CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of Chinese Patent Application No. 201810734273.7, filed on Jul. 6, 2018 and Chinese Patent Application No. 201810734190.8 filed on Jul. 6, 2018, and Chinese Patent Application No. 201910321614.2, filed on Apr. 22, 2019, the disclosures of which are incorporated herein by reference in their entirety.

SEQUENCE STATEMENT

Incorporated by reference herein in its entirety is the Sequence Listing entitled “0503.13_ST25.txt”, size of 76 kilobytes, created Feb. 3, 2021.

FIELD OF THE INVENTION

The invention relates to a long-chain dibasic acid with low content of monobasic acid impurity and a preparation method thereof; as well as to a method for preparing a long-chain dibasic acid producing strain by using directed evolution and homologous recombination, and a method for producing a long-chain dibasic acid with low content of monobasic acid impurity by using said strain.

BACKGROUND

Long-chain dibasic acid (LCDA; also referred to as long chain dicarboxylic acid or long chain diacid) is a dibasic acid comprising the formula HOOC(CH2)nCOOH, where n≥7. As important monomer raw materials, long-chain dibasic acids are widely used in the synthesis of nylon, resins, hot-melt adhesives, powder coatings, preservatives, perfumes, lubricants, plasticizers, and the like.

For a long time, long-chain dibasic acids are synthesized via classical chemical synthesis pathway from the petroleum, such as butadiene multiple-step oxidation process. The Chemical synthesis methods face many challenges. Dibasic acid obtained by the chemical synthesis method is a mixture of long-chain dibasic acid and short-chain dibasic acid, and thus the subsequent complicated extraction and purification steps are necessary, which become a huge obstacle for production technique and production cost. Microbiological fermentation technology of producing a long-chain dibasic acid is advantageous over classical chemical synthesis method because it produces less pollution, is environment friendly, and is capable of synthesizing a long-chain dibasic acid such as the one having more than 12 carbon atoms which is difficult to be produced by chemical synthesis methods and has high purity and the like.

But using the microbiological fermentation method for producing a long-chain dibasic acid, there are residual impurities in the product sometimes and the reduction in the product purity affects the quality of the product significantly, which greatly affects its later application. In particular, the impurity which is characteristically similar to long-chain dibasic acid not only generates technical challenges to the later extraction and purification, but also produces great negative effects on the production cost control. Therefore, to genetically modify a strain for producing a long-chain dibasic acid so as to reduce the content of certain impurity during fermentation is important and valuable in industry to dibasic acid production via biological synthesis.

Previously, the improvement of a dibasic acid producing strain was mostly achieved through conventional random mutagenesis or genetic engineering methods. Due to the characteristic of random mutagenesis, there is a high requirement for screening throughput, and a new round of mutagenesis screening is required for each changed trait, which has become an important limiting factor technically. The genetic engineering means can be used to perform targeted genetic modification of a strain, so as to obtain a good strain with higher yield. The microbiological fermentation method of a long-chain dibasic acid is mainly based on ω-oxidation of alkane, which can then be degraded by β-oxidation pathway. Previous studies have shown that the yield of a long-chain dibasic acid can be increased by means of enhancing the ω-oxidation pathway and inhibiting the β-oxidation pathway. Pictaggio et al. of Coginis company (Mol. Cell. Biol., 11(9), 4333-4339, 1991) reported that knocking out two alleles of each of POX4 and POX5 could effectively block the β-oxidation pathway to achieve 100% conversion rate of the substrate. Further overexpression of the genes of two key enzymes, P450 and oxidoreductase CPR-b, of the rate-limiting step in the ω-oxidation pathway could effectively increase production. Xiaoqin Lai et al. (Chinese patent CN103992959B) reported that the introduction of one copy of the CYP52A14 gene into a dibasic acid producing strain could also effectively increase the conversion rate and production efficiency of the dibasic acid. In addition, Zhu'an Cao et al. (Biotechnol. J., 1, 68-74, 2006) from Tsinghua University found that knocking out a copy of the key gene CAT in the transportation of acetyl coenzyme A from peroxisome to mitochondria could partially block acetyl coenzyme A entering the citric acid cycle, and also effectively reduce the degradation of dibasic acids.

Error-prone PCR is the technique proposed by Leung et al. (Technical, 1, 11-15, 1989) to construct a gene library for directed studies. By changing PCR conditions, such as adjusting the concentration of four deoxyribonucleic acids in the reaction system, changing the concentration of Mg2+, and using a low-fidelity DNA polymerase and the like, the bases are mismatched so as to introduce a mutation. Too high or too low mutation rate will affect the effect of constructing mutant libraries. The ideal base mutation ratio is 1-3 per DNA fragment. Therefore, the beneficial mutations that contribute to further improvement of the strain productivity can be screened through gene-directed genetic modification by using error-prone PCR of generating random mutations in combination with homologous recombination.

Previous studies on dibasic acid producing strains focused on random mutagenesis or overexpression of a gene in the upstream synthetic pathway or blocking the downstream β-oxidation pathway, and the directed evolution of a gene in the metabolic pathway has not been reported or applied. There is still a need in the art for a strain that significantly increases the yield of a long-chain dibasic acid and significantly reduces the content of some impurities, as well as preparation methods thereof.

SUMMARY OF THE INVENTION

The invention relates to an isolated mutated CYP52A12 gene, homologous gene or variant thereof, which, relative to GenBank Accession Number AY230498 (e.g. set forth in SEQ ID NO: 21, in particular nucleotides 1-1176 or 265-1176) and taking the first base (e.g. the base “C” at position 1176 of SEQ ID NO: 21) upstream of the start codon ATG (wherein “A” is position 1) as −1, comprises any one or more of the following base mutations: −876A>G; −853A>T; −831delT; −825C>A; −823delG; −579A>G; −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), wherein the variant has at least 70% sequence identity to the mutated CYP52A12 gene or homologous gene thereof.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutation −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), e.g. set forth in SEQ ID NO: 22.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutations −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), e.g. set forth in SEQ ID NO: 23.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutations −579A>G; −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), e.g. set forth in SEQ ID NO: 24.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutations −831delT; −825C>A; −823delG; −579A>G; −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), e.g. set forth in SEQ ID NO: 25.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutations −876A>G; −853A>T; −831delT; −825C>A; −823delG; −579A>G; −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), e.g. set forth in SEQ ID NO: 26.

The invention relates to an isolated DNA molecule, which, relative to nucleotides 1-1176 or 265-1176 of SEQ ID NO: 21, comprises any one or more of the following base mutations 301A>G, 324A>T, 346delT, 352C>A, 354delG, 598A>G, 765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC (SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40)(e.g. 1170_1173delACCA).

In an embodiment, the isolated DNA molecule comprises the base mutation 1162_1176ACCAACCAACCAACC (SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40) (e.g. 1170_1173delACCA), e.g. set forth in SEQ ID NO: 27 or 32.

In an embodiment, the isolated DNA molecule comprises the base mutations 765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC (SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40) (e.g. 1170_1173delACCA), e.g. set forth in SEQ ID NO: 28 or 33.

In an embodiment, the isolated DNA molecule comprises the base mutations 598A>G, 765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC (SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40) (e.g. 1170_1173delACCA), e.g. set forth in SEQ ID NO: 29 or 34.

In an embodiment, the isolated DNA molecule comprises the base mutations 346delT, 352C>A, 354delG, 598A>G, 765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC (SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40) (e.g. 1170_1173delACCA), e.g. set forth in SEQ ID NO: 30 or 35.

In an embodiment, the isolated DNA molecule comprises the base mutations 301A>G, 324A>T, 346delT, 352C>A, 354delG, 598A>G, 765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC(SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40) (e.g. 1170_1173delACCA), e.g. set forth in SEQ ID NO: 31 or 36.

In an embodiment, the sequence of the mutated CYP52A12 gene according to the invention is set forth in any of SEQ ID NOs. 16 and 22-26 or a sequence having at least 70% sequence identity thereto, for example at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, or 99.96% sequence identity.

The invention further relates to a microorganism containing the isolated DNA molecule (for controlling the expression of CYP52A12 gene, e.g. as a promoter) or the mutated CYP52A12 gene, homologous gene or variant thereof according to the invention, which produces a long-chain dibasic acid with a reduced content of monobasic acid impurity, compared to a microorganism containing a non-mutated CYP52A12 gene or homologous gene thereof.

The invention further relates to a method of producing a long-chain dibasic acid by fermentation with a microorganism containing the isolated DNA molecule or the mutated CYP52A12 gene, homologous gene or variant thereof according to the invention, comprising a step of culturing the microorganism, and optionally a step of isolating, extracting and/or purifying the long-chain dibasic acid from the culture.

In some embodiments, after the completion of the process of producing a long-chain dibasic acid by fermentation with a microorganism according to the invention, the mass ratio of monobasic acid impurity in fermentation broth is below 5%, wherein the mass ratio is the mass percentage of the monobasic acid impurity to the long-chain dibasic acid in the fermentation broth.

In some embodiments, after the completion of the process of producing a long-chain dibasic acid by fermentation with a microorganism according to the invention, the content of monobasic acid impurity in the fermentation broth, compared with that of the monobasic acid impurity in the long-chain dibasic acid produced by fermentation method with a conventional microorganism, such as by fermentation with a non-mutant microorganism according to the invention, is decreased by at least 5%.

The invention also relates to a long-chain dibasic acid with low content of monobasic acid impurity, wherein the content of monobasic acid impurity is greater than 0, and less than 12,000 ppm, preferably less than 10,000 ppm or 6,000 ppm, more preferably less than 3,000 ppm, more preferably less than 1,000 ppm, more preferably less than 500 ppm, more preferably less than 200 ppm; and wherein the monobasic acid impurity comprises a long-chain monocarboxylic acid impurity, i.e. containing only one carboxyl group (—COOH) in the carboxylic acid molecule.

In some embodiments, the long-chain dibasic acid producing microorganism strain according to the invention contains the isolated DNA molecule (for controlling the expression of CYP52A12 gene, e.g. as a promoter) or the mutated CYP52A12 gene, homologous gene or variant thereof according to the invention. In some embodiments, the long-chain dibasic acid producing microorganism strain is the microorganism according to the invention which contains the isolated DNA molecule or the mutated CYP52A12 gene, homologous gene or variant thereof according to the invention.

In some embodiments, the microorganism of the invention is selected from the group consisting ofCorynebacterium, Geotrichum candidum, Candida, Pichia, Rhodotroula, SaccharomycesandYarrowia, more preferably the microorganism is yeast, and more preferably the microorganism isCandida tropicalisorCandida sake. In a particular embodiment, the microorganism is CCTCC M2011192 or CCTCC M203052.

In some embodiments, the long-chain dibasic acid is selected from C9 to C22 long-chain dibasic acids, preferably selected from C9 to C18 long-chain dibasic acids, more preferably one or more of C10 dibasic acid, C11 dibasic acid, C12 dibasic acid, C13 dibasic acid, C14 dibasic acid, C15 dibasic acid and C16 dibasic acid. More preferably, the long-chain dibasic acid is at least one or more of C10 to C16 dibasic acids, or at least one or more of n-C10 to C16 dibasic acids, e.g. at least one or more selected from the group consisting of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

In some embodiments, the monobasic acid impurity according to the invention has the formula of CH3—(CH2)n-COOH, where n≥7, and/or CH2OH—(CH2)n-COOH, where n≥7;

preferably the monobasic acid impurity comprises, but not limited to, a long-chain monobasic acid with the number of carbon atoms in the carbon chain being greater than 9; preferably the monobasic acid impurity comprises any one or more of a monobasic acid having 9 carbon atoms, a monobasic acid having 10 carbon atoms, a monobasic acid having 11 carbon atoms, a monobasic acid having 12 carbon atoms, a monobasic acid having 13 carbon atoms, a monobasic acid having 14 carbon atoms, a monobasic acid having 15 carbon atoms, a monobasic acid having 16 carbon atoms, a monobasic acid having 17 carbon atoms, a monobasic acid having 18 carbon atoms, and a monobasic acid having 19 carbon atoms.

In some embodiments, where the long-chain dibasic acid is C12 dibasic acid e.g. dodecanedioic acid, the monobasic acid impurity is predominantly a monobasic acid impurity having 12 carbon atoms, and the content of the monobasic acid impurity having 12 carbon atoms is less than 8,000 ppm.

In some embodiments, where the long-chain dibasic acid is C10 dibasic acid e.g. decanedioic acid, the monobasic acid impurity is predominantly a monobasic acid impurity having 10 carbon atoms, and the content of the monobasic acid impurity having 10 carbon atoms is less than 2,500 ppm.

In some embodiments, where the long-chain dibasic acid is C16 dibasic acid e.g. hexadecanedioic acid, the monobasic acid impurity is predominantly a monobasic acid impurity having 16 carbon atoms, and the content of the monobasic acid impurity having 16 carbon atoms is less than 12,000 ppm.

The invention further relates to a method of modifying a long-chain dibasic acid producing microorganism strain, comprising a step of directed evolution of a key gene in the pathway of the long-chain dibasic acid synthesis, wherein, compared to the microorganism strain before modified, the modified long chain dibasic acid producing microorganism strain is capable of producing the long chain dibasic acid with substantially decreased content of monobasic acid impurity, e.g. under the same conditions. In some embodiments, the key gene in the pathway of the long-chain dibasic acid synthesis is CYP52A12 gene.

In some embodiments, the microorganism of the invention is selected from the group consisting ofCorynebacterium, Geotrichum candidum, Candida, Pichia, Rhodotroula, SaccharomycesandYarrowia, more preferably the microorganism is yeast, and more preferably the microorganism isCandida tropicalisorCandida sake. In a particular embodiment, the microorganism is CCTCC M2011192 or CCTCC M203052.

In some embodiments, the long-chain dibasic acid according to the invention is selected from C9 to C22 long-chain dibasic acids, preferably selected from C9 to C18 long-chain dibasic acids, more preferably one or more selected from the group consisting of C10 dibasic acid, C11 dibasic acid, C12 dibasic acid, C13 dibasic acid, C14 dibasic acid, C15 dibasic acid and C16 dibasic acid. More preferably, the long-chain dibasic acid is at least one or more of C10 to C16 dibasic acids, or at least one or more of n-C10 to C16 dibasic acids, e.g. at least one or more selected from the group consisting of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

In some embodiments, the content of monobasic acid impurity, compared with that of the monobasic acid impurity in the long-chain dibasic acid produced by fermentation method with a conventional microorganism, is decreased by at least 5%, preferably at least 10%, more preferably at least 20%, more preferably at least 40%, and more preferably at least 50% or more.

In some embodiments, the method of modifying a long-chain dibasic acid producing microorganism strain comprises steps of:

1) preparing a target gene (CYP52A12 gene) fragment having a mutation by error-prone PCR;

2) preparing fragments upstream and downstream of the target gene (CYP52A12 gene) necessary for homologous recombination as templates for homologous recombination with a resistance marker gene, preferably the resistance marker gene is hygromycin B;

3) preparing a complete recombination fragment by PCR overlap extension;

4) introducing the recombination fragment into a strain by homologous recombination;

5) screening positive strains by means of the resistance marker;

6) screening strains wherein the content of monobasic acid impurity in the fermentation broth after completion of fermentation is significantly decreased;

7) optionally, removing the resistance marker in the screened strains by further homologous recombination.

The invention further relates to a fermentation broth during a process of producing a long-chain dibasic acid by fermentation with a microorganism, wherein the fermentation broth contains monobasic acid impurity, and the mass ratio of monobasic acid impurity is less than 5%, preferably less than 1.5%, more preferably less than 1.0%, less than 0.9%, wherein the mass ratio is the mass percentage of monobasic acid impurity to the long-chain dibasic acid in the fermentation broth.

Preferably, the long-chain dibasic acid is selected from C9 to C22 long-chain dibasic acids, and the monobasic acid impurity comprises, but not limited to, a long-chain monobasic acid with the number of carbon atoms in the carbon chain being greater than 9.

In some embodiments, the microorganism contains the isolated DNA molecule (for controlling the expression of CYP52A12 gene, e.g. as a promoter) or the mutated CYP52A12 gene, homologous gene or variant thereof according to the invention. In some embodiments, the microorganism is the microorganism according to the invention which contains the isolated DNA molecule or the mutated CYP52A12 gene, homologous gene or variant thereof according to the invention. In some embodiments, the fermentation broth is obtained by a method of producing a long-chain dibasic acid by fermentation with a microorganism containing the isolated DNA molecule or the mutated CYP52A12 gene, homologous gene or variant thereof according to the invention. In some embodiments, the fermentation broth is obtained in a process of producing a long-chain dibasic acid with a microorganism obtained by the method of modifying a long-chain dibasic acid producing microorganism strain according to the invention.

The invention further relates to a method of producing a long-chain dibasic acid as described in the invention, comprising obtaining a long-chain dibasic acid producing microorganism strain containing a mutated CYP52A12 gene, a homologous gene or a variant thereof by directed evolution of the CYP52A12 gene in the long-chain dibasic acid synthesis pathway; culturing the strain to produce the long-chain dibasic acid by fermentation; optionally, further comprising the steps of isolating, extracting and/or purifying the long-chain dibasic acid from the culture product;

wherein, the mutated CYP52A12 gene, homologous gene or variant thereof, which, relative to GenBank Accession Number AY230498 and taking the first base upstream of the start codon ATG (in which the “A” is 1) as −1, comprises any one or a combination of some of the following base mutations occurred in the promoter region thereof: −876A>G; −853A>T; −831delT; −825C>A; −823delG; −579A>G; −412_−411AC>TT; −402insTT; −15_1 ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA); and the variant has at least 70% sequence identity to the mutated CYP52A12 gene or homologous gene thereof.

Preferably, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutation −15_1 ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA) in the promoter region thereof.

Preferably, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutations −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA) in the promoter region thereof.

Preferably, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutations −579A>G; −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA) in the promoter region thereof.

Preferably, the mutated CYP52A12 gene, homologous gene or variant thereof comprises the base mutations −831delT; −825C>A; −823delG; −579A>G; −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA) in the promoter region thereof.

Preferably, the sequence of the mutated CYP52A12 gene is set forth in any of SEQ ID NOs. 16 and 22-26 or a sequence having at least 70% sequence identity thereto, for example at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, or 99.96% sequence identity.

Preferably, the long-chain dibasic acid is selected from C9-C22 long-chain dibasic acids, preferably selected from C9-C18 long-chain dibasic acids, more preferably one or more selected from the group consisting of C10 dibasic acid, C11 dibasic acid, C12 dibasic acid, C13 dibasic acid, C14 dibasic acid, C15 dibasic acid and C16 dibasic acid. More preferably, the long-chain dibasic acid is at least one of C10 to C16 dibasic acids, or at least one of n-C10 to C16 dibasic acids, e.g. at least one selected from the group consisting of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

Preferably, the chemical formula of the monobasic acid impurity comprises CH3—(CH2)n-COOH, where n≥7, and/or CH2OH—(CH2)n-COOH, where n≥7.

In some embodiments, the microorganism is yeast; more preferably the microorganism is selected fromCandida tropicalisorCandida sake.

In some embodiments, to obtain a long-chain dibasic acid producing microorganism strain with a mutated CYP52A12 gene, a homologous gene or a variant thereof, comprises the following steps:

1) preparing a CYP52A12 gene fragment having a mutation by error-prone PCR;

2) preparing fragments upstream and downstream of the CYP52A12 gene necessary for homologous recombination as templates for homologous recombination with a resistance marker gene, preferably the resistance marker gene is hygromycin B;

3) preparing a complete recombination fragment by PCR overlap extension;

4) introducing the recombination fragment into a strain by homologous recombination;

5) screening positive strains by means of the resistance marker;

6) screening strains wherein the content of monobasic acid impurity in the fermentation broth after fermentation is significantly reduced;

7) optionally, removing the resistance marker in the screened strains by further homologous recombination.

Preferably, in the present invention, the existingCandida tropicalisstrain CATN145 (Deposit No. CCTCC M2011192) is used as the starting strain, hereinafter referred to as CCTCC M2011192, and error-prone PCR method is used to randomly mutate the CYP52A12 gene. The gene is subjected to directed evolution by homologous recombination method to screen for a long-chain dibasic acid producing strain with significantly reduced content of impurity during the production of dibasic acid, wherein the impurity is monobasic acid impurity.

By screening, the present invention obtains a strain in which the content of monobasic acid impurity in the fermentation product is significantly reduced, and is named as mutant strain 430. By sequencing analysis, it is found that, compared to parental strain CCTCC M2011192 and taking the first base upstream of the start codon ATG (wherein “A” is 1) as −1, following base mutations occurred in the promoter region of the CYP52A12 gene of the mutant strain ofCandida tropicalisscreened in the invention: −876A>G, −853A>T, −831delT, −825C>A, −823delG, −579A>G, −412_−411AC>TT, −402insTT, and −15_1 ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA). A promoter sequence containing different combinations of mutation sites was synthesized by the method of whole-genome synthesis (Sangon) (Genbank Accession No. AY230498), comprising 912 bp upstream of the start codon and adjacent 23 bp CDS sequence. Taking the first base upstream of the start codon ATG as −1, any one or combination of several of the above base mutation sites can also achieve the effect of significantly decreased content of monobasic acid impurity.

According to the present invention, the sequence of theCandida tropicalisCYP52A12 gene is set forth in SEQ ID NO: 16.

After further removing the resistance marker from the mutant strain, compared to the original strain, the mass ratio of monobasic acid impurity in the fermentation broth after fermentation is significantly reduced, and the content of monobasic acid impurity in the long-chain dibasic acid product obtained after extracting and purifying the fermentation broth can be reduced to 200 ppm or less, particularly during fermentation production of long-chain dodecanedioic acid.

The present invention screens a strain which has base mutations in the promoter region of the CYP52A12 gene by directed evolution of said gene, and the content of monobasic acid impurity in the fermentation broth is significant reduced for different fermentation substrates. After extraction and purification, compared to the parental strain, the content of monobasic acid impurity is decreased by nearly 50%, further improving the purity of the fermentation product long-chain dibasic acid, making the dibasic acid product which is used as important raw materials for nylon filament, engineering plastic, synthetic fragrance, cold-resistant plasticizer, advanced lubricants and polyamide hot melt adhesives, favorable for the production of and the quality improvement of downstream products. More importantly, this greatly reduces the difficulty of the extraction and purification processes at later stages of dibasic acid production, simplifies the process and saves energy.

