ω-oxidation pathway I do. Furthermore, the use of such mutants prevents undesired chain modifications associated with the β-oxidation pathway, such as unsaturation, hydroxylation or chain shortening. In Candida maltosa, it encodes an acyl-CoA oxidase (Masuda et al., Gene, 16
7: 157-161 (1995)) by inactivating both POX4 genes to functionally inhibit the β-oxidation pathway and redirect metabolic flow back to the microsomal ω-oxidation pathway, thereby Carboxylate yield can be increased.

[0010] A method for disrupting a target gene of a yeast belonging to the genus Pichia is disclosed in EP2266752. In addition, US 5,254,466 includes Candida tropicalis (C
A method for completely inhibiting the consumption of carboxylate through genetic engineering of C. andida tropicalis has been claimed. There is a great deal of scientific evidence demonstrating that there are significant differences between Candida tropicalis and Candida maltosa as commercial biocatalysts (see below). In addition, a number of strains of Candida maltosa that metabolize aliphatic hydrocarbons for growth have been described (Bos et al., Antoni van Leeuwenh).
oek, 39: 99-107, (1973)). However, the prior art includes
There are no reports of using Candida maltosa to produce mono- or di-terminal carboxylate.

In addition to inhibiting the β-oxidation pathway, another strategy has recently been reported as another possible route to increasing excess carboxylate production in yeast. Rather than inhibiting consumption, increasing carboxylate production was done by increasing cytochrome P450 hydroxylation activity. DE1
9507546 discloses the expression of an alkane hydroxylated cytochrome P450 system in Saccharomyces cerevisiae, a yeast that cannot normally hydroxylate aliphatic hydrocarbons or fatty acids. By increasing the alkane cytochrome P450 monooxygenase activity in this yeast, if the normal pathway for rapid carboxylate consumption is lacking,
The carboxylate accumulates naturally. However, the cytochrome P450 monooxygenase activity in this strain appears to be abnormally sensitive to the toxic effects of oxygen (Zimmeret et al., DNA & Cell Biolog).
y, 14: 619-628, (1995)), presumably indicating the lack of structural integrity required for this genetically engineered strain.

[0012] WO 9114781 includes Candida tropicalis by genetic engineering.
A method for amplifying a cytochrome P450 hydroxylation system in D. tropicalis is described. Although some increase in carboxylate production was observed, expression of the cytochrome P450 enzyme was inadequate and improvement in activity was not completely successful (Picataggio et al., Bio / Techno).
10: 894-898 (1992)). In addition, German patent DE3
929337 has a selective reagent, cytochrome P45 using 1-dodecine.
Limited success in selecting mutants with improved monooxygenase activity and dicarboxylate production has been described.

[0013] Wild-type Candida maltosa strains IAM12247 and ATCC2814
0 is an equivalent organism. They are available from the Institute for Applied Microbiology (University of Tokyo, Tokyo, Japan) and the American Type Culture Collection (American Type Culture).
re Collection) (Manassas, VA, USA). Strain ATCC90625 and ATCC90677 are IAM
12247, a trophic marker mutation ade1, his5 (90625)
And ade1, his5, ura3 (90677). Both of these strains are available from the 19th Edition of the Yeast Reference Guide 1995 of the American Type Culture Collection.

[0014] Recent reports include Candida maltosa IAM12247 / ATCC2814.
There is a description of DNA sequence information for a number of alkane cytochrome P450 monooxidases, as well as cytochrome P450 reductase derived from O.0 (Ohkuma et al., DNA & Cell Biology, 14: 163-1).
73, (1995), and Kargel et al., Yeast, 12: 333-3.
48, (1996)). For Candida maltosa, at least eight structurally different cytochrome P450s with different substrate specificities have been identified. Each of these intact membrane proteins is responsible for catalyzing the monooxygenase reaction,
Require electron transfer from NADPH via cytochrome P450-NADPH reductase.

[0015] Usually, such mutant marker strains are used for gene transformation. However, Candida tropicalis
Given the limited success reported to date for homologous expression of the P450 monooxygenase system in Candida maltosa, it was uncertain whether such a biocatalyst could be developed in Candida maltosa.

A biocatalyst for producing dodecanedioic acid (DDDA) based on Candida maltosa is Candida tropicalis.
There is a great deal of evidence that it is clearly different from that based on). These yeasts are two species that differ in the field of yeast taxa, and there are significant differences between the two species at the molecular and biochemical levels (Meyer et al., Arch. Micr.
obiol. , 104: 225-231 (1975)). These differences, as well as the importance of their classification, have practical implications.

[0017] Candida maltosa cannot grow on starch. Candida tropicalis is capable of growing on starch. Candida maltosa is usually resistant to high concentrations of cyclohexamide, whereas Candida tropicalis is not. Candida tropicalis (Candida tropicalis)
) Is often associated with human disease, but not with Candida maltosa.

These differences affect the usefulness of organisms as biocatalysts in industrial processes. Starch is an inexpensive raw material that slowly releases glucose and is an effective co-substrate in producing DDDA by Candida tropicalis. Candida
For maltosa, starch is not a substance that can be selected as a co-substrate. The insensitivity of Candida maltosa to cyclohexamide makes it impossible to use one of the few antibiotic selection techniques available in yeast genetic engineering.

In molecular comparison, the two species are different from each other. One widely accepted method for assessing the evolutionary gap between species is the small ribosomal RNA subunit (18S
) Is based on a DNA sequence comparison. To date, comparisons between Candida maltosa and Candida tropicalis have shown high similarity (ie, ≧ 94%) in these sequences (Ohkuma et al., Biosci. Biotech. Biochem., 57).
: 1793-1794 (1993); Pesole et al., Genetics, 14
1: 903-907 (1995); Cai et al., Internal J. Med. Sys.
Bacterial. 46: 542-549 (1996)). However,
Comparing the GenBank sequences of the key enzymes in the alkane oxidation process (cytochrome P450 monooxygenase and cytochrome P450 reductase)
It can be seen that the difference in these genes is greater than the difference by 18S RNA gene comparison. For cytochrome P450 reductase, DNA sequence similarity is only 83%. For cytochrome P450 monooxygenase, the maximum DNA sequence similarity of the seven genes by comparing the seven genes is only 77%. Most of the cytochrome P450 monooxygenase sequence comparisons are less than 70%. This suggests that genes important for the alkane oxidation process are under selection pressure and have evolved separately in these two different species. In fact, Meyer et al. (Arch. Microbiol, 104: 225-
231 (1975)) report that the two species appear to occupy different ecological status. Candida maltosa is found only in hydrocarbon-contaminated environments. Candida tropicalis (Candida tropicalis)
) Can grow in environments contaminated with hydrocarbons, but are often found in relation to warm-blooded animals. Finally, a whole genomic DNA recombination test showed that Candida maltosa and Candida tropicalis.
alis) had a total DNA similarity of less than 40% (M
Eyer et al., Arch. MicrobioL, 104: 225-231 (197
5)). Candida maltosa and Candida tropicalis
Such differences in the molecular biology of C. tropicalis obscure whether genes from two organisms act in the same manner. Therefore, P in Candida tropicalis
The limited success that resulted in increased activity of the 450 system does not guarantee that increased activity in Candida maltosa will be successful. It has been demonstrated that the homologous expression of some P450 genes was increased (Ohkuma et al., Bioch.
im. Biophys. Acta. , 1236: 163-169 (1995)).
There is no report that P450 monooxygenase activity was increased in Candida maltosa.

The very good expression of the active P450 monooxygenase system in genetically engineered Candida maltosa can be a useful biocatalyst for carboxylate production. To date, reports on Candida maltosa transformants capable of expressing alkane P450 monooxygenase, fatty acid monooxygenase, and cytochrome P450-NADPH reductase in combination are not known in the art.

SUMMARY OF THE INVENTION [0021] The present invention provides genetically designed increased alkane hydroxylation activity.
Characterized transformed Pichia pastoris (Pichia pastori)
s) is replaced by at least one chemical formula CH_Three(CH_Two)_nCH_Three(However, n = 4 to 20
) C₆~ C_{twenty two}By contacting a linear hydrocarbon under aerobic conditions,₆~ C _{twenty two} Bioproduction of mono- or dicarboxylates
How to do.

[0022] Another aspect of the invention provides a transformed Candida maltosa, which has been genetically engineered to have increased alkane hydroxylation activity, using at least one chemical formula CH ₃ (CH ₂ ) _n The C ₆ -C ₂₂ monocarboxylate or dicarboxylate is bio-produced by contacting the C ₆ -C ₂₂ linear hydrocarbon with CH ₃ (where n = 4-20) under aerobic conditions. How to

A further aspect of the invention comprises at least one foreign gene encoding cytochrome P450 monooxygenase and at least one foreign gene encoding cytochrome P450 reductase, each of which increases alkane hydroxylation activity. A transformed Pichia pastoris operably linked to a suitable control element. Genes encoding cytochrome P450 include P450 Alk1-A (D12475), Alk2-A (X5
881), Alk3-A (X55881), Alk4-A (D12716), A
lk5-A (D12717), Alk6-A (D12718), Alk7 (D1
2719) and Alk8 (D12719) or a gene substantially similar thereto.

A further aspect of the invention provides that at least one copy of the gene encoding cytochrome P450 monooxygenase and / or cytochrome P45
Transformed Candida maltosa, which further comprises at least one copy of the gene encoding the C.0 reductase, each of which is operably linked to appropriate regulatory elements to increase alkane hydroxylation activity. It is. In addition, the present invention provides a primary alkane monooxygenase (P450Alk1-A), fatty acid monooxygenase (P450Al
k3-A) and the construction of an expression cassette designed to deregulate the expression of cytochrome P450-NADPH reductase.

The present invention relates to a method of transforming Candida maltosa, which has been genetically engineered to inhibit the β-oxidation pathway, using at least one compound of the formula CH ₃ (CH ₂ ) _n CH ₃ ( However, by contacting with C ₆ -C ₂₂ linear hydrocarbon and aerobic conditions of n = 4 to 20), to a method of bioproduction a monocarboxylate or Jikarubokishire DOO C ₆ -C ₂₂ .

The present invention further provides a transformed Candida maltosa characterized by genetically engineered inhibition of the β-oxidation pathway and increased alkane hydroxylation activity by at least one chemical formula CH ₃ (CH ₂ ) _n CH ₃ (where n = 4-2)
By contacting with C ₆ -C ₂₂ linear hydrocarbon and aerobic conditions 0), C ₆ ~
The monocarboxylate or dicarboxylate of C ₂₂ relates to a method for bioproduction.

A further aspect of the present invention is a genetically engineered Candida maltosa strain that has increased cytochrome P450 activity and / or has been disrupted in the β-oxidation pathway.

A further aspect of the present invention is a novel DNA fragment. These fragments include (a
A) a first Candida maltosa promoter operably linked to a DNA encoding at least one Candida maltosa-derived polypeptide; and (b) a DNA encoding at least one Candida maltosa-derived polypeptide. An operably linked second Candida maltosa promoter is included. The gene binding to the first Candida maltosa promoter encodes a cytochrome P450 monooxygenase, and the gene binding to the second Candida maltosa promoter encodes a cytochrome P450 reductase. More preferably, the first Candida maltosa promoter is PGK and the genes encoding cytochrome P450 monooxygenase are Alk1-A (D12475), Alk2-A (X
55881), Alk3-A (X55881), Alk4-A (D12716)
, Alk5-A (D12717), Alk6-A (D12718), Alk7 (
D12719) and Alk8 (D12719).

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS, BIOLOGICAL DEPOSITIONS AND FIGURES The present invention provides detailed descriptions, biological deposits,
It can be understood in detail from the sequence notation and the drawings.

[0030] Applicants filed a 37C. F. R. §1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequence and / or Amino Acid Sequence Descriptions—Sequence Rules”), standard WIPO ST. 2
5 (1998) and provided a sequence listing that also met the requirements of the Sequence Listing for PCT and EPO.

SEQ ID NO: 1 represents the sense primer for cytochrome P450-NADPH reductase.

[0032] SEQ ID NO: 2 represents an antisense primer for cytochrome P450-NADPH reductase.

[0033] SEQ ID NO: 3 represents the sense primer for the cytochrome P450Alk1-A gene.

[0034] SEQ ID NO: 4 represents an antisense primer for the cytochrome P450Alk1-A gene.

[0035] SEQ ID NO: 5 represents the sense primer for the cytochrome P450Alk3-A gene.

[0036] SEQ ID NO: 6 represents an antisense primer for the cytochrome P450Alk3-A gene.

[0037] SEQ ID NO: 7 represents a sense primer for the PGK promoter.

[0038] SEQ ID NO: 8 represents an antisense primer for binding of the PGK promoter to the P450Alk1-A gene.

SEQ ID NO: 9 represents a sense primer for the 5 ′ end of P450Alk1-A gene.

[0040] SEQ ID NO: 10 represents an antisense primer to the 5 'end of the P450Alk1-A gene.

SEQ ID NO: 11 represents a sense primer for the 3 ′ end of P450Alk1-A gene.

[0042] SEQ ID NO: 12 represents an antisense primer to the 3 'end of P450Alk1-A gene.

[0043] SEQ ID NO: 13 represents the sense primer for the fusion of the PGK terminator to the P450Alk1-A gene.

[0044] SEQ ID NO: 14 represents an antisense primer for the PGK terminator.

[0045] SEQ ID NO: 15 represents an antisense primer for fusion of the PGK promoter to the P450Alk3-A gene.

SEQ ID NO: 16 represents the sense primer for the 5 ′ end of P450Alk3-A gene.

[0047] SEQ ID NO: 17 represents an antisense primer to the 5 'end of the P450Alk3-A gene.

[0048] SEQ ID NO: 18 represents the sense primer for the 3 'end of the P450Alk3-A gene.

[0049] SEQ ID NO: 19 represents an antisense primer to the 3 'end of P450Alk3-A gene.

[0050] SEQ ID NO: 20 represents the sense primer for the fusion of the PGK terminator to the P450Alk3-A gene.

[0051] SEQ ID NO: 21 is a fragment of the P to the cytochrome P450-ADPH reductase gene.
Represents the antisense primer for the fusion of the GK promoter.

[0052] SEQ ID NO: 22 is a fragment of the cytochrome P450-NADPH reductase gene
'Represents the sense primer for the terminus.

SEQ ID NO: 23 is a fragment of the cytochrome P450-NADPH reductase gene
'Indicates an antisense primer for the terminus.

SEQ ID NO: 24 is a fragment of the cytochrome P450-NADPH reductase gene
'Represents the sense primer for the terminus.

SEQ ID NO: 25 is a fragment of the cytochrome P450-NADPH reductase gene
'Indicates an antisense primer for the terminus.

SEQ ID NO: 26 represents the sense primer for the fusion of the PGK terminator to the cytochrome P450-NADPH reductase gene.

SEQ ID NO: 27 represents a sense primer for the Candida maltosa POX4 gene.

SEQ ID NO: 28 represents an antisense primer for the Candida maltosa POX4 gene.

SEQ ID NO: 29 represents the sense primer for the Candida maltosa URA3 gene.

[0060] SEQ ID NO: 30 represents an antisense primer for the Candida maltosa URA3 gene.

SEQ ID NO: 31 represents a sense primer for the Candida maltosa ADE1 gene.

[0062] SEQ ID NO: 32 represents an antisense primer for the Candida maltosa ADE1 gene.

[0063] SEQ ID NO: 33 represents the sense primer for the Candida maltosa HIS5 gene.

SEQ ID NO: 34 represents an antisense primer for the Candida maltosa HIS5 gene.

Applicants have made the following biological deposits in accordance with the provisions of the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Procedures. As used herein, "ATCC" is 10801 University Street, Manassas, Virginia, USA, 10801.
International Depositary, American Tyrant Conservation Agency (American Ty)
Pe Culture Collection). "ATCC
"Number" is the accession number for the culture at the time of deposit with the ATCC.

[0066]

Pichia pastoris SW64 / 65 is capable of producing active alkane cytochrome P450, which, when induced by the presence of methanol, converts C ₆ -C ₂₂ alkanes to the corresponding monobasic and dibasic acids. It can be characterized as a Pichia pastoris strain having a possible unusual ability.

Candida maltosa SW81 / 82 is an unusual Candida maltosa in that it cannot grow on C ₆ -C ₂₂ alkanes or mono-fatty acids, and also has a suitable carbon and energy source such as glycerol. in ability in the presence generates a diacid from a monobasic acid or alkane C ₆ -C ₂₂ can be characterized as Candida Marutosa unusual. This strain contains the disrupted POX4 gene and other auxotrophic markers are distant. This strain is β
-Oxidation is inhibited.

Candida maltosa SW84 / 87.2 is an unusual Candida maltosa in that it cannot grow on C ₆ -C ₂₂ alkanes or monofatty acids, and also contains carbon and glycerol as appropriate. it can be characterized as Candida Marutosa unusual in a capability of generating diacid from a monobasic acid or an alkane in the presence of an energy source C ₆ ~ C _22. SW84 /
87.2 is unusual in its ability to oxidize C ₆ -C ₂₂ alkanes or monobasic acids to dibasic acids in the presence of glucose at concentrations above 5 g / L. This strain expresses increased alkane hydroxylation activity and contains a disrupted POX4 gene.

[0070] DETAILED DESCRIPTION OF THE INVENTION The present invention uses genetic engineering techniques it has been subjected organisms, aliphatic compounds of the formula _{CH 3 (CH 2) n C} H 3 ( however, n = 4 to 20) To a monoterminal and diterminal carboxylate. The present invention relates to cytochrome P4.
50 transformed with increased activity (including the combined simultaneous expression of alkane P450 monooxygenase, fatty acid monooxygenase and cytochrome P450-NADPH reductase expression) and / or having a disrupted β-oxidation pathway First description of Candida maltosa strain. Furthermore, it is economical to improve the volumetric productivity (g product / L / hr) of either the P450-enhancing strain or the β-inhibiting strain based on the rate of growth and alkane utilization of the wild-type strain. Bioprocesses are required. Thus, the combination of these two concepts provides an excellent biocatalyst for producing mono- and di-terminal carboxylate from aliphatic substrates. The present invention provides the desired carboxylate in sufficient quantity and conversion efficiency to be commercially viable.

Certain recombinant organisms express increased alkane hydroxylation activity. Hydroxylation of the terminal methyl group occurs through alkane hydroxylation activity.
Increased hydroxylation activity results from increased alkane monooxygenase, fatty acid monooxygenase or cytochrome P450 reductase, separately or in various combinations. Additional enzymatic steps are necessary to further oxidize to the carboxylate form. Alcohol oxidase (Kemp et al., Appl.
. Microbiol. And Biotechnol. , 28: 370 (19)
88)), and two further oxidation steps catalyzed by alcohol didrogenase, yield the corresponding carboxylate.

Another recombinant organism has a disruption of the β-oxidation pathway. The diploid yeast Candida maltosa grows on alkanes as a single carbon source by extracting carbon and energy through the β-oxidation pathway. Because this pathway is so efficient, wild-type strains usually do not produce dicarboxylic acids by ω-oxidation while growing on alkanes. Inhibiting the β-oxidation pathway so as to increase the metabolic flux to the ω-oxidation pathway increases the yield and selectivity of the bioprocess for the conversion of alkanes to mono- and di-terminal carboxylate.

A third recombinant organism has both increased alkane hydroxylation activity and gene disruption of the β-oxidation pathway. Increased hydroxylation activity results from increased alkane monooxygenase, fatty acid monooxygenase or cytochrome P450 reductase, separately or in various combinations. The products of the present invention are useful as intermediates in the formation of corrosion inhibitor compounds and surfactants. More particularly, the methods and materials of the present invention are useful for bioproduction of dodecandionic acid. Bioprocesses provide greater flexibility in the manufacture and sale of intermediates related to the current chemical route to polymer and chemical grade dodecanedioic acids. In particular, high yields are obtained with good selectivity. further,
The environmental impact of performing the commercial bioprocess is expected to be more favorable than current chemical processes.

The terms and abbreviations used herein are specified as follows.

“Reduced nicotinamide adenine dinucleotide” is abbreviated NADPH.

“Reduced nicotinamide adenine dinucleotide phosphate” is abbreviated NADPH.

[0077] "Candida mallosa IAM 12247 cytochrome P45
"0Alk1-A gene" is abbreviated as Alk1-A.

"Candida mallosa IAM 12247 cytochrome P45
"0Alk3-A gene" is abbreviated as Alk3-A.

“Candida mallosa cytochrome P450-NADPH reductase gene” is abbreviated as P450 reductase or CPR.

“Candida maltosaacyl CoA gene” is abbreviated as POX4.

“Candida maltosa IAM12247 URA3 gene code encoding the enzyme orotidine-5′-monophosphate decarboxylase” is abbreviated as URA3.

“Phosphoglycerol kinase” is abbreviated PGK.

“Alcohol oxidase I” is abbreviated AOX1.

“Gas chromatography” is abbreviated GC.

“Polymerase chain reaction” is abbreviated PCR.

“Autonomously replicating sequence” is abbreviated as ARS.

“Dodecanedioic acid” is abbreviated DDDA.

The term “genetically engineered” refers to the insertion of a nucleic acid molecule, created or removed by any means extracellular, into any viral, bacterial plasmid or other vector system to remove genetic material. Forming new combinations, which are taken up by the host organism and propagated in the host organism and expressed to alter the phenotype of the host organism.

The term “transformation” refers to a genetic engineering technique that transfers a nucleic acid fragment into the genome of a host organism to produce a genetically stable heritable trait. Host organisms containing the transferred nucleic acid fragments are referred to as "transgenic" or "transformed" organisms or transformants.

The term “nucleic acid” refers to a high molecular weight complex compound present in living cells, the basic unit to which nucleotides are linked together with phosphate bridges. Nucleic acids are divided into two types: ribonucleic acids (RNA) and deoxyribonucleic acids (DNA).

An “isolated nucleic acid fragment” is a polymer of single- or double-stranded RNA or DNA, optionally containing synthetic or unnatural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a DNA polymer may consist of one or more cDNAs, genomic DNAs or synthetic DNAs.

The term “cytochrome P450” refers to a widely distributed monooxygenase that is active in many different biological hydroxylation reactions and is a component of the cytochrome P450 hydroxylation system.

The term “cytochrome P450 reductase” refers to a widely distributed reductase that is active in many different biological hydroxylation reactions and is a component of the cytochrome P450 hydroxylation system.

The term “inhibited β-oxidation pathway” or “β-inhibition” refers to wild-type β-oxidation.
Gene disruption that effectively removes acyl-CoA oxidase, the first enzyme in the oxidation pathway.

“Altered level” refers to the production of a gene product in an organism that differs in quantity or ratio from the production of the gene product of a normal, wild-type, or non-transformed organism. Production may be specifically described as "increased" or "decreased" relative to production by normal, wild-type or non-transformed organisms.

[0096] The term "augmented" refers to an improvement or increase over what is inherently observed or over native function. The increase in alkane hydroxylation activity is due to cytochrome P450 monooxygenase and / or cytochrome P450-
It is associated with at least one additional copy of the gene encoding NADPH reductase (compared to wild type).

The terms “cassette” and “gene cassette” refer to a large number of nucleotide sequences that have been purposely joined or linked in vitro to a unique construct. An "expression cassette" specifically contains a promoter fragment, a DNA sequence for a selected gene product, and a transcription terminator.

The terms “plasmid” and “cloning vector” relate to an extrachromosomal element usually having the form of a circular double-stranded DNA molecule and carrying a gene that is not part of the central metabolism of the cell. Such elements may be linear or circular, autonomously replicating sequences, genomic integration sequences, phage sequences of single-stranded or double-stranded DNA or RNA from any source. The term "autonomously replicating sequence" refers to a chromosomal sequence capable of autonomously replicating a plasmid in yeast.

The term “expression” refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment of the invention. Further, expression may refer to translation of the mRNA into a polypeptide. "Overexpression" refers to the production of a gene product in a transgenic organism that is in excess of the level of production in a normal or non-transformed organism. "Co-suppression" refers to the generation of a sense RNA transcript that can suppress the expression of the same or substantially similar foreign or endogenous gene (US 5,231,020).

The term “mutation” relates to a chemical change in the DNA of an organism that alters the genetic traits of the organism. Strains exhibiting such altered properties are referred to as "mutants."

The term “oligonucleotide primer” relates to short oligonucleotides that are base pairs to a region of a single-stranded template oligonucleotide. Primers are necessary to form a starting point for DNA polymerase in generating complementary strand synthesis with single-stranded DNA.

The terms “restriction enzyme” and “restriction endonuclease” refer to double-stranded DNA
And enzymes that catalyze hydrolytic cleavage within a particular nucleotide sequence of

The term “linear hydrocarbon” relates to C ₆ -C ₂₂ aliphatic hydrocarbons, fatty acids and fatty acid esters containing zero, one or two double bonds in the carbon skeleton. In addition, the term includes any of the above straight chain compounds wherein one of the terminal carbons has been replaced with a phenyl group. Particularly preferred hydrocarbons are nonane, decane,
Undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, or their respective monocarboxylic acids. Preferably, an alkane of C ₁₂ -C _14. Particularly, dodecane is preferred.

The term “alkane hydroxylation activity” relates to the ability of an organism, such as yeast, to enzymatically hydroxylate the terminal methyl group of a linear hydrocarbon using a cytochrome P450 hydroxylation system. The term "cytochrome P450 hydroxylation system" refers to a hydroxylation system consisting of at least three biological components: 1) cytochrome P450 monooxygenase, 2) cytochrome P450-NADPH reductase, and 3) reduced nicotinamide. Adenine dinucleotide (NADPH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH).

“Gene” refers to a nucleic acid fragment encoding a particular protein, including regulatory sequences before the coding sequence (5 ′ non-coding sequences) and regulatory sequences after the coding sequence (3 ′ non-coding sequences). "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, including regulatory and coding sequences that are not found together in nature. Thus, a chimeric gene may contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source, but where the sequences are different from those found in nature. It was made. "Endogenous gene" refers to a native gene that is in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but which is introduced into the host organism by introduction of the gene. Foreign genes can include native genes inserted into a non-native organism, or chimeric genes. "
A “transgene” is a gene that has been introduced into the genome by a transformation method.

“Coding sequence” is a DNA sequence that codes for a specific amino acid sequence. An "appropriate regulatory sequence" is a nucleotide sequence that is located upstream (5 'non-coding sequence), within a coding sequence, or downstream (3' non-coding sequence) of a coding sequence and is associated with the relevant coding sequence. Affects sequence transcription, RNA processing or stability, or translation. Regulatory sequences may include promoters, translation leader sequences, introns and polyadenylation recognition sequences.

"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is 3 ′ to the promoter sequence.
Located on the side. Promoter sequences consist of proximal and more distal upstream regions, the latter often referred to as enhancers. An "enhancer" is a DNA sequence capable of stimulating promoter activity, which may be an innate element of the promoter, or a heterologous heterologous inserted to increase the level of the promoter or the tissue specificity of the promoter. May be an element. A promoter may be entirely derived from the native gene, may be composed of different elements derived from different promoters found in nature, or may contain synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cells, or at different stages of development, or in response to different environmental conditions. Promoters that cause a gene to be expressed at most times under most growth conditions are commonly referred to as "constitutive promoters". A variety of new promoters useful in plant cells are constantly being discovered. Numerous examples can be found in the compilation by Okamaro and Goldberg (Biochemis
try of Plants 15: 1-82 (1989)). Furthermore, in most cases, the exact boundaries of the regulatory sequences have not been completely specified, so that DNA fragments of different lengths may have the same promoter activity.

The term “operably linked” relates to the binding of nucleic acid sequences on a single nucleic acid fragment such that one function acts on another. For example, a promoter is operably linked to a coding sequence if the promoter affects the expression of the coding sequence (ie, the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. A “mature” protein is a polypeptide that has been post-translationally processed, ie, one in which the pre- or pro-peptides present in the primary translation product have been removed. "Precursor" proteins are mRNA
Is the primary product of translation, ie, the pre- and pro-peptides are still present. However, pre- and propeptides are not restricted to intracellular localization signals.

Construction of Recombinant Pichia pastoris Another aspect of the present invention relates to the genetic engineering of Pichia pastoris that results in the expression of an active P450 system from a heterologous source. Alk1-A gene (or Alk3-
A or P450 reductase gene) followed by a transcription terminator (AO
The expression cassette is constructed to include a promoter, such as, but not limited to, the strong methanol-inducible promoter of alcohol oxidase I (AOX1) fused to such an X1 derived promoter. The expression cassette was transferred to Zeocin.
Subcloning into a vector containing a suitable transformation marker, such as, but not limited to, the HIS4, ARG4, SUC2 or shble gene encoding resistance (Invitrogen, San Diego, CA, USA). Subsequent transformation of the appropriate Pichia pastoris strain by established methods (US 4,855,231) results in the integration of the expression cassette for the gene into the Pichia pastoris genome. By various methods, including but not limited to PCR and Southern blot analysis, transformants carrying multiple copies of the expression cassette can be identified.

Another embodiment of designing a Pichia pastoris for expression of an active P450 system from a heterologous source requires subcloning multiple expression cassettes on one or two plasmids. For example, the expression cassette for the Alk1-A and Alk3-A genes can be subcloned on one plasmid, and the expression cassette for the P450 reductase gene can be subcloned on another plasmid. Alternatively, the expression cassette for the Alk1-A and P450 reductase genes can be subcloned on one plasmid and the expression cassette for the Alk3-A gene can be subcloned on another plasmid. Alternatively, the expression cassette for the Alk3-A and P450 reductase genes can be subcloned on one plasmid and the expression cassette for the Alk1-A gene can be subcloned on another plasmid. Alternatively, the expression cassette for the Alk1-A and Alk3-A and P450 reductase genes can be subcloned on a plasmid. The plasmid is then used to transform a suitable Pichia pastoris host, either sequentially or simultaneously. By various methods, such as, but not limited to, PCR and Southern blot analysis, transformants carrying multiple copies of the expression cassette can be identified.

A further aspect of designing Pichia pastoris for expression of an active P450 system from a heterologous source is as described above for Alk1-A, Alk3-A and P4
It requires subcloning the expression cassette for the 50 reductase gene, individually or in multiple copies, onto a replicating plasmid. The replicating plasmid is then used to transform, either sequentially or simultaneously, the appropriate Pichia pastoris hosts. By various methods, such as, but not limited to, PCR and Southern blot analysis, transformants carrying multiple copies of the expression cassette can be identified.

Genetically engineered Pichia pastoris cells containing multiple copies of the expression cassette for the Alk1-A, Alk3-A and P450 reductase genes are saturated in minimal medium containing glycerol (or glucose) as a carbon source. And then induction of the AOX1 promoter with methanol. This produces high levels of P450-based components and increases hydroxylation activity. Aliphatic substrates can be added prior to induction, at the beginning of induction, or at any time during induction, and after an appropriate time, and the medium is analyzed for carboxylate as described above.

PCR Amplification of Genomic DNA Obtained from Candida maltosa Candida maltosa cytochrome P450Alk1-A of CAM. Gene Bank (Nati
onal Center for Biotechnology Inform
Oligonucleotide primers are prepared based on sequences available from Co., Bethesda, MD, USA). Appropriate unique restriction sites are designed into the primers to allow convenient ligation to the cloning vector as well as construction of the gene expression cassette (eg, Sambrook et al., Molecular Cloning: A Laboratory Manner).
ual, Second Edition, Cold Spring Harbo
r Laboratory Press, (1989)). In a similar manner, for the URA3 gene of Candida maltosa IAM12247,
Design oligonucleotide primers. Using the polymerase chain reaction (PCR) US Pat. No. 4,683,202 (1987, Mullis et al.) And US Pat. No. 4,683,195 (1986, Mullis et al.)
Genome DN obtained from Candida maltosa IAM12247 corresponding to 77
Amplify the appropriate DNA sequence from A. By similar procedures and appropriate primers, cytochrome P450 Alk2-A (X5881), Alk4-A (D
12716), Alk5-A (D12717), Alk6-A (D12718)
), Alk7 (D12719) and Alk8 (D12719), including, but not limited to, other Candida maltosa IAs available from the gene bank
The M12247 sequence can be amplified by PCR.

Construction of Recombinant Candida maltosa-Chromosomal Integration The following description is an embodiment of the present invention using integration by integration of a gene of interest into a transformed host.

[0115] The DNA fragment synthesized by PCR was converted into a compound of the formula Alk1-A / Alk3-A.
A convenient cloning vector such as pUC18 or Lambda Zap (Invitrogen, San Diego, CA, USA) resulting in a vector containing the / P450 reductase / URA3 / Alk1-A gene cassette is inserted. After cloning the vector containing the gene cassette in Escherichia coli, the cassette fragment is linearized by cutting with an appropriate restriction enzyme. Candida maltosa using methods known in the art (Sambrook et al., Supra).
IAM 12247 (corresponding to ATCC 28140) is transformed and transformants that have obtained a functional copy of the URA3 gene are selected by growing on minimal medium supplemented with histidine and adenine sulfate. Genomic DNA is isolated from the transformed strain using techniques known in the art. Genomic DNA is cut with the appropriate restriction enzymes and subsequently analyzed using the Southern blot method.
In this way, clones with the maximum number of gene copies inserted into the chromosome are detected.
Larger gene copy numbers generally lead to higher levels of enzyme activity.

A further aspect of the invention is the sequential addition of P450 family genes to the Candida maltosa chromosome. Chemical formula X / URA3 / X (where X is Alk1-A described above)
Gene, Alk3-A gene, P450 reductase gene or other P450
In order to insert any of the cassettes represented by the system gene) into the host genome, following the same procedure of PCR amplification, cloning, linearization, transformation, minimal medium selection and Southern blot screening, each original copy is inserted into the chromosome. Generating a clone further comprising at least one copy of gene X against Since different cytochrome P450 enzymes will have different substrate specificities, Alk
By inserting the 1-A gene, the Alk3-A gene and the P450 reductase gene in any combination, or alternatively by inserting one or more genes, any suitable having 9 to 18 carbon atoms A set of biocatalysts that are useful for producing mono- or di-terminal carboxylate from various substrates.

[0117] Multigene copy number is increased through successive integrated transformations by inserting a reproducible marker gene with the gene of interest during each transformation. In one aspect of the invention, the URA3 gene is used repeatedly. After each transformation, the ura3-genotype is regenerated by selective growth on 5-fluoroorotic acid and can be used in subsequent transformations as the same marker gene. This method is repeated for each additional transformation. In another embodiment of the present invention, the marker gene is his5 (GenBank Accession No. X17310).
) Or ade1 (GenBank accession number D00855) marker gene. Since Candida maltosa strain ATCC 90677 is auxotrophic for three different marker genes (URA3, HIS5 and ADE1), up to three interests before it is necessary to regenerate an auxotrophic mutation. Certain genes can be inserted.

Construction of Recombinant Candida maltosa-Autonomous Replication In another aspect of the invention, an autonomously replicating sequence (ARS) is added to a vector containing a cassette having a gene encoding a cytochrome P450 system. The host Candida maltosa is transformed with this construct. The vector is stably maintained in the host as a result of ARS and selection pressure in media without uracil.
As a result of the extra copy of the gene of interest held in the vector, the active P
The expression of the 450 system is increased, which produces more carboxylate. However, the present invention relates to the genes Alk1-A, Alk3-
A, should not be considered limited by the use of P450 reductase and URA3. Any P45 identified in this Candida maltosa strain
The 0-line gene can also be included alone or in combination within the replicating plasmid construct and can be transformed into Candida maltosa to produce useful biocatalysts. Particularly useful in the present invention are the Alk1-A gene, Alk3
-A gene and P450 reductase gene. High levels of carboxylate are produced by increasing the level of expression of appropriate P450-based genes.

Reaction Conditions for Candida maltosa Clones containing high levels of cytochrome P450 hydroxylating activity are grown on a suitable medium, optionally containing an effective amount of an aliphatic substrate, for 2-3 days.
At the end of this period, additional substrate is added and the cells are cultured for another 1-2 days. Removal of the cells and acidification of the supernatant results in precipitation of the mono- and di-terminal carboxylate. The precipitate and any dissolved carboxylate were extracted from the supernatant into methyl tertiary butyl ether (MTBE) and extracted with MTBE.
It is recovered in a significantly purified state after evaporation of the solvent.

Reaction Substrates The use of dodecane as a carboxylate producing substrate is included for purposes of illustration, but should not be considered as limiting the scope of the invention. Alternative substrates that are suitable for production of carboxylate, linear hydrocarbon with carbon number C _6s -C ₂₂ are contained in a single or in combination. Fatty acids having a carbon number of C ₆ -C ₂₂ are also useful as substrates for the generation of di terminal carboxylate. In addition, aliphatic hydrocarbons or fatty acids containing one or two double bonds in the carbon skeleton are:
It is useful as a substrate for carboxylate production when one or two additional terminal carboxylate groups appear in the product. Any of the above straight chain compounds wherein one of the terminal carbons has been replaced by a phenyl group is useful for the production of carboxylate.

Cell Lines and Growth Conditions Candida maltosa strains ATCC90625 and ATCC90677 (AT
CC (see catalog of yeast) is used for transformation and expression of alkane hydroxylation activity. Pichia pastoris strain GTS115 was obtained from Invitro.
gen (San Diego, CA, USA). These strains were prepared in YEPD medium (10 g / L yeast extract, 20 g / L peptone, 2 g glucose).
0 g / L) and grow normally at 30 ° C. with shaking at 250 rpm. Candida maltosa ATCC further having a functional copy of the URA3 gene
90677 transformants are selected for by growth on minimal medium supplemented with histidine and adenine sulfate. The minimal medium was Y containing amino acids + 50 mg / L histidine and 20 mg / L adenosine sulfate + 10 g / L glucose.
NB medium (DIFCO Laboratories, Detroit, MI, U
SA).

GC Conditions Using an SE54 capillary column (15 m × 0.53 mm), MSTFA was used.
The concentration of DDDA was determined by gas chromatography of the + 1% TMCS derivative. 1.2 μm coating, temperature program at 150 ° C. for 1.5 minutes, 5 ° C./min to 200 ° C., 200 ° C. for 5 minutes; injector: 310 ° C .; detector:
320 ° C .; FID detection.

EXAMPLES The present invention is further defined by the following examples, in which all parts and percentages are by weight and temperatures are in degrees Celsius, unless otherwise specified. It should be understood that these examples illustrate preferred embodiments of the present invention and are provided for illustrative purposes only. From the above discussion and these examples, those skilled in the art will be able to ascertain the essential characteristics of the present invention and to apply the present invention to various uses and conditions without departing from the spirit and scope of the invention. Various alterations and modifications can be made to.

General Methods Methods for enzymatic digestion of DNA with restriction endonucleases, phosphorylation, ligation and transformation are well known in the art. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and include
ambrook, J.M. Fritsch, E .; F. And Maniatis,
T. Molecular Cloning: A Laboratory Man
ual- Cold Spring Harbor Laboratory P
res: Cold Spring Harbor (1989), Silhav
y, T. J. Bennan, M .; L. And Enquist, L .; W. Experiments with Gene Fusions, Cold by
Spring Harbor Press: Cold Spring Har
bor (1984), and Ausubel, F .; M. Curre et al.
nt Protocols in Molecular Biology, Gr
eene Publishing Assoc. and Wiley-inte
rscience (1987) has a detailed description.

[0125] Materials and methods suitable for the management and growth of bacterial and yeast cultures are well-known in the art. Techniques suitable for use in the examples below are Manual
of Methods for General Bacteriology (
Phillipp Gerhardt, R.A. G. FIG. E. FIG. Murray, Ralph
N. Costilow, Eugene W.W. Nester, Willis A
. Wood, Noel R. Krieg and G.W. Briggs Phil
edited by Lips), American Society for Micr
obiology, Washington, DC. (1994), or Th
omas D. Block in Biotechnology: A Tex
tbook of Industrial Microbiology, Sec
on Edition (1989) Sinauer Associates
, Inc. , Sunderland, MA. Unless otherwise specified, all reagents and materials used to grow and control bacterial cells
rich Chemicals (Milwaukee, Wl, USA), DI
FCO Laboratories (Detroit, MI, USA), Gib
coBRL (Gaithersburg, MD, USA) or Sigma C
Chemical Company (St. Louis, MO, USA).

The meanings of the abbreviations are as follows. “H” represents time, “min” represents minutes, “sec” represents seconds, “d” represents days, “mL” represents milliliters,
“L” stands for liter, “μL” stands for microliter, and “mm” stands for millimeter.

Example 1 Construction of Pichia pastoris Strains Expressing Alkane Hydroxylating Activity Primer 1 (SEQ ID NO: 1) and Primer 2 (SEQ ID NO: 1) each incorporating terminal BamHI and AvrII sites (shown in lowercase) Using 2),
Cytochrome P450-NADPH from Candida maltosa ATCC 90677
Reductase was PCR amplified. Primer 1- (SEQ ID NO: 1): 5′-AgatccATGGCATTAGATAAATTAG
-3 ′ primer 2- (SEQ ID NO: 2): 5′-ActagggCTACCAAAACATCTTCTG-
3 ′ This DNA fragment was subcloned between the BamHI and AvrII sites of the vector pPIC3K (Invitrogen, San Diego, Calif., USA) to produce pSW64 in which the AOXI promoter controls the expression of the cytochrome P450Alk1-A gene. Terminal KpnI and Ap respectively
Cytochrome P450Alk1-A was amplified by PCR from Candida maltosa ATCC 90677 using primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4) incorporating an aI site (shown in lowercase). Primer 3- (SEQ ID NO: 3): 5'-CggtaccATGGCTATAGAACAAATTA
-3 'primer 4- (SEQ ID NO: 4): 5'-AggcccTTTAGCAGAAATAAAACAC-
3 'This DNA fragment was subcloned between the KpnI and ApaI sites of the vector pPICZA (Invitrogen, San Diego, Calif., USA) to create pSW65 in which the AOXI promoter controls the expression of the cytochrome P450Alk1-A gene. Terminal XhoI and ApaI respectively
Cytochrome P450Alk3-A from Candida maltosa ATCC 90677 was PCR amplified using Primer 5 (SEQ ID NO: 5) and Primer 6 (SEQ ID NO: 6) incorporating the site (shown in lowercase). Primer 5- (SEQ ID NO: 5): 5′-ActcgagATGCCGGTTTCCTTTGTTTC
-3 'primer 6- (SEQ ID NO: 6): 5'-AggcccGTACATTTGGGATATTGG-3
'This DNA fragment was subcloned between the XhoI and ApaI sites of the vector pPICZA (Invitrogen, San Diego, CA, USA) to create pSW72 in which the AOXI promoter controls the expression of the cytochrome P450 Alk3-A gene. PSW7 containing Alk3-A expression cassette into BamHI site of pSW65 containing Alk1-A expression cassette
The BgIII / BamHI fragment from No. 2 was subcloned to generate pSW73 containing an expression cassette for Alk1-A and Alk3-A genes.

The spheroplast method (Cregg), which is one step of integrating the plasmid into the genome
Et al., Mol. Cell Biol. 5: 3376-3385, (1985))
By Pichia pastoris GTS11
5 (his4) was transformed with pSW64 to HIS prototrophy. A high copy number transformant, designated SW64, was described by Scorer et al. (Bio / Tec).
hnology, 12: 181-184, (1994)) high concentration (> 1 mg / m)
(L) by growing on G418. Strain SW64 was retransformed with pSW65 to zeocin resistance by electroporation (Invitrogen, San Diego, Calif., USA), one step of integrating the plasmid into the genome. PCR analysis confirmed the integration of the expression cassettes for both P450 reductase and P450Alk1-A genes into the genome of the double transformant. The double transformant is designated SW64 / 65 and is identified as ATCC Accession No. 74409.

The double transformant SW64 / 65 (ATCC 74409) was saturated with 20 mL of MGY (1.34% yeast nitrogen base without amino acids, 1% glycerol, 0.00004% biotin) at 30 ° C. with shaking. (48 h). After centrifugation, cells were induced by resuspension in 20 mL of MM + Fe (1.34% yeast nitrogen base without amino acids, 0.5% methanol, 0.00004% biotin, 1 mM Fe ⁺³ ) and 48 The culture was shaken at 30 ° C. for up to an hour. Cells were washed twice with PBS (S
wash with sucrose buffer (0.25M sucrose, 0.0
Washed once with 5M Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT and resuspended in 2 mL sucrose buffer supplemented with 0.3% BSA (Sambrook et al., Supra). Extracts were prepared by vortexing the cells with 容量 volume of 0.5 mm glass beads (approximately 1 mL) for 1 minute in 1 minute steps on ice for a total of 4 minutes. Translucent lysates were obtained by centrifugation of glass beads (3000 × g) and necrotic debris was centrifuged at 12000 × g. The resulting supernatant was added to 250
Centrifuge at 00xg. The resulting supernatant was centrifuged at 45,000 xg,
. The microsomal pellet was resuspended in 1 mL sucrose buffer supplemented with 3% BSA.

An equal volume (1 mL) of a NADPH / NADPH mixture (0.5 mM NADPH and 0.5 mM NADPH in sucrose buffer) was added to the microsomal sample. ¹⁴ C-lauric acid (50 mCi / mmole; ICN, Costa M)
(esa, CA, USA) was added and the mixture was incubated for 0-60 minutes with shaking at 150 rpm. The reaction was quenched by the addition of 0.1 mL of sulfuric acid, then extracted three times with 5 mL of ether and collected. Air dry the sample and add
Resuspend in mL and measure 2 μL by liquid scintillation. 200,000 dp of TLC plate (Kodak, Rochester, NY, USA)
and TLC was performed in a sealed bottle with toluene: acetic acid (9: 1) for about 2.5 hours. The plate was exposed to X-rays overnight. Conversion of lauric acid to 12-hydroxylauric acid and DDDA from genetically engineered Pichia pastoris strain SW64 / 65 (ATCC 74409) was determined according to laboratory standards (Aldrich Chemical Co., Milwaukee. Wl. U
SA) and confirmed. No conversion to DDDA was observed from the control Pichia pastoris.

Example 2 Construction of Candida maltosa P450 Alk1-A Expression Cassette The primary alkane monooxygenase (P450Alk1-A) gene is isolated after PCR amplification and Candida maltosa PGK promoter and terminator by PCR-mediated overlap extension. Exactly fused. In this manner, P45 can be altered without altering the DNA sequence that could alter PGK-mediated expression.
PGK to translation start codon and stop codon of OAlk1-A structural gene
The promoter and terminator could be correctly fused. SpeI restriction enzyme cleavage site (shown in lowercase) and P45 required for subsequent subcloning
Using primer 7 (SEQ ID NO: 7) and primer 8 (SEQ ID NO: 8) to introduce a 15 bp DNA sequence corresponding to the 5'-end of the 0Alk1-A gene (the relevant nucleotides are underlined) About 100ng of Candida maltosa A
5 of 766 bp upstream from the TCC90677 [adel, his5, ura3 / ura3] PGK structural gene (positions 56 to 756, but without primers)
The PGK promoter consisting of the 'side DNA sequence was amplified. Primer 7- (SEQ ID NO: 7): 5′-AactagtGGTAGAGCGGATGGTTACATACGAC-3
'Primer 8- (SEQ ID NO: 8): 5'- TTGTTCTATAGCCAT TCTAGTTTAAGGCAATTGA
Using primers 9 (SEQ ID NO: 9) and 10 (SEQ ID NO: 10) to introduce a 15 bp DNA sequence corresponding to the T-3 '3'-terminal PGK promoter (the relevant nucleotides are underlined) , About 20ng Candida maltosa P450Al
From pGEM-Alk1-A DNA containing k1-A gene, P450Alk1-
A 998 bp DNA fragment corresponding to the 5'-end of the A gene (positions 47 to 977) was amplified. Primer 9- (SEQ ID NO: 9): 5'- GCCTTAACTTAGAATG GCTATAGAACAAATTATT
GAAGAA-3 ′ Primer 10- (SEQ ID NO: 10): Primer 11 (SEQ ID NO: 11) to introduce a 15 bp DNA sequence corresponding to 5′-TAAACCTGCAGTGGTATCTCTACGGCA-3 ′ PGK terminator (the relevant nucleotides are underlined) ) And 12 (
Using SEQ ID NO: 12), approximately 20 ng of pGEM-Alkl-A DNA
6 corresponding to the 3'-end of the 450Alkl-A gene (positions 1004 to 1596)
A 63 bp DNA fragment was amplified. Primer 11- (SEQ ID NO: 11): 5'-TGCCGGTAGAGATACCACTGCAGGTTTTA-3 'Primer 12- (SEQ ID NO: 12): 5'- CATAAAAAAATCAATT CTATTTAGCAGAAAATAAA
AACACC-3 'Nhel restriction site required for subsequent subcloning (shown in lowercase) and 15 bp DN corresponding to the 3'-end of the P450Alk1-A gene.
Primer 13 to introduce the A sequence (the relevant nucleotides are underlined)
(SEQ ID NO: 13) and 14 (SEQ ID NO: 14) were used to convert the PGK structural gene (205
A PGK terminator containing 588 bp of the downstream 3 ′ DNA sequence (positions 0 to 2571) was amplified. Primer 13- (SEQ ID NO: 13): 5'- ATTTCTGCTAAATAG AATTGATTTTTTATGACA
CTTG-3 ′ Primer 14- (SEQ ID NO: 14): 5′-AAAG CTAGC TTTGAAACAATCTGTGGTTTG-3 ′ These PCRs were performed in 50 μL using Perkin Elmer Amplitaq kit. Amplification was performed by Perkin Elmer Gene.
Amp PCR System 9600, 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C
One cycle of 2 minutes was performed for 35 cycles. The last cycle was followed by a 5 minute extension at 72 ° C, after which the sample was held at 4 ° C before analysis by gel electrophoresis. After preparative gel electrophoresis, the desired DNA fragment was isolated and purified using Gene Clean kit (Bio101, Vista, CA).

A 998 bp DNA fragment containing the PGK promoter and P450Alk
A 766 bp DNA fragment corresponding to the 5'-end of the lA gene was ligated in a second PCR that anneals to the complementary 3'-end of the PGK promoter and the complementary 5 'end of the P450Alk1-A gene. 5'-PGK and 3'-P450A
By adding the lk1-A primer, primer 7 and primer 10, respectively, a 1749 bp DNA fragment containing the correct fusion of the PGK promoter to the 5 ′ end of the P450Alk1-A gene could be amplified. P450
A 663 bp DNA fragment corresponding to the 3'-end of the ALK1A gene, and PG
The 588 bp DNA fragment containing the K terminator was ligated in a second PCR that anneals the complementary 3'-end of the P450Alk1-A gene and the complementary 5 'end of the PGK terminator. A 1236 bp DNA fragment containing the correct fusion of the 3'-end of the P450Alk1-A gene to the PGK terminator was amplified by adding 5'-P450Alk1-A and 3'-PGK primers, primer 11 and primer 14, respectively. We were able to. PCR is Pe
Performed in a volume of 50 μL using the rkin Elmer Amplitaq kit. Perkin Elmer GeneAmp PCR System
Amplification was performed with em 9600 by 35 cycles of 1 cycle consisting of 94 ° C. for 1 minute, 45 ° C. for 1 minute, and 72 ° C. for 2 minutes. The last cycle was followed by a 5 minute extension at 72 ° C, after which the sample was held at 4 ° C before analysis by gel electrophoresis. Following preparative gel electrophoresis, the desired DNA fragment was isolated and
Purification was performed using a lean kit (Bio101).

A 1749 bp DNA fragment containing the correct fusion of the PGK promoter to the 5 ′ end of the P450Alk1-A gene was digested with SpeI and PstI, and similarly digested pLitmus38 (New England Biolabs, Be.
(Very, MA). Escherichia coli DH5α (G
ibcoBRL, Gaithersberg, MD) and transformed into LB medium (
1% (w / v) tryptone, 1% (w / v) containing X-gal (40 ng / mL)
/ D) NaCl and 0.5% (w / v) yeast extract (Difco (Det
The presence of the desired plasmid was confirmed by analyzing a plasmid DNA obtained from an ampicillin-resistant transformant showing a white colony color by the method of Roit, MI)) and named pLPA1. P450ALKI to PGK terminator
A 1236 bp DNA fragment containing the correct fusion of the 3'-end of the A gene was
And NheI, and ligated into similarly digested pLitmus38. The ligated DNA was used to transform Escherichia coli DH5α, and the desired plasmid was analyzed by analyzing plasmid DNA obtained from ampicillin-resistant transformants showing a white colony color in LB medium containing X-gal. Was confirmed and named pLA1T. Next, pLPA1 was linearized by digestion with PstI and NheI, and a 1236 bp PstI / Nh derived from pLA1T.
ligated to the eI DNA fragment. The ligated DNA was used to transform Escherichia coli DH5α, and the presence of the desired plasmid was confirmed by analyzing the plasmid DNA obtained from the ampicillin-resistant transformant, which was named pLPA1T. This plasmid was digested with SpeI and NheI and contains the Alk1-A gene correctly fused to the PGK promoter and terminator.
A 5 bp expression cassette was made.

Example 3 Construction of a Candida maltosa P450 Alk3-A Expression Cassette In addition, the major fatty acid monooxygenase (P450Alk3-A) gene was isolated and amplified by PCR-mediated overlap extension with the Candida maltosa PGK promoter and terminator. Exactly fused. SpeI restriction enzyme cleavage site (shown in lowercase) and P45 required for subsequent subcloning
Using primers 7 (SEQ ID NO: 7) and 15 (SEQ ID NO: 15) to introduce a 15 bp DNA sequence corresponding to the 5 'end of the 0Alk3-A gene (the relevant nucleotides are underlined), about 100 ng Candida maltosa A
From the genomic DNA of TCC90677, a 766 bp PGK promoter (56 to 7)
56, no primer). Primer 7- (SEQ ID NO: 7): 5′-AactagtGGTAGAGCGGATGGTTACATACGAC-3
'Primer 15- (SEQ ID NO: 15): 5'- AAAGGAAAACCGACAT TCTAGTTTAAGGCAATTGA
Using primers 16 (SEQ ID NO: 16) and 17 (SEQ ID NO: 17) to introduce a 15 bp DNA sequence corresponding to the 3'-end of the T-3 'PGK promoter (the relevant nucleotides are underlined) PGEM-Alk3-A DNA containing about 20 ng of Candida maltosa P450Alk3-A gene
Corresponding to the 5'-end of the P450Alk3-A gene (positions 62 to 655)
A 28 bp DNA fragment was amplified. Primer 16- (SEQ ID NO: 16): 5'- GCCTTAACTTAGAATG TCGGTTTCCTTTGTTCAC
AACGTTT-3 ′ Primer 17- (SEQ ID NO: 17): Primer 18 to introduce a 15 bp DNA sequence corresponding to the 5′-end of 5′-TCTTGGATATCCGAAGTTTTACCTTGAC-3 ′ PGK terminator (the relevant nucleotide is underlined) (SEQ ID NO: 18) and primer 19 (SEQ ID NO: 19) using approximately 20 ng of pGEM-Al
3 of P450Alk3-A gene (positions 652 to 1632) from k3-A DNA
A 1058 bp DNA fragment corresponding to the '-end was amplified. Primer 18- (SEQ ID NO: 18): 5'-GTCAAGGGTAAAACTTTCGATATCCAAGA-3 'Primer 19- (SEQ ID NO: 19): 5'- CATAAAAAATCAAATT TTAGTACATTTGGAATT
GGCACC-3 'Nhel restriction site required for subsequent subcloning (shown in lowercase) and 15 bp DNA corresponding to the 3'-end of P450Alk3-A gene
Approximately 100 ng of Candida maltosa ATCC 90677 genomic DNA from 588 bp PGK terminator (2050) using primer 20 (SEQ ID NO: 20) and primer 14 (SEQ ID NO: 14) to introduce the sequence (the relevant nucleotides are underlined) ２５2571) was amplified. Primer 20- (SEQ ID NO: 20): 5'- ATCCAAATGTACTAA AATTGAATTTTTTATGACA
CTTG-3 ′ Primer 14- (SEQ ID NO: 14): 5′-AAAgtagtagcTTTTGAAACAATCTGTGGTTTG-3 ′ These PCRs were performed in 50 μL using Perkin Elmer Amplitaq kit. Amplification was performed by Perkin Elmer Gene.
Amp PCR System 9600, 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C
One cycle of 2 minutes was performed for 35 cycles. The last cycle was followed by a 5 minute extension at 72 ° C, after which the sample was held at 4 ° C before analysis by gel electrophoresis. Gene Clean kit (Bio101)
Was used to purify the desired DNA fragment.

A 766 bp DNA fragment consisting of the PGK promoter and P450Alk3
A 628 bp DNA fragment corresponding to the 5'-end of the -A gene was ligated in a second PCR in which the complementary 3'-end of the PGK promoter and the 5'-end of the P450Alk3-A gene were annealed. 5'-PGK primer and 3'-P450
By adding the Alk3-A primer, primer 7 and primer 17, respectively, a 1379 bp DNA fragment containing the correct fusion of the PGK promoter to the 5 'end of the P450Alk3-A gene could be amplified. P45
A 1058 bp DNA fragment corresponding to the 3'-end of the 0Alk3-A gene and a 588 bp DNA fragment containing the PGK terminator were converted to P450Alk3-A.
The complementary 3'-end of the gene and the 5 'end of the PGK terminator were joined in a second PCR annealing. 5'-P450Alk3-A primer and 3 '
By adding the -PGK primer, primer 18 and primer 14, respectively, a 1631 bp DNA fragment containing the correct fusion of the 3'-end of the P450Alk3-A gene to the PGK terminator could be amplified. These PCRs were performed at 50 μL using a Perkin Elmer Amplitaq kit. Amplification is performed by Perkin Elmer GeneAmp
One cycle consisting of 94 ° C. for 1 minute, 45 ° C. for 1 minute, and 72 ° C. for 2 minutes was performed on the PCR System 9600 in 35 cycles. Following the last cycle, 72 ° C
The sample was kept at 4 ° C., and then analyzed by gel electrophoresis. After preparative gel electrophoresis, the desired DNA fragment is isolated and
Purification was performed using ne Clean kit (Bio101).

Digesting a 1379 bp DNA fragment containing the correct fusion of the PGK promoter to the 5 ′ end of the P450Alk3-A gene using SpeI and EcoRV,
Ligation to pLitmus38 digested in the same manner. The ligated DNA is used to transform Escherichia coli DH5α and the presence of the desired plasmid is determined by analyzing plasmid DNA obtained from ampicillin-resistant transformants that exhibit white colony color in LB medium containing X-gal. And it was named pLPA3.
A 1631 bp DNA fragment containing the correct fusion of the 3'-end of the P450Alk3-A gene to the PGK terminator was digested with EcoRV and NheI and ligated to similarly digested pLitmus 38. The ligated DNA is used to transform Escherichia coli DH5α and the presence of the desired plasmid is determined by analyzing plasmid DNA obtained from ampicillin-resistant transformants that exhibit white colony color in LB medium containing X-gal. And it was named pLA3T. Next, pLPA3 was linearized by digestion with EcoRV and NheI,
It was ligated to a 1631 bp EcoRV / Nhel DNA fragment from LA1T.
The ligated DNA was used to transform Escherichia coli DH5α, and the presence of the desired plasmid was confirmed by analyzing the plasmid DNA obtained from the ampicillin-resistant transformant, which was named pLPA3T. Digestion of this plasmid with SpeI and NheI created a 3010 bp expression cassette containing the Alk3-A gene fused exactly to the PGK promoter and terminator.

Example 4 Construction of a Candida maltosa Cytochrome P450-NADPH Reductase Expression Cassette Further isolating the cytochrome P450-NADPH reductase (CPR) gene, the Candida maltosa PGK promoter and terminator were isolated by PCR-mediated overlap extension. Accurately fused. Spel restriction enzyme cleavage site (shown in lowercase) required for subsequent subcloning, and CP
Primer 7 (SEQ ID NO: 7) and Primer 2 to introduce a 15 bp DNA sequence corresponding to the 5 'end of the R gene (the relevant nucleotides are underlined)
1 (SEQ ID NO: 21) using approximately 100 ng of Candida maltosa ATCC90
A 766 bp PGK promoter (positions 56-756, without primers) was amplified from 677 genomic DNA. Primer 7- (SEQ ID NO: 7): 5′-AactagtGGTAGAGCGGATGGTTACATACGAC-3
'Primer 21- (SEQ ID NO: 21): 5'- TTTTATCTAATGCCAT TCTAGTTAAGGCAATTGA
Using primers 22 (SEQ ID NO: 22) and 23 (SEQ ID NO: 23) to introduce a 15 bp DNA sequence corresponding to the 3′-end of the T-3 ′ PGK promoter (the relevant nucleotides are underlined) The pGEM-CPR DNA containing about 20 ng of Candida maltosa CPR gene was
A 1038 bp DNA fragment corresponding to the 5'-end of (positions 8 to 1065) was amplified. Primer 22- (SEQ ID NO: 22): 5'- GCCTTAACTTAGAATG GCATTAGATAAAATTTAGAT
TT-3 ′ primer 23- (SEQ ID NO: 23): Primer 24 for introducing a 15 bp DNA sequence corresponding to the 5′-end of the 5′-AAGTGGAATCTAAAGCTTTTAATTCG-3 ′ PGK terminator (the relevant nucleotides are underlined) (SEQ ID NO: 24) and Primer 25 (SEQ ID NO: 25) using about 20 ng of pGEM-CPR
1 corresponding to the 3′-end of the CPR gene (positions 1090 to 2089) from DNA
A 062 bp DNA fragment was amplified. Primer 24- (SEQ ID NO: 24): 5'-CGAATTAAAAGCTTTAGATTCCACTT-3 'Primer 25- (SEQ ID NO: 25): 5'- CATAAAAAAATCAATT CTACCAACACATCTTCTGTG
GTA-3 'Introduces the NheI restriction enzyme cleavage site required for subsequent subcloning (shown in lowercase) and the 15 bp DNA sequence corresponding to the 3' end of the CPR gene (the relevant nucleotides are underlined). For primer 26 (SEQ ID NO: 26)
) And primer 14 (SEQ ID NO: 14), approximately 100 ng of Candida
A 588 bp PGK terminator (positions 2050 to 2571) was amplified from the genomic DNA of Maltosa ATCC 90677. Primer 26- (SEQ ID NO: 26): 5'-GAAGATGTTTGGTAGAATTGATTTTTTATGACA
CTTG-3 'Primer 14- (SEQ ID NO: 14): 5'-AAAgctagcTTTTGAAACAATCTGTGGTTTG-3' These PCRs are available from Perkin Elmer Amplitaq®
The kit was used to perform 50 μL. Amplification was performed by Perkin Elmer
GeneAmp® PCR System 9600, 94 ° C. for 1 minute,
One cycle consisting of 50 ° C. for 1 minute and 72 ° C. for 2 minutes was performed for 35 cycles. Following the last cycle, there was a 5 minute extension at 72 ° C., after which the sample was
After keeping at ℃, analysis by gel electrophoresis was performed. Copy the desired DNA fragment to Gene
Purification was performed using Clean kit (Bio101).

A 766 bp DNA fragment consisting of the PGK promoter and 5 of the CPR gene
A 1038 bp DNA fragment corresponding to the '-end was ligated to the complementary 3 of the PGK promoter.
The '-end and the 5' end of the CPR gene were joined in a second PCR annealing. A 1789 bp DNA fragment containing the correct fusion of the PGK promoter to the 5 'end of the CPR gene was amplified by adding 5'-PGK primer and 3'-CPR primer, primer 7 and primer 23, respectively. Further, a 1062 bp DNA fragment corresponding to the 3′-end of the CPR gene and a 588 bp DNA fragment consisting of the PGK terminator were ligated to the second 3′-end that anneals to the complementary 3′-end of the CPR gene and the 5 ′ end of the PGK terminator. PC
Linked with R. By adding 5'-CPR and 3'-PGK primers, primer 24 and primer 14, respectively, a 1635 bp DNA containing the correct fusion of the 3'-end of the CPR gene to the PGK terminator
The fragment could be amplified. These PCRs are available from Perkin Elmer
Performed in 50 μL using Amplitaq® kit. Amplification was performed using Perkin Elmer GeneAmp® PCR System.
m 9600, one cycle consisting of 94 ° C for 1 minute, 45 ° C for 1 minute, 72 ° C for 2 minutes
The test was performed for 5 cycles. The last cycle was followed by a 5 minute extension at 72 ° C, after which the sample was held at 4 ° C before analysis by gel electrophoresis. After preparative gel electrophoresis, the desired DNA fragment was isolated and Gene Clean
The kit was purified using kit (Bio101).

A 1789 bp DNA fragment containing the correct fusion of the PGK promoter to the 5 ′ end of the CPR gene was digested with SpeI and HindIII and similarly digested p
Litmus 38. The ligated DNA is used to transform Escherichia coli DH5α and the presence of the desired plasmid is determined by analyzing plasmid DNA obtained from ampicillin-resistant transformants that exhibit white colony color in LB medium containing X-gal. And it was named pLA1T. A 1635 bp DNA fragment containing the correct fusion of the 3'-end of the CPR gene to the PGK terminator was digested with HindIII and NheI and similarly digested pLitmus.
38. Escherichia coli DH5α was transformed with the ligated DNA, and
The presence of the desired plasmid was confirmed by analyzing plasmid DNA obtained from ampicillin-resistant transformants showing a white colony color in the LB medium containing gal and named pLRT. Next, HindIII and Nhe
The pLPR was linearized by digestion with I and ligated to the 1635 bp HindIII / NheI DNA fragment from pLRT. The ligated DNA was used to transform Escherichia coli DH5α, and the presence of the desired plasmid was confirmed by analyzing the plasmid DNA obtained from the ampicillin-resistant transformant, which was named pLPRT. This plasmid was digested with SpeI and NheI to create a 3424 bp expression cassette containing the CPR gene correctly fused to the PGK promoter and terminator.

Example 5 Construction of Candida maltosa strains expressing enhanced alkane hydroxylation activity Candida maltosa ATCC 2814 using two primer sets
From 0, cytochrome P450 reductase was PCR amplified. One set is
A BamHI site at the 5 'end and a PstI site at the 3'-end
The fragment was amplified to extend 2616 bases from 340 bases upstream of the reductase start codon. Another set incorporated a SphI site at the 3 ′ end with a PstI site at the 5 ′ end and an XhoI site immediately upstream from the SphI site at the 3 ′ end, and amplified the same DNA fragment. Thereafter, the BamHI of the pUC18 cloning vector (GibcoBRL, Baltimore, MD, USA) was used.
And the first DN containing the P450 reductase gene between the sites of PstI
The A fragment was cloned to obtain plasmid pRDF1. The latter DNA fragment plasmid containing the P450 reductase gene was digested with a PstI site and a SphI site.
Subcloning between the sites yielded RDF2. Furthermore, the XhoI at the 5 'end
Site and a primer that incorporates a SphI site at the 3 ′ end, Cand
The ade1 gene was amplified from ida mallosa ATCC 28140. These primers amplified the DNA fragment and extended the site 1396 bases from the site 284 bases upstream of the phosphoribosylamidoimidazole succinocarboxamide synthetase start codon. The XhoI-ade1-SphI DNA fragment was cloned into the plasmid pRDF2 between the XhoI site and the SphI site to prepare a plasmid pRDF3. The same DNA segment containing the ade1 gene was then re-amplified from Candida maltosa ATCC 28140 using primers that incorporated a 5 'Smal site and a 3' BamHI site. This D was added to pRDF3 between the SmaI and BamHI sites.
The NA fragment was cloned to obtain plasmid pRDF4.

Using primers incorporating a BamHI site at the 5 ′ end and an XbaI site at the 3 ′ end, URA from Candida maltosa ATCC 28140 by PCR
A second plasmid construct was created by first amplification of the three genes. These primers amplified the DNA fragment and extended 1171 bases from 285 bases upstream of the orotidine-5'-phosphate decarboxylase start codon. In addition, the URA
Another set of primers incorporating a SphI site at the 5 'end and a HindIII site at the 3' end was used to amplify the same DNA fragment containing the three genes. Ba
The pUC18 cloning vector between the mHI and XbaI sites (Gib
The BamHI-URA3 gene-XbaI fragment was cloned into coBRL, Baltimore, MD, USA) to obtain pRDF5. SphI of pRDF5
The latter DNA fragment containing the URA3 gene between the site and the HindIII site was subcloned to obtain pRDF6. Then, Sa at the 5 'end of the DNA fragment was
The Alk1-A gene was amplified from Candida maltosa ATCC 28140 using primers that incorporate an I1 site and a SphI site at the 3 ′ end. These primers amplified the DNA fragment and extended 1958 bases from 291 bases upstream of the P450Alk1-A start codon. The SalI-P450Alk1-A-SphI fragment was cloned into the plasmid pRDF6 between the SalI and SphI sites to give pRDF7. Subsequently, Candida maltosa was used with primers incorporating an XbaI site at the 5 'end and a SalI site at the 3' end.
The Alk3-A gene was amplified from ATCC 28140. These primers amplified the DNA fragment and extended 2063 bases from 276 bases upstream of the P450Alk3-A start codon. PRD between XbaI and SalI sites
This DNA fragment was cloned into F6 to obtain plasmid pRDF8.

The spheroplast method (Cregg et al., Mol. Cell Biol., 5:
3376-3385, (1985)) according to Candida maltosa ATCC 906.
The plasmid was integrated into the genome using plasmid RDF4 to transform 77 into adenine prototrophy [ade1 and his5]. Transformants with high copy number were selected by screening the transformants using the Southern blot method. The clone giving the strongest signal contained the highest copy number of the integrated copies of the P450 reductase gene. High copy number transformants were again transformed to uracil prototrophy with plasmid RDF8 and the plasmid was integrated into the genome. With increasing amounts of 1-dodecin, transformants growing on dodecane were selected. Clones expressing high levels of P450 activity are able to grow at the highest I-dodecin concentrations. In addition, PCR and / or Southern blot analysis was used to confirm the integration of the expression cassette for Alk1-A. In addition, PCR and / or Southern blot analysis was used to confirm that the expression cassettes for the Alk1-A, Alk3-A and P450 reductase genes had been integrated into the genome of the double transformant.

Double-transformed Candida maltosa ATCC 90677 was grown in YEP medium (10 g / L yeast extract + 20 g / L peptone (pH 8)) supplemented with 0.05% Tween 80 and 10 g / L dodecane. Growing to late log phase (増 殖 48 hours) at 250 ° C. with shaking at 250 rpm. Centrifuge the cells,
Washed once with 10% YEP, pH 8. Cells were resuspended in 10% YEP (pH 8) supplemented with 0.05% Tween 80 and 10 g / L dodecane and shaken at 250 rpm for 24 hours at 30 ° C. Dodecane loss and dodecanedioic acid formation were determined by GC analysis after extraction. It was confirmed that the double-transformed strain had an increased production of dodecanedioic acid as compared with the original ATCC 90677 strain.

Example 6 Construction of a POX4 disruption cassette Candida maltosa P from genomic DNA using gene-specific primers
In addition to amplifying the OX4 gene, unique restriction sites were added to their flanking regions for later ligation to the plasmid. Perkin Elm
er Amplitaq kit and Ba required for subsequent subcloning
Standard PCR mixture 5 using primer 27 (SEQ ID NO: 27) and primer 28 (SEQ ID NO: 28) to introduce an mHI cleavage site (shown in lowercase).
In 0 μL, about 100 ng of Candida maltosa ATCC 90677 [ade
1, his5, ura3 / ura3] A 1567 bp Candida maltosa POX4 gene fragment (positions 908 to 2412) was amplified by PCR from genomic DNA. Primer 27- (SEQ ID NO: 27): 5'-GGGTCACggatccAATGTTGCTGGG-3 'Primer 28- (SEQ ID NO: 28): 5'-GCAGCAGTGTATggatccTTAGGTGTCTTTTGGT
GGG-3 ′ amplification was performed using a Perkin Elmer GeneAmp PCR System.
At 9600, one cycle consisting of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was performed in 35 cycles. The last cycle was followed by a 5 minute extension at 72 ° C, after which the sample was held at 4 ° C before analysis by gel electrophoresis. The reaction containing the desired 1567 bp DNA fragment was extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1 v / v), the DNA was precipitated with ethanol, and TE buffer (10 mM Tris, pH 7.5) was used. 1 mM EDTA
). A 1567 bp DNA fragment was digested with BamHI and BamHI was digested.
Digested pBR322 (New England Biolabs. Beverly
, MA). Using the ligated DNA, Escherichia coli DM1 (Gibc
oBRL, Gaithersberg, MD) and the presence of the desired plasmid was confirmed by analyzing plasmid DNA from ampicillin-resistant, tetracycline-sensitive transformants, which was named pBR-CMPOX4.

Using primers 29 (SEQ ID NO: 29) and 30 (SEQ ID NO: 30) to introduce the Perkin Elmer Amplitaq kit and the BclI cleavage site required for subsequent subcloning (shown in lowercase), the standard Approximately 100 ng of Candida maltosa ATCC in 50 μL of PCR mixture
1184b containing the Candida maltosa URA3 gene (positions 8 to 1192) from 90625 [ade1, his5, ura3 / ura3] genomic DNA
The p DNA fragment was amplified by PCR. Primer 29- (SEQ ID NO: 29): 5'-GACTTTgatcaATTTTGGTACCAT-3 'Primer 30- (SEQ ID NO: 30): 5'-AGGGTACCATGAAGTTTTAGACTCTtgatacaCT
The -3 'amplification was performed using a Perkin Elmer GeneAmp PCR System.
At 9600, one cycle consisting of 94 ° C. for 1 minute, 50 ° C. for 1 minute, and 72 ° C. for 2 minutes was performed in 35 cycles. The last cycle was followed by a 5 minute extension at 72 ° C, after which the sample was held at 4 ° C before analysis by gel electrophoresis. The reaction containing the desired 1184 bp DNA fragment was extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1 v / v), the DNA was precipitated with ethanol, and TE buffer (10 mM Tris (pH 7.0)) was used. 5) 1 mM E
DTA).

A 1184 bp PCR fragment containing the URA3 selectable marker was digested with BclI, then digested with BclII, and treated with calf intestinal phosphatase-treated pB.
Connected to R-CMPOX4. The ligated DNA was used to transform E. coli DH5α competent cells (GibcoBRL, Gaithersberg, MD), and the presence of the desired plasmid was confirmed by analyzing the ampicillin-resistant plasmid DNA, confirming that pBR-pox4 :: It was named URA3. Bam
This plasmid was digested with HI and the 5 '770 homologous to the POX4 target gene was digested.
A 2.8 kb linear POX4 disruption cassette containing the bp and the URA3 selectable marker flanked by the 3 '734 bp was released.

Example 7 Construction of Candida maltosa Strains with Disrupted POX4 Gene A β-oxidation inhibiting strain of Candida maltosa was developed by sequentially disrupting both genes of POX4, which encode acyl CoA oxidase. In addition,
Acyl CoA oxidase is an enzyme that catalyzes the first reaction in the β-oxidation pathway. Candida maltosa ATCC 90677 is a product of the URA3 gene orotidine-
It lacks 5'-monophosphate decarboxylase and requires uracil for growth. Molecular Genetics by Gietz and Woods
of Year: A Practical Approach (edit, J
Ohnson, J .; R. ) Pp. 121-134, Oxford University
plasmid pBR-pox, as described in Sitency Press (1994).
4: Candida maltosa ATCC 90677 was transformed using a 2.8 kb linear POX4 disruption cassette from URA3 and rendered uracil prototrophic. 0.
67 g / L yeast nitrogen source base (Difco, Detroit, MI), 2% (
w / v) Ura ⁺ transformants were selected on glucose, 2% Bacto agar medium (Difco) and minimal medium supplemented with 20 mg / L each of adenine sulfate and L-histidine.

Southern hybridization of XmnI digested genomic DNA from 20 independent Ura ⁺ transformants to the POX4 probe indicated that each contained a disrupted single copy of the desired POX4. (See FIG. 1, lane marked Ura ⁺ ). These results indicate that the ends of the linear DNA are highly transgenic and define the exact site of integration into the Candida maltosa genome. Because Candida maltosa is a diploid yeast, these transformants also contained another functional copy of the POX4 gene that had to be disrupted to inactivate the β-oxidation pathway. To subsequently disrupt both copies of the POX4 gene in a single strain, an additional 2 mg of 5-fluoroorotic acid (5-FOA), a toxic analog of the uracil biosynthetic pathway intermediate that is incorporated only into Ura ⁺ cells. Uracil auxotrophic revertants were selected reverse to the first step in supplemented minimal medium containing 1 / mL. Therefore, only Ura ^- cells survive and grow in the presence of a mixture of 5-FOA and uracil. F
OA resistant, uracil auxotrophic derivatives were isolated and the original POX
4 broken holds, further uracil prototrophy those which show also low revertant frequency with respect to (see FIG. 1, lanes describing the FOA ^R), the plasmid pBR-p
A second transformation to uracil prototrophy was performed using the same pox4 :: URA3 disruption cassette from ox4: URA3. After transformation, about half of the resulting Ura ⁺ transformants failed to grow on dodecane as the sole carbon source, which impaired the β-oxidation pathway of those transformants. Suggest that. These transformants were analyzed by Southern hybridization to determine POX
It was confirmed that both genome copies of the four genes were disrupted (see FIG. 1, lane labeled β-inhibition). Subsequent analysis confirmed that these transformants lacked the extra acyl CoA oxidase activity and the ability to convert dodecane to DDDA. Each URA3-mediated gene disruption was followed by a subsequent cytochrome P450
In amplification of the monooxygenase and cytochrome P450-NADPH reductase genes, it is convenient to provide a distinct integration target.

Example 8 Construction of Candida maltosa Strains with Disrupted POX4 Gene and Separate Other Auxiliary Markers Primer 31 (SEQ ID NO: 31) incorporating an NdeI site (shown in lowercase)
) And primer 32 (SEQ ID NO: 32) by PCR
The maltosa ADE1 gene was isolated, and pUC18m (SpeI and Nh
The restriction enzyme cleavage site of eI was subcloned into the NdeI site of pUC18 derivative inserted between SalI and XbaI in the polylinker region, and pSW81 was cloned.
Was prepared. Primer 31- (SEQ ID NO: 31): 5'-CTTCTTCAAACCTTcatatgACATTGTTTCG-3
'Primer 32- (SEQ ID NO: 32): 5'-CTAATGGTCAAGcatatg TTGCATTATC-3' Primer 33 incorporating NdeI site (shown in lowercase) (SEQ ID NO: 33)
Using the primer 34 (SEQ ID NO: 34), the Candida maltosa HIS5 gene was isolated by PCR and subcloned into the NdeI site of pUC18m to create pSW82. Primer 33- (SEQ ID NO: 33): 5'-TTTGGTTGACTcatatgTGAGCGCGGGTAAAG-3
'Primer 34- (SEQ ID NO: 34): 5'-GTTTGTTCTGGCcatatgTTGAACTGGATGG-3
'One of the β-inhibiting Candida maltosa transformants described in Example 7 (Cand
ida mallosa 11-11), further comprising ATCC 906
It was modified to remove the remaining two auxotrophy from 77, adenine and histidine. This removal is substantially as described by Gietz et al. (Methods).
Mol. Cell. Biol. 5: 255-269, (1996)), co-transformation of Candida maltosa 11-11 with pSW81 and pSW82, and minimal plate medium without supplementation of adenine or histidine (1.34). % Yeast nitrogen source base without amino acids, 2% (w / v)
(Glucose) at 30 ° C. The resulting strain is called Candida maltosa SW81 / 82 and is identified as ATCC Accession No. 74431.

Example 9 Construction of a Candida maltosa strain expressing increased alkane hydroxylation activity and a disrupted POX4 gene Candida maltosa cytochrome P450 Alk as described in Example 2
The 1-A expression cassette was subcloned into pSW81 between SpeI and NheI (described in Example 8) to generate pSW83 (see FIG. 2). The expression cassette of Candida maltocytochrome P450-NADPH reductase described in Example 4 was subcloned into pSW83 NheI to generate pSW84. It consists of cytochrome P450Alk1-A and cytochrome P4
It contains an expression cassette for both 50-NADPH reductase, as well as an adel selectable marker (see Figure 4). Candida described in Example 3
Maltocytochrome P450Alk3-A expression cassette was constructed with SpeI and Nh
It was subcloned into pSW82 during eI to create pSW85. The expression cassette for Candida maltocytochrome P450-NADPH reductase described in Example 4 was subcloned into pSW85 NheI to produce pSW87.
It is cytochrome P450Alk3-A and cytochrome P450-NA
It contains an expression cassette for both DPH reductase, as well as a his5 selectable marker.

[0151] Substantially the description of Gietz et al. (Methods Mol. Cell. Biol)
. 5: 255-269, (1996)), using pSW84 (see FIG. 4) and pSW87 (see FIG. 5).
Candida maltosa β-inhibitory strain designated 1-11 and co-transformed with minimal plate medium supplemented with adenine or histidine (1.34% amino acid free yeast nitrogen source base, 2% (w / v) glucose) Or at 30 ° C. on minimal plate medium without supplementation. By PCR and / or Southern analysis, cytochrome P450Alk1-A and cytochrome P450-NADPH reductase (a strain called Candida maltosa SW84), cytochrome P450A
lk3-A and cytochrome P450-NADPH reductase (Candida.
A strain called Maltosa SW87) or cytochrome P450Alk1-A
, Cytochrome P450Alk3-A and cytochrome P450-NADPH
Chromosomal integration of the expression cassette into reductase (strain designated Candida maltosa SW84 / 87) was confirmed. Candida maltosa SW84 / 87.
One Candida maltosa SW84 / 87 double transformant designated as AT2
Identified as CC accession number 74430. Candida maltosa SW84, Candida maltosa SW87 and Candida maltosa SW84 in YEPD (1% yeast extract, 2% peptone, 2% glucose) at 30 ° C. until saturation (24 hours).
After growing / 87, cells were harvested by centrifugation and disrupted with glass beads to produce a translucent lysate as described in Example 1, and hydroxylated as described in Example 1. (Hydroxylation) activity was analyzed. Candida Maltosa SW
84, Candida maltosa SW87 and Candida maltosa 84/87 each showed conversion of lauric acid to DDDA.

Example 10 Production of Dodecanedioic Acid (DDDA) from Dodecane by Candida maltosa Strain SW81 / 82 (ATCC 74431) 5 mL of Candida maltosa SW81 / 82 (ATCC 74431) inoculum was transformed into YEPD medium (10 g / L) Yeast extract, 20 g / L peptone, and 20
g / L of glucose) and grown at 30 ° C. with shaking at 250 rpm for 24 hours. The resulting mixture was added to a yeast minimal medium (pH 3) (3 g / L of (NH ₄ ) ₂ S).
O ₄ , 6.6 g / L KH ₂ PO ₄ , 0.4 g / L K2HPO ₄ , 0.6 g / L anhydrous MgSO ₄ , 4 g / L yeast extract, 75 g / L glucose, 100 μg / L biotin, FeSO ₄ · 7H ₂ O of _{13mg / L, CuSO 4 · 5H} 2 O of _{2mg / L, ZnSO 4 · 7H} 2 O of _{20mg / L, MnSO 4 · H} 2 O of 6 mg / L, of 2 mg / L 350 mL of Co (NO ₃ ) 2 .6H ₂ O, 3 mg / L NaMoO ₄ .2H ₂ O and 1.6 mg / L KI) were inoculated and grown at 30 ° C. with shaking at 250 rpm for 24 hours. A fermenter (Braun) containing 7 L of pH 5 yeast minimal medium was inoculated with the overnight culture. The fermentor was maintained with minimal airflow and agitation until dissolved oxygen was 20% of the atmosphere. Thereafter, the dissolved oxygen was raised to about 80% of the atmosphere, and the fermenter was aerated at 30 ° C. to 2 vvm at 1400 rpm.
m by controlling stirring. 10% w / v NH ₄ OH was added to supply nitrogen for cell growth and the medium was maintained at pH 5. About 14
After time, the glucose concentration had decreased to near zero. Thereafter, dodecane was added to a final concentration of about 20 g / L. PH by adding 20% w / v KOH
The pH of the medium was adjusted to 7.5. Additional 20% w / v KOH was added to the medium to keep the pH at 7.5 for the remainder of the fermentation. Dodecane concentration was monitored periodically and kept above 3 g / L. In addition, 0.2 to 0.1 glucose per minute.
Glucose was fed at a slow rate of 8 g and the glucose concentration was monitored. The glucose feed was at a slow rate to keep the glucose concentration below 1 g glucose / L. About 69 hours after dodecane addition, material was collected from the fermentor and analyzed for DDDA.

DDDA was recovered from the whole fermentor liquor (cells and supernatant) by acidifying the liquor to pH 2 with 2M phosphoric acid and extracting the resulting precipitate three times into 5 mL of methyl tert-butyl ether. . A portion of the ether extract is evaporated to dryness and the recovered DDDA is reacted with MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide) to form a derivative detectable by GC under standard conditions specified above. did.

DDDA from the fermentor was 28.8 g / L, or 187 g total yield. The average production rate was 2.7 g DDDA / hour.

Example 11 Dodeca according to Candida maltosa 84 / 87.2 (ATCC 74430)
Of Dodecandionic Acid (DDDA) from Candida maltosa strain 84 / 87.2 (ATCC 74430)
The inoculum was used in a YEPD medium (10 g / L yeast extract, 20 g / L peptone, and
And 20 g / L glucose) at 30 ° C. with shaking at 250 rpm.
Proliferated for 24 hours. The resulting mixture was diluted with a pH 5 yeast minimal medium (3 g / L (N
H_Four)_TwoSO_Four6.6 g / L KH_TwoPO_Four, 0.4g / L K2HPO_Four, 0.6
g / L anhydrous MgSO_Four4 g / L yeast extract, 75 g / L glucose, 100 μg / L biotin, 13 mg / L FeSO_Four・ 7H_TwoO, 2 mg / L C
uSO_Four・ 5H_TwoO, 20 mg / L ZnSO_Four・ 7H_TwoO, 6 mg / L MnSO _Four ・ H_TwoO, 2 mg / L Co (NO_Three) 2.6H_TwoO, 3mg / L NaMoO_Four ・ 2H_TwoO and 1.6 mg / L KI) were inoculated into 2 × 350 mL and grown for 24 hours at 30 ° C. with shaking at 250 rpm. Contains 7L of yeast minimum medium of pH5
The fermenter (Braun) was inoculated with 525 mL of the overnight culture. Dissolved oxygen
The fermentor was maintained with minimal airflow and agitation until 20% of the atmosphere. Then large
The dissolved oxygen is raised to about 80% of the air and the fermenter is air-raised at 30 ° C to 2 vvm.
The agitation was maintained by controlling the agitation to 1400 rpm. 10% w
/ V of NH_FourOH was added to supply nitrogen for cell growth and the medium was maintained at pH5. After about 18 hours, the glucose concentration had decreased to near zero. That
Thereafter, dodecane was added to a final concentration of about 20 g / L. Add 20% w / v KOH
The pH of the medium was adjusted to pH 7.5 by addition. 20% w / v
KOH was added to maintain the pH of the medium at 7.5 for the remainder of the fermentation. Dodeca
Concentration was monitored periodically and maintained at 3 g / L or more. In addition, per minute
Supply glucose at a slow rate of 0.2-0.8g and monitor glucose concentration
did. To maintain a glucose concentration lower than 1 g glucose / L, glucose
Feeding was at a slow rate. Collect substance from fermenter about 51 hours after dodecane addition
And analyzed for DDDA.

DDDA was recovered from all fermenter liquors (cells and supernatant) by acidifying the liquor to pH 2 with 2M phosphoric acid and extracting the resulting precipitate three times into 5 mL of methyl tert-butyl ether. . A portion of the ether extract was evaporated to dryness and the recovered DDDA was reacted with MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide) and 1% TMCS (trimethylchlorosilane) under standard conditions specified above. A derivative was formed which was detectable by GC.

DDDA from the fermentor was 21.6 g / L, or a total yield of 173 g. The average production rate in Candida maltosa SW84 / 87.2 is 3.4 gDDD
A / hour, which is 20 times higher than the production rate in Candida maltosa SW81 / 82.
% Production improved.

Example 12 Lau by Candida maltosa strain 84 / 87.2 (ATCC 77430)
Production of Dodecanedioic Acid (DDDA) from Phosphate Methyl Ester 10 mL of Candida maltosa strain 84 / 87.2 (ATCC 74430)
The inoculum was used in a YEPD medium (10 g / L yeast extract, 20 g / L peptone, and
And 20 g / L glucose) at 30 ° C. with shaking at 250 rpm.
Proliferated for 24 hours. This seed culture was transformed into a yeast minimal medium at pH 5 (3 g / L of (NH _Four )_TwoSO_Four6.6 g / L KH_TwoPO_Four, 0.4g / L K2HPO_Four, 0.6g
/ L anhydrous MgSO_Four4 g / L yeast extract, 75 g / L glucose, 100 μg / L biotin, 13 mg / L FeSO_Four・ 7H_TwoO, 2mg / L Cu
SO_Four・ 5H_TwoO, 20 mg / L ZnSO_Four・ 7H_TwoO, 6 mg / L MnSO_Four ・ H_TwoO, 2 mg / L Co (NO_Three) 2.6H_TwoO, 3mg / L NaMoO_Four・
2H_TwoO and 1.6 mg / L KI) were inoculated twice into 350 mL and grown at 30 ° C. with shaking at 250 rpm for 24 hours. including 7L of pH5 yeast minimal medium
A fermenter (Braun) was inoculated with 525 mL of the overnight culture. Large dissolved oxygen
The fermenter was maintained with minimal airflow and agitation until 20% of the air. Then the atmosphere
The dissolved oxygen is raised to about 80% of the
Was maintained by controlling the agitation to 1400 rpm. 10% w /
NH of v_FourOH was added to supply nitrogen for cell growth and the medium was maintained at pH5. When the glucose concentration in the fermenter drops below 1 g / L,
Phosphate methyl ester was added to a final concentration of about 5 g / L. 20% w / v KO
The pH of the medium was adjusted to 7.5 by adding H. Further 20% w / v
KOH was added to maintain the pH of the medium at 7.5 for the remainder of the fermentation. Lau
The concentration of the phosphoric acid methyl ester was periodically monitored and maintained at 3 g / L or more.
Further, glucose is supplied at a slow rate of 0.2 to 0.8 g per minute,
The course concentration was monitored. Maintains glucose concentration lower than 1g glucose / L
Glucose supply was at a slow rate. After 48 hours, methyl laurate
Stop adding steal and repeat until no more lauric acid methyl ester can be detected.
Priority was given to response. Material was collected from the fermentor and DDDA was recovered.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a pedigree of Candida maltosa strains in which β-oxidation was inhibited by Southern blot of XmnI digested genomic DNA probed with the POX4 gene.

FIG. 2 is a restriction map of pSW83.

FIG. 3 is a restriction map of pSW84.

FIG. 4 is a restriction map of pSW85.

FIG. 5 is a restriction map of pSW87.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] January 21, 2000 (2000.1.21)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:84) (C12N 1/15 (C12N 1/15 C12R 1:72) C12R 1:72) C12N 15 / 00 ZNAA (72) Inventor Pain Mark S. United States 19808 Wilmington Newell Drive, Delaware 2408 (72) Inventor Picatagio Stephen K. United States 19350 Landenburg, Meadow Wood Lane 17 (72) Inventor Wu Sijun United States 19720 Delaware New Castle Cornstoke Drive 834

Claims (27)

[Claims] 1. A a) Genetic engineering has been applied, it has been transformed, characterized by alkanes hydroxylation activity, linear hydrocarbon and aerobic conditions of at least one C ₆ -C ₂₂ Pichia pastoris and contacting under, b), characterized in that it comprises and recovering the mono- and dicarboxylic acids of C ₆ -C _22, raw Monogaku of mono- and dicarboxylic acids of C ₆ -C ₂₂ Production method. 2. The transformed Pichia pastoris is strain SW64 / 65 identified as ATCC 74409, wherein at least one C ₆ -C ₂₂ linear hydrocarbon is dodecane, and the recovered product is dodecane. 2. The method according to claim 1, wherein it is dionic acid. 3. A) at least one copy of a foreign gene encoding cytochrome P450 monooxygenase, and optionally b) at least one copy of a foreign gene encoding cytochrome P450 reductase.
One of a Pichia pastoris transformed and a copy, each gene is suitably such that at least one of A Le Kang hydroxylation activity in contact with the linear hydrocarbons C ₆ -C ₂₂ increases Pichia pastoris operably coupled to various control elements. 4. The foreign gene encoding cytochrome P450 monooxygenase is Alk1-A (D12475), Alk2-A (X55881), or Alk1-A (X55881).
k3-A (X55881), Alk4-A (D12716), Alk5-A (D
12717), Alk6-A (D12718), Alk7 (D12719), and Alk8 (D12719). The transformed Pichia pastoris according to claim 3, characterized in that it is selected from the group consisting of: 5. The transformed Pichia pastoris according to claim 3, wherein the foreign gene encoding cytochrome P450 reductase is cytochrome P450 reductase (D25327). 6. a) Cytochrome P450 monooxygenase Alk1-A.
And DN encoding cytochrome P450 monooxygenase Alk3-A
At least one DNA fragment from Candida maltosa ATCC 90677 selected from the group of A fragments and, optionally, b) Candida maltosa ATC encoding cytochrome P450 reductase
C90677 and at least one DNA fragment derived from a stable characterized by an increase in alkane hydroxylation activity to a Pichia pastoris strains transformed, each DNA fragment, at least one C ₆ -C _A Pichia pastoris strain, operably linked to a suitable control element such that upon contact with the ₂₂ linear hydrocarbons, the alkane hydroxylation activity is increased. 7. The transformed Pichia pastoris strain identified as ATCC 74409 is SW64 / 65. 8. a) linear hydrocarbon and aerobic conditions Candida Marutosa transformed characterized by an increase in the genetic engineering techniques applied alkane hydroxylation activity by at least one C ₆ -C ₂₂ the method comprising contacting under, b), characterized in that it comprises and recovering the mono- and dicarboxylic acids of C ₆ -C _22, raw Monogaku of mono- and dicarboxylic acids of C ₆ -C ₂₂ Production method. 9. a) i) Alk1-A (D12475), Alk2-A (
X55881), Alk3-A (X55881), Alk4-A (D12716)
), Alk5-A (D12717), Alk6-A (D12718), Alk7
(D12719), and at least one copy of a gene encoding a cytochrome P450 monooxygenase selected from the group consisting of Alk8 (D12719), or ii) at least one copy of a gene encoding a cytochrome P450 reductase (D25327). Or iii) an increase in genetically engineered alkane hydroxylation activity caused by at least one copy of both the i) and ii) genes;
b) linear hydrocarbon of at least one C ₆ -C ₂₂ are dodecane, characterized in that c) the recovered product is a dodecanedioic acid, The method of claim 8. 10. A) at least one copy of a gene encoding a cytochrome P450 monooxygenase, or b) an at least one copy of a gene encoding a cytochrome P450 reductase, or c) an encoding of a P450 monooxygenase. Genes and cytochrome P45
0 A further Candida Marutosa transformed comprising at least one copy of the gene both encoding reductase, each gene is A in contact with the linear hydrocarbon of at least one C ₆ -C ₂₂ Candida maltosa, characterized in that it is operably linked to a suitable control element so as to increase lucan hydroxylation activity. 11. The genes encoding cytochrome P450 monooxygenase are Alk1-A (D12475), Alk2-A (X55881), Alk
3-A (X55881), Alk4-A (D12716), Alk5-A (D1
2717), selected from the group consisting of Alk6-A (D12718), Alk7 (D12719) and Alk8 (D12719).
0. The transformed Candida maltosa according to 0. 12. The gene encoding cytochrome P450 reductase is cytochrome P450 reductase (D25327).
A transformed Candida maltosa according to claim 10. 13. a) Cytochrome P450 monooxygenase ALK1
-A and at least one DNA fragment from Candida maltosa (ATCC 90677) selected from the group of DNA fragments encoding the cytochrome P450 monooxygenase ALK3-A; and b) cytochrome P450 from Candida maltosa (ATCC 90677).
A transformed Candida Marutosa and at least one DNA fragment encoding the reductase, each gene is an alkane with hydroxyl and child contact with a straight chain hydrocarbon of at least one C ₆ -C ₂₂ Candida maltosa characterized by the fact that it is operably linked to a suitable control element so as to increase its activating activity. 14. a) transforming a transformed Candida maltosa characterized by being genetically engineered and inhibiting the β-oxidation pathway into at least one C ₆ -C ₂₂ linear carbon and contacting with hydrogen and aerobic conditions, b), characterized in that it comprises and recovering the mono- and dicarboxylic acids of C ₆ -C _22, monocarboxylic acids and dicarboxylic of C ₆ -C ₂₂ Methods for increasing the biological production of acids. 15. The method according to claim 14, wherein the β-oxidation pathway of the transformed Candida maltosa has been functionally inhibited by disrupting both POX4 genes encoding acyl-CoA oxidase. . 16. A transformed Candida maltosa characterized by a β-oxidation pathway that was functionally inhibited by disrupting both POX4 genes encoding acyl CoA oxidase. 17. A β-Function Functionally Blocked by Disruption of Both POX4 Genes Encoding Acyl-CoA Oxidase Using a Single URA3 Selectable Marker
Transformed Candida maltosa, characterized by an oxidative pathway. 18. A transformed Candida maltosa strain SW81 / 82 identified as ATCC74431. 19. Transformation characterized by a) i) genetically engineered, increased alkane hydroxylation activity and ii) genetically engineered, inhibited β-oxidation pathway. and causing the Candida Marutosa which is contacted with linear hydrocarbon and aerobic conditions of at least one C ₆ ~C _22, b) and recovering the mono- and dicarboxylic acids of C ₆ -C ₂₂ A method for increasing the biological production of C ₆ -C ₂₂ monocarboxylic and dicarboxylic acids, characterized by comprising: 20) a) i) Alk1-A (D12475), Alk2-A
(X55881), Alk3-A (X55881), Alk4-A (D1271)
6), Alk5-A (D12717), Alk6-A (D12718), Alk
7 (D12719), and at least one copy of a gene encoding a cytochrome P450 monooxygenase selected from the group consisting of Alk8 (D12719), or ii) at least one further copy of a gene encoding a cytochrome P450 reductase (D25327). Iii) increased alkane hydroxylation activity caused by at least one further copy of both genes i) and ii), and b) functionally by disrupting both POX4 genes encoding acyl CoA oxidase A transformed Candida maltosa, characterized by an inhibited β-oxidation pathway. 21. a) Cytochrome P450 monooxygenase ALK1-
A and D encoding cytochrome P450 monooxygenase ALK3-A
21. The transformed Candida maltosa strain of claim 20, characterized by an increase in alkane hydroxylation activity caused by the NA fragment. 22. A transformed Candida maltosa strain SW84 / 87.2, identified as ATCC 74430. 23. The at least one C ₆ -C ₂₂ linear hydrocarbon is hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane,
20. The method according to claim 1, 8, 14 or 19, characterized in that it is selected from the group consisting of nonadecane, eicosane, renecosane, docosane and their respective monocarboxylic acids and esters. . 24. a) a first Candida maltosa promoter operably linked to DNA encoding at least one polypeptide from Candida maltosa; and b) at least one poly from Candida maltosa. DN encoding peptide
A second Candida maltosa promoter operably linked to A. 25. a) a first Candida maltosa promoter operably linked to a gene encoding Candida maltosa cytochrome P450 monooxygenase; and b) a gene encoding a Candida maltosa cytochrome P450 reductase. And a second Candida maltosa promoter operably linked to: a DNA fragment. 26) a) Alk1-A (D12475), Alk2-A (X5
5881), Alk3-A (X55881), Alk4-A (D12716),
Alk5-A (D12717), Alk6-A (D12718), Alk7 (D
12719), and a first Candida maltosa PGK promoter operably linked to a gene encoding a cytochrome P450 monooxygenase selected from the group consisting of Alk8 (D12719); b) Candida maltosa cytochrome P450 A second Candida maltosa PGK promoter operably linked to a gene encoding reductase. 27. A plasmid, which is selected from the group consisting of pSW84 and pSW87.