阅读说明：本技术 一种低含量脂肪酸杂质的长链二元酸及其生产方法 (Long-chain dibasic acid with low content of fatty acid impurities and production method thereof ) 是由 刘文波 徐敏 杨晨 周豪宏 刘修才 于 2019-04-01 设计创作，主要内容包括：本发明涉及一种低含量脂肪酸杂质的长链二元酸及其生产方法,具体涉及利用定向进化和同源重组方法制备长链二元酸菌株、利用该菌株发酵生产低含量脂肪酸杂质的长链二元酸。本发明涉及一种分离的突变的CPR-b基因、其同源基因或其变体,其相对于GenBank登录号AY823228,以起始密码子ATG上游第一位碱基为-1计,具有碱基突变-322G>A以及以终止密码子TAG下游第一位碱基为1计,具有突变3’UTR.19C>T和3’UTR.76_77insT。本发明还涉及含有所述突变的CPR-b基因、同源基因或变体的菌株,该菌株发酵生产长链二元酸时,其发酵产物中脂肪酸杂质的含量显著降低。(The invention relates to a long-chain dibasic acid with low content of fatty acid impurities and a production method thereof, in particular to a long-chain dibasic acid strain prepared by using directed evolution and homologous recombination methods and a long-chain dibasic acid with low content of fatty acid impurities produced by fermenting the strain. The present invention relates to an isolated mutated CPR-b gene, a homologous gene thereof or a variant thereof having the mutation-322G > a, calculated as the first base upstream of the start codon ATG of-1 and the mutation 3'utr.19c > T and 3' utr.76_77insT, calculated as the first base downstream of the stop codon TAG of 1, relative to GenBank accession No. AY 823228. The invention also relates to a strain containing the mutated CPR-b gene, homologous gene or variant, wherein the content of fatty acid impurities in a fermentation product is obviously reduced when the strain is used for producing the long-chain dibasic acid by fermentation.)

1. An isolated mutated CPR-b gene, homologous gene thereof or variant thereof having the base mutation-322G > a, relative to GenBank accession No. AY823228, based on the first base upstream of the start codon ATG being-1; the gene has mutation 3'UTR.19C > T and 3' UTR.76_77insT based on the first base at the downstream of a stop codon TAG as 1; wherein the variant has at least 70% sequence identity to a mutated CPR-b gene, a homologous gene thereof,

preferably, the mutated CPR-b gene has the sequence set forth in SEQ ID NO: 13 or 23 or at least 70% sequence identity thereto, e.g., a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.18%, 99.21%, 99.25%, 99.28%, 99.32%, 99.36%, 99.39%, 99.43%, 99.46%, 99.50%, 99.53%, 99.57%, 99.61%, 99.64%, 99.68%, 99.72%, 99.75%, 99.79%, 99.82%, 99.86%, 99.89%, 99.93%, or 99.96% identity thereto.

2. A microorganism containing the mutated CPR-b gene of claim 1, a homologous gene thereof, or a variant thereof, which has a reduced content of fatty acid impurities when producing long chain dibasic acids relative to a microorganism containing the unmutated CPR-b gene and homologous gene thereof;

preferably, the microorganism is selected from the group consisting of corynebacterium, geotrichum candidum, candida, pichia, rhodotorula, saccharomyces, yarrowia; more preferably, the microorganism is a yeast; more preferably, the microorganism is selected from Candida tropicalis (Candida tropicalis) or Candida sake (Candida sake);

preferably, the long-chain dibasic acid is selected from C9-C22 long-chain dibasic acids, preferably from C9-C18 long-chain dibasic acids, more preferably from one or more of dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid; more preferably, the long-chain dibasic acid is selected from at least one of deca to hexadecanedioic acid or at least one of n-deca to hexadecanedioic acid, for example from at least one of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

3. A method for producing a long-chain dicarboxylic acid, comprising the step of culturing the microorganism of claim 2, optionally further comprising the step of isolating, extracting and/or purifying the long-chain dicarboxylic acid from the culture product,

preferably, the mass ratio of fatty acid impurities contained in the fermentation liquor produced by the microbial fermentation is below 1.50%, and/or the content of the fatty acid impurities is reduced by at least 5% relative to the content of the fatty acid impurities in the long-chain dibasic acid produced by the conventional microbial fermentation method, such as the non-mutation microbial fermentation, wherein the mass ratio is the mass percentage of the fatty acid impurities in the fermentation liquor to the long-chain dibasic acid.

4. A long-chain dibasic acid having a low content of fatty acid impurities, which is characterized in that the content of fatty acid impurities contained in the long-chain dibasic acid is more than 0 and less than 4000ppm, preferably less than 1000ppm, more preferably less than 200ppm, wherein the fatty acid impurities comprise a saturated straight-chain organic acid having one terminal carboxyl group,

preferably, the long-chain dibasic acid is one or more of C9-C22 long-chain dibasic acid, preferably C9-C18 long-chain dibasic acid, more preferably dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid; more preferably, the long-chain dibasic acid is at least one of deca to hexadecanedioic acid or at least one of n-deca to hexadecanedioic acid, for example at least one selected from sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

5. The long chain dicarboxylic acid of claim 4, wherein the fatty acid impurity has the formula CH₃-(CH₂) n-COOH, wherein n is not less than 7, preferably the fatty acid impurities comprise long chain fatty acids having 9 or more carbon atoms in the carbon chain and containing 1 carboxyl end group, preferably the fatty acid impurities comprise one or more of nine-carbon fatty acids, ten-carbon fatty acids or capric acid, undecanoic fatty acids, dodecanoic fatty acids or lauric acid, tridecanoic fatty acids, tetradecanoic fatty acids or myristic acid, pentadecanoic fatty acids, hexadecanoic fatty acids or palmitic acid, heptadecanoic fatty acids, octadecanoic fatty acids or stearic acid, or nineteen-carbon fatty acids.

6. The long-chain dibasic acid of claim 4 or 5, wherein:

when the long chain dibasic acid is dodecanedioic acid, the fatty acid impurity is predominantly lauric acid and the lauric acid impurity is present in an amount of less than 3000ppm, preferably less than 500ppm, 400ppm, 300ppm, 200ppm or less;

when the long chain diacid is dodecanedioic acid, the fatty acid impurities are predominantly decanoic acid and the decanoic acid impurities are present in an amount less than 2000ppm, preferably less than 500ppm, 400ppm, 300ppm, 200ppm or less; or

When the long chain diacid is hexadecanedioic acid, the palmitic acid impurity is predominantly palmitic acid and the content of the palmitic acid impurity is less than 4000ppm, preferably less than 500ppm, 400ppm, 300ppm or less.

7. A fermentation liquor in the process of producing long-chain dibasic acid by a microbial fermentation method is characterized in that the fermentation liquor contains fatty acid impurities, the content of the fatty acid impurities is less than 1.5%, such as less than 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3% or less, and the percentage is the mass percentage of the fatty acid impurities in the fermentation liquor in the long-chain dibasic acid;

preferably, the long-chain dibasic acid is a C9-C22 long-chain dibasic acid, and the fatty acid impurities include a saturated straight-chain organic acid containing one terminal carboxyl group;

more preferably, the long-chain dibasic acid is C9-C18 long-chain dibasic acid, more preferably one or more of dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid; more preferably, the long-chain dibasic acid is at least one of deca to hexadecanedioic acid or at least one of n-deca to hexadecanedioic acid, for example at least one selected from sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid;

more preferably, the fatty acid impurity has the chemical formula CH₃-(CH₂) n-COOH, wherein n is not less than 7, more preferably the fatty acid impurities comprise long chain fatty acids having 9 or more carbon atoms in the carbon chain and containing 1 carboxyl end group, and more preferably the estersThe fatty acid impurities include one or more of nine-carbon fatty acid, ten-carbon fatty acid or capric acid, eleven-carbon fatty acid, twelve-carbon fatty acid or lauric acid, thirteen-carbon fatty acid, fourteen-carbon fatty acid or myristic acid, fifteen-carbon fatty acid, sixteen-carbon fatty acid or palmitic acid, seventeen-carbon fatty acid, eighteen-carbon fatty acid or stearic acid, or nineteen-carbon fatty acid.

8. The long chain dicarboxylic acid according to any one of claims 4 to 6 or the fermentation broth according to claim 7, which is obtained by the process according to claim 3 or obtainable by the process according to claim 3.

9. A method for transforming long-chain dibasic acid to produce a microbial strain comprises the step of directionally evolving key genes of a long-chain dibasic acid synthesis way, wherein the content of fatty acid impurities in the long-chain dibasic acid produced by the transformed long-chain dibasic acid-producing microbial strain is substantially reduced relative to the content of fatty acid impurities in the microbial strain before transformation;

preferably, the key gene of the long chain dibasic acid synthesis pathway is the CPR-b gene;

preferably, the microorganism is selected from corynebacterium, geotrichum candidum, candida, pichia, rhodotorula, saccharomyces, yarrowia, more preferably the microorganism is a yeast, more preferably the microorganism is selected from candida tropicalis or candida sake;

preferably, the long-chain dibasic acid is selected from C9-C22 long-chain dibasic acids, preferably C9-C18 long-chain dibasic acids, more preferably one or more of dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, more preferably the long-chain dibasic acid is selected from at least one of deca-to-hexadecanedioic acid or at least one of n-deca-to-hexadecanedioic acid, for example from at least one of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid;

preferably, the fatty acid impurities comprise long chain fatty acids with a carbon chain having a number of carbon atoms greater than 9, more preferably C10 acid (capric acid), C12 acid (lauric acid), C14 acid (myristic acid), C16 acid (palmitic acid) and/or C18 acid (stearic acid);

preferably, the level of fatty acid impurities is reduced to less than 300ppm, such as 290ppm, 270ppm, 250ppm, 200ppm, 150ppm, 140ppm, 130ppm, 120ppm, 110ppm, 100ppm or less.

10. The method of claim 9, comprising the steps of:

1) preparing a target gene segment with mutation by error-prone PCR;

2) preparing upstream and downstream segments of a target gene required by homologous recombination as a template of the homologous recombination and a resistance marker gene, wherein the resistance marker gene is preferably hygromycin B;

3) preparing complete recombinant fragments by PCR overlap extension;

4) introducing the recombinant fragment into a strain using homologous recombination;

5) screening positive strains by using a resistance marker;

6) screening strains with obviously reduced content of fatty acid impurities in fermentation liquor after fermentation is finished;

7) optionally, the selected strain is further subjected to homologous recombination to remove the resistance selection marker.

11. A method for producing a long-chain dibasic acid, characterized by obtaining a strain of a long-chain dibasic acid-producing microorganism containing a mutated CPR-b gene, a homologous gene thereof, or a variant thereof by directed evolution of a CPR-b gene of a long-chain dibasic acid synthesis pathway, a homologous gene thereof, or a variant thereof, culturing the strain to produce a long-chain dibasic acid by fermentation, and optionally, further comprising the step of isolating, extracting and/or purifying a long-chain dibasic acid from the culture product;

the mutated CPR-b gene, homologous gene thereof or variant thereof, having a base mutation of-322G > a, relative to GenBank accession No. AY823228, with the first base upstream of the start codon ATG being-1; (ii) has a mutation, based on 1 as the first base downstream of the stop codon TAG: 3'UTR.19C > T and 3' UTR.76_77 insT; the variant has at least 70% sequence identity to a mutated CPR-b gene, a homologous gene thereof;

preferably, the mutated CPR-b gene has the sequence set forth in SEQ ID NO: 13 or 23 or at least 70% sequence identity thereto, e.g., a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.18%, 99.21%, 99.25%, 99.28%, 99.32%, 99.36%, 99.39%, 99.43%, 99.46%, 99.50%, 99.53%, 99.57%, 99.61%, 99.64%, 99.68%, 99.72%, 99.75%, 99.79%, 99.82%, 99.86%, 99.89%, 99.93%, or 99.96% identity thereto;

preferably, the long-chain dibasic acid is one or more of C9-C22 long-chain dibasic acid, preferably C9-C18 long-chain dibasic acid, more preferably dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid; preferably, the long-chain dibasic acid is at least one of deca to hexadecanedioic acid or at least one of n-deca to hexadecanedioic acid, for example, at least one selected from sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid;

preferably, the long-chain dicarboxylic acid is the long-chain dicarboxylic acid according to any one of claims 4 to 6.

12. The method according to claim 11, wherein the microorganism is a yeast, more preferably selected from candida tropicalis or candida sake.

13. The method according to claim 11 or 12, wherein the long-chain dibasic acid-producing microbial strain containing the mutated CPR-b gene, homologous gene thereof or variant thereof is obtained by the method of claim 9 or 10 or is obtainable by the method of claim 9 or 10.

Technical Field

The invention relates to a long-chain dibasic acid with low content of fatty acid impurities and a production method thereof, as well as a method for preparing a long-chain dibasic acid strain by utilizing an directed evolution and homologous recombination method and a method for producing the long-chain dibasic acid with low content of the fatty acid impurities by utilizing the strain.

Background

The long chain dibasic acid (LCDA; also known as long chain dicarboxylic acid or long chain diacid) comprises the formula HOOC (CH)₂)_nA dibasic acid of COOH, wherein n is more than or equal to 7. The long-chain dibasic acid is used as an important monomer raw material and widely used for synthesizing nylon, resin, hot melt adhesive, powder coating, preservative, spice, lubricant, plasticizer and the like.

Long chain diacids have long been synthesized via petroleum by conventional chemical synthetic routes such as the multi-step oxidation of butadiene. However, the chemical synthesis method faces various challenges, and the dibasic acid obtained by the chemical synthesis method is a mixture of long-chain dibasic acid and short-chain dibasic acid, so that complicated subsequent extraction and purification steps are required, and the method is a huge obstacle to the production process and the production cost. The long-chain dibasic acid is produced by adopting a microbial fermentation technology, and has obvious advantages compared with the traditional chemical synthesis method due to the characteristics of low pollution, environmental friendliness, capability of synthesizing products which are difficult to synthesize by the chemical synthesis method, such as long-chain dibasic acid with more than 12 carbon atoms, high purity and the like.

However, the long-chain dibasic acid produced by the microbial fermentation technology sometimes leaves impurities in the product, and the reduction of the product purity seriously affects the product quality and greatly affects the later application. Especially, the impurities with characteristics similar to those of the long-chain dibasic acid not only bring great technical challenges to the later extraction and purification, but also cause serious negative effects on the production cost control. Therefore, the method carries out genetic modification on strains for producing the long-chain dibasic acid so as to reduce the content of certain impurities in the fermentation process, and has important significance and production value for producing the dibasic acid by a biosynthesis method.

The improvement of the diacid strains is mostly realized by the traditional random mutagenesis or a genetic engineering method, and due to the randomness of the mutagenesis, the requirement on screening flux is high, and each time a new round of mutagenesis screening is required for character change, the improvement becomes an important limiting factor in the technology. The bacterial strain can be subjected to targeted genetic modification by adopting a genetic engineering means, so that an excellent bacterial strain with higher yield can be obtained. The production method of the long-chain dibasic acid by the microbial fermentation method mainly comprises the step of oxidizing alkane by omega. Which in turn can be degraded via the beta-oxidation pathway. Previous studies have shown that the yield of long chain diacids can be increased by means of enhancing the omega-oxidation pathway and inhibiting the beta-oxidation pathway. Pictaggio et al (mol. cell. biol.,11 (9)), 4333-. Further over-expressing two key enzyme P450 and oxidoreductase CPR-b genes in the rate-limiting step in the omega-oxidation pathway can effectively improve the yield. The invention reports that the conversion rate and the production efficiency of the dibasic acid can be effectively improved by introducing a copy of CYP52A14 gene into a dibasic acid producing strain by virtue of the fact that the invention is carried out by Ministry of Living Engineers and so on (Chinese patent CN 103992959B). In addition, the inventor of Hua university Cao Zhuan et al (Biotechnol. J.,1,68-74,2006) finds that the knock-out of one copy of key gene CAT in the process of transporting acetyl coenzyme A from peroxisome to mitochondria can partially block the entry of acetyl coenzyme A into the citric acid cycle, and can also effectively reduce the degradation of dibasic acid.

Error-prone PCR was the first Technique proposed by Leung et al (Technique,1,11-15,1989) to construct gene libraries for targeted studies. By changing PCR reaction conditions, e.g. adjusting the concentration of four kinds of DNA in the reaction system, changing Mg²⁺The mutation is introduced by mismatching bases by a method such as DNA polymerase with low fidelity. The effect of constructing mutation library can be influenced by too high or too low mutation rate, and the ideal base mutation ratio is 1-3 per DNA fragment. Therefore, random mutation is generated by error-prone PCR, and the directional genetic modification of genes is carried out by combining a homologous recombination method, so that the screening of beneficial mutation which is helpful for further improving the productivity of the strain can be facilitated.

However, no report has been made on the studies of modifying a dibasic acid-producing strain by genetic engineering to reduce the fatty acid content. There remains a need in the art for long chain diacid products having low levels of impurities, as well as strains, and methods of making the same, that ferment to produce such products.

Disclosure of Invention

The invention relates to an isolated mutated CPR-b gene, a homologous gene thereof or a variant thereof, which has a base mutation of-322G > A in a promoter region thereof relative to GenBank accession number AY823228 (shown in SEQ ID NO: 22, for example) with the first base upstream of the ATG start codon (shown in SEQ ID NO: 22, for example, base "C" 763) as-1; based on the number 1 of the first base (such as the 2804 th base "A" shown in SEQ ID NO: 22) at the downstream of the stop codon TAG, the mutation of the terminator region is as follows: 3'UTR.19C > T and 3' UTR.76_77 insT; wherein the variant has at least 70% sequence identity to a mutated CPR-b gene, a homologous gene thereof.

In some embodiments, the mutated CPR-b gene of the invention has the sequence set forth in SEQ ID NO: 13 or 23 or at least 70% sequence identity thereto, e.g., a sequence having at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.18%, 99.21%, 99.25%, 99.28%, 99.32%, 99.36%, 99.39%, 99.43%, 99.46%, 99.50%, 99.53%, 99.57%, 99.61%, 99.64%, 99.68%, 99.72%, 99.75%, 99.79%, 99.82%, 99.86%, 99.89%, 99.93% or 99.96% identity thereto.

The invention further relates to a microorganism containing a mutated CPR-b gene, homologous gene thereof or variant thereof according to the invention, which has a reduced content of fatty acid impurities in the production of long chain dibasic acids relative to a microorganism containing an unmutated CPR-b gene and homologous gene thereof.

The present invention further relates to a method for producing a long-chain dicarboxylic acid by fermentation using a microorganism containing the mutated CPR-b gene, homologous gene thereof or variant thereof of the present invention, which comprises the step of culturing the microorganism, optionally further comprising the step of isolating, extracting and/or purifying the long-chain dicarboxylic acid from the culture product.

In some embodiments, after the process of producing the long-chain dibasic acid by microbial fermentation is finished, the fermentation broth contains fatty acid impurities, and the mass ratio of the fatty acid impurities in the fermentation broth is less than 1.50%, wherein the mass ratio is the mass percentage of the fatty acid impurities in the fermentation broth to the long-chain dibasic acid.

In some embodiments, after the process of producing the long-chain dicarboxylic acid by microbial fermentation according to the present invention is completed, the fermentation broth contains fatty acid impurities, and the content of the fatty acid impurities in the fermentation broth is reduced by at least 5% relative to the content of the fatty acid impurities in the long-chain dicarboxylic acid produced by conventional microbial fermentation, such as fermentation by a non-mutated microbial fermentation according to the present invention.

The invention further relates to a long-chain dibasic acid with low content of fatty acid impurities, wherein the content of the fatty acid impurities contained in the long-chain dibasic acid is more than 0 and less than 4000ppm, preferably less than 1000ppm, more preferably less than 200ppm, and the fatty acid impurities comprise saturated straight-chain organic acid containing one terminal carboxyl group. Preferably, the long-chain dicarboxylic acid is obtained by culturing a long-chain dicarboxylic acid-producing microbial strain and performing fermentation production.

In some embodiments, the long-chain dibasic acid-producing microbial strain comprises a mutated CPR-b gene, homologous gene thereof, or variant thereof of the present invention. In some embodiments, the long chain dibasic acid producing microbial strain is a microorganism of the invention comprising a mutated CPR-b gene of the invention, a homologous gene thereof, or a variant thereof.

In some embodiments, the microorganism of the present invention is selected from the group consisting of corynebacterium, geotrichum, candida, pichia, rhodotorula, saccharomyces, yarrowia; more preferably, the microorganism is a yeast; more preferably, the microorganism is selected from the group consisting of Candida tropicalis (Candida tropicalis) and Candida sake (Candida sake). In a particular embodiment, the microorganism is selected from CCTCC M2011192 and CCTCC M203052.

In some embodiments, the long chain dibasic acid of the present invention is selected from C9 to C22 long chain dibasic acids, preferably from C9 to C18 long chain dibasic acids, more preferably from one or more of dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid. More preferably, the long-chain dibasic acid is selected from at least one of deca to hexadecanedioic acid or at least one of n-deca to hexadecanedioic acid, for example from at least one of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

In some embodiments, the fatty acid impurity is of the formula CH₃-(CH₂) n-COOH, wherein n is not less than 7, preferably the fatty acid impurities comprise long chain fatty acids having 9 or more carbon atoms in the carbon chain and containing 1 carboxyl end group, preferably the fatty acid impurities comprise one or more of nine-carbon fatty acids, ten-carbon fatty acids or capric acid, undecanoic fatty acids, dodecanoic fatty acids or lauric acid, tridecanoic fatty acids, tetradecanoic fatty acids or myristic acid, pentadecanoic fatty acids, hexadecanoic fatty acids or palmitic acid, heptadecanoic fatty acids, octadecanoic fatty acids or stearic acid, or nineteen-carbon fatty acids.

In some embodiments, when the long chain diacid is dodecanedioic acid (e.g., dodecanedioic acid), the fatty acid impurity is predominantly lauric acid and the lauric acid impurity is present in an amount less than 3000ppm, preferably less than 400ppm, 300ppm, 200ppm or less.

In some embodiments, when the long chain diacid is a dodecanedioic acid (e.g., sebacic acid), the fatty acid impurity is predominantly decanoic acid and the decanoic acid impurity is present in an amount less than 2000ppm, preferably less than 500ppm, 400ppm, 300ppm, 200ppm or less.

In some embodiments, when the long chain diacid is hexadecanedioic acid (e.g., hexadecanedioic acid), the fatty acid impurities are predominantly palmitic acid and the palmitic acid impurities are present in an amount of less than 4000ppm, preferably less than 500ppm, 400ppm, 300ppm or less.

The invention further relates to a method for modifying a long-chain dicarboxylic acid-producing microbial strain, comprising the step of directed evolution of key genes of a long-chain dicarboxylic acid synthesis pathway, wherein the modified long-chain dicarboxylic acid-producing microbial strain produces a substantially reduced content of fatty acid impurities, e.g. under the same conditions, in the produced long-chain dicarboxylic acid compared to the microbial strain before modification. In some embodiments, a key gene of the long chain diacid synthesis pathway of the invention is the CPR-b gene.

In some embodiments, the microorganism of the present invention is selected from the group consisting of corynebacterium, geotrichum, candida, pichia, rhodotorula, saccharomyces, yarrowia, more preferably wherein the microorganism is a yeast, more preferably wherein the microorganism is selected from the group consisting of candida tropicalis or candida sake. In a particular embodiment, the microorganism is selected from CCTCC M2011192 and CCTCC M203052.

In some embodiments, the long chain dibasic acid of the present invention is selected from C9 to C22 long chain dibasic acids, preferably from C9 to C18 long chain dibasic acids, more preferably from one or more of dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid. More preferably, the long-chain dibasic acid is selected from at least one of deca to hexadecanedioic acid or at least one of n-deca to hexadecanedioic acid, for example from at least one of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

In some embodiments, the fatty acid impurities of the present invention include long chain fatty acids with a carbon chain having a number of carbon atoms greater than 9, more preferably C10 acid (capric acid), C12 acid (lauric acid), C14 acid (myristic acid), C16 acid (palmitic acid), and/or C18 acid (stearic acid). Preferably, the level of fatty acid impurities is reduced to less than 300ppm, such as 290ppm, 270ppm, 250ppm, 200ppm, 150ppm, 140ppm, 130ppm, 120ppm, 110ppm, 100ppm or less.

In some embodiments, the method of engineering a long chain dibasic acid producing microbial strain comprises the steps of:

1) preparing a target gene segment with mutation by error-prone PCR;

2) preparing upstream and downstream segments of a target gene required by homologous recombination as a template of the homologous recombination and a resistance marker gene, wherein the resistance marker gene is preferably hygromycin B;

3) preparing complete recombinant fragments by PCR overlap extension;

4) introducing the recombinant fragment into a strain using homologous recombination;

5) screening positive strains by using a resistance marker;

6) screening strains with obviously reduced content of fatty acid impurities in fermentation liquor after fermentation is finished; and

7) optionally, the selected strain is further subjected to homologous recombination to remove the resistance selection marker.

The invention further relates to a fermentation liquor in the process of producing the long-chain dibasic acid by a microbial fermentation method, wherein the fermentation liquor contains fatty acid impurities, the content of the fatty acid impurities is less than 1.5%, such as less than 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3% or less, and the percentage is the mass percentage of the fatty acid impurities in the fermentation liquor in the long-chain dibasic acid.

Preferably, the long-chain dibasic acid is selected from C9-C22 long-chain dibasic acids, and the fatty acid impurities include saturated straight-chain organic acids having one terminal carboxyl group.

In some embodiments, the microorganism contains a mutated CPR-b gene, homologous gene thereof, or variant thereof according to the invention. In some embodiments, the microorganism is a microorganism of the invention that contains a mutated CPR-b gene of the invention, a homologous gene thereof, or a variant thereof. In some embodiments, the fermentation broth is obtained by a method of producing a long chain dibasic acid by fermentation with a microorganism comprising a mutated CPR-b gene of the invention, a homologous gene thereof, or a variant thereof, as described herein. In some embodiments, the fermentation broth is obtained when a long-chain dicarboxylic acid is produced using a microorganism obtained by the method of modifying a long-chain dicarboxylic acid-producing microorganism strain of the present invention.

The present invention further relates to a method for producing a long-chain dibasic acid, comprising obtaining a strain of a long-chain dibasic acid-producing microorganism containing a mutated CPR-b gene, a homologous gene thereof or a variant thereof by directed evolution of a CPR-b gene of a long-chain dibasic acid synthesis pathway, culturing said strain to produce a long-chain dibasic acid by fermentation, and optionally, further comprising the step of isolating, extracting and/or purifying the long-chain dibasic acid from the culture product.

The mutated CPR-b gene, homologous gene thereof or variant thereof, has a base mutation of-322G > A in the promoter region relative to GenBank accession number AY823228 (e.g., SEQ ID NO: 22) with the first base upstream of the ATG start codon (e.g., base "C" 763 as shown in SEQ ID NO: 22) as-1; based on the number 1 of the first base (such as the 2804 th base "A" shown in SEQ ID NO: 22) at the downstream of the stop codon TAG, the mutation of the terminator region is as follows: 3' UTR.19C > T; 3' UTR.76_77 insT; the variant has at least 70% sequence identity to a mutated CPR-b gene, its cognate gene.

Preferably, the mutated CPR-b gene has the sequence set forth in SEQ ID NO: 13 or 23 or at least 70% sequence identity thereto, e.g., a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.18%, 99.21%, 99.25%, 99.28%, 99.32%, 99.36%, 99.39%, 99.43%, 99.46%, 99.50%, 99.53%, 99.57%, 99.61%, 99.64%, 99.68%, 99.72%, 99.75%, 99.79%, 99.82%, 99.86%, 99.89%, 99.93%, or 99.96% identity thereto.

In some embodiments, the long chain dibasic acid is selected from one or more of C9 to C22 long chain dibasic acids, preferably C9 to C18 long chain dibasic acids, more preferably dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid. In some embodiments, the long chain diacid is at least one of deca to hexadecanedioic acid or at least one of n-deca to hexadecanedioic acid, for example at least one selected from sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

In some embodiments, the fatty acid impurity is of the formula CH₃-(CH₂) n-COOH, wherein n is not less than 7, preferably the fatty acid impurities comprise long chain fatty acids with carbon number of 9 or more on the carbon chain and 1 carboxyl end group.

In some embodiments, the microorganism is a yeast, more preferably, the microorganism is selected from candida tropicalis or candida sake.

In some embodiments, obtaining a long-chain dibasic acid-producing microbial strain containing a mutated CPR-b gene, a homologous gene thereof, or a variant thereof comprises the steps of:

1) preparing a target gene (CPR-b gene) fragment with mutation by error-prone PCR;

2) preparing target gene (CPR-B gene) upstream and downstream fragments required by homologous recombination as a template for homologous recombination and a resistance marker gene, preferably, the resistance marker gene is hygromycin B;

3) preparing complete recombinant fragments by PCR overlap extension;

4) introducing the recombinant fragment into a strain using homologous recombination;

5) screening positive strains by using a resistance marker;

6) screening strains with obviously reduced content of fatty acid impurities in fermentation liquor after fermentation is finished;

7) optionally, the selected strain is further subjected to homologous recombination to remove the resistance selection marker.

The invention takes the existing candida tropicalis strain CATN145 (the preservation number is CCTCC M2011192) as a starting strain, adopts error-prone PCR to randomly mutate CPR-b genes, and carries out directed evolution on the genes by a homologous recombination method, so as to screen out the strain for producing the long-chain dibasic acid, wherein the content of fatty acid impurities is obviously reduced. Through screening, the strain with the remarkably reduced content of fatty acid impurities in the fermentation product is obtained and named as mutant 5473. Sequencing analysis shows that compared with a parental strain CCTCC M2011192, the CPR-b gene of the mutant 5473 is counted by taking the first base at the upstream of an initiation codon ATG as-1, and the Candida tropicalis mutant screened by the invention has base mutation-322G > A in the promoter region; the mutation of the terminator region is calculated by taking the first base downstream of the stop codon TAG as 1: 3'UTR.19C > T and 3' UTR.76_77 insT.

According to the invention, the sequence of the mutated candida tropicalis CPR-b gene comprises or is as set forth in SEQ ID NO: shown at 13.

After the resistance screening marker is further removed from the mutant strain, compared with the parent strain, the mass ratio of fatty acid impurities in the fermentation liquid after fermentation is obviously reduced, and the content of the fatty acid impurities in the long-chain dicarboxylic acid finished product obtained after the fermentation liquid is extracted and purified can be reduced to below 300 ppm.

The invention screens a strain which generates base mutation in the promoter region and the terminator region of the gene by performing directed evolution on the CPR-b gene, obviously reduces the content of fatty acid impurities in fermentation liquor aiming at different fermentation substrates, reduces the content of fatty acid by nearly 40 percent compared with a parental strain, further improves the purity of the fermentation product long-chain dibasic acid, ensures that the dibasic acid product is used as an important raw material of products such as nylon filament, engineering plastics, synthetic spices, cold-resistant plasticizers, high-grade lubricating oil, polyamide hot melt adhesive and the like, is more beneficial to the production and manufacture of downstream products, and improves the quality of the downstream products. More importantly, the difficulty of the later-stage extraction and purification process of the dibasic acid is greatly reduced, the process is simplified, and the energy consumption is saved.

Drawings

FIG. 1 is a schematic representation of the incorporation of the CPR-b gene with mutation sites by homologous recombination and the removal of the hygromycin selection marker, ". indicates mutations that may be present in any region of CPR-b, including the promoter, coding region and terminator.

FIG. 2 shows the result of alignment of the nucleotide sequences of the CPR-b gene of the mutant strain of the present invention (represented by CPR-b', represented by nucleotides 295 and 3087 in SEQ ID NO: 23) with the nucleotide sequence of the CPR-b gene of the original strain (represented by nucleotides 295 and 3086 in SEQ ID NO: 22), the mutation sites being indicated by black boxes.

Detailed Description

Defining:

unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, e.g., Singleton et al, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY2nd ed., J.Wiley & Sons (New York, NY 1994); sambrook et al, Molecular clone, ALABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

Long-chain alkanes: the fermentation substrate comprises long-chain alkane, the long-chain alkane belongs to saturated chain hydrocarbon, is saturated hydrocarbon under hydrocarbon, and the whole structure of the fermentation substrate is mostly only composed of carbon, hydrogen, carbon-carbon single bond and carbon-carbon single bond, and comprises a chemical formula CH₃(CH₂)_nCH₃Wherein n.gtoreq.7. Preferably, the n-alkanes are C9-C22, more preferably C9-C18, and most preferably C10, C11, C12, C13, C14, C15 or C16.

Long chain dibasic acids (LCDA; also known as long chain dicarboxylic acids or long chain diacids, hereinafter or simply dibasic acids) include the formula HOOC (CH)₂)_nA dibasic acid of COOH, wherein n is more than or equal to 7. Preferably, the long-chain dibasic acid comprises a long-chain dibasic acid of C9-C22, preferably comprises a long-chain dibasic acid of C9-C18, and more preferably comprises one or more of dodecanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid. Preferably, the long-chain dibasic acid is at least one of deca to hexadecanedioic acid, preferably at least one of n-deca to hexadecanedioic acid, for example at least one selected from sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

A long-chain dicarboxylic acid-producing microorganism: strains that have been reported to produce and accumulate dibasic acids include bacteria, yeast, and mold, among others, such as: corynebacterium (Corynebacterium), Geotrichum candidum (Geotrichum candidum), Candida (Candida), Pichia (Pichia), Rhodotorula (Rhodotroula), Saccharomyces (Saccharomyces), Yarrowia (Yarrowia), and the like. Many of the species of Candida are superior species for the fermentative production of dibasic acids. The species for fermentation preferably comprises: candida tropicalis or Candida sake.

In the process of producing the long-chain dibasic acid by fermenting the fermentation substrate long-chain alkane, the alkane is firstly oxidized into fatty acid and then oxidized into dibasic acid, but the inventor finds that partial fatty acid is remained in the fermentation liquor if the alkane is not completely oxidized. Because of their very similar properties to long chain diacids, they are difficult to separate efficiently by conventional means. Fatty acid as an impurity can enter into the final dibasic acid product along with the post-treatment process, and the purity and the quality of the product are greatly influenced.

The fatty acid impurities described in the present invention include saturated linear organic acids containing one terminal carboxyl group (-COOH). The chemical formula of the fatty acid impurity is CH₃-(CH₂) n-COOH, wherein n is more than or equal to 7. Preferably, the fatty acid impurities include long-chain fatty acids having 9 or more carbon atoms in the carbon chain and containing 1 terminal carboxyl group, such as one or more of nine-carbon fatty acids, ten-carbon fatty acids or capric acid, undecanoic fatty acids, dodecanoic fatty acids or lauric acid, tridecanoic fatty acids, tetradecanoic fatty acids or myristic acid, pentadecanoic fatty acids, hexadecanoic fatty acids or palmitic acid, heptadecanoic fatty acids, octadecanoic fatty acids or stearic acid, or nineteen-carbon fatty acids.

As used herein, a substantial or significant reduction in the level of a fatty acid impurity in accordance with the present invention means a reduction in the level of a fatty acid impurity by at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, preferably by at least 10%, more preferably by at least 20%, more preferably by at least 40%, more preferably by at least 50%, more preferably by at least 70% or more, as compared to a reference.

When the long-chain dibasic acid is produced by fermentation according to the present invention, the fermentation broth after the fermentation is finished contains fatty acid impurities, and the content of the fatty acid impurities is significantly reduced, such as at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, preferably at least 10%, more preferably at least 20%, more preferably at least 40%, more preferably at least 50%, more preferably at least 70% or more, relative to the content of the fatty acid impurities produced by conventional microbial fermentation, such as non-mutated microbial fermentation according to the present invention.

In some embodiments, the long-chain dibasic acid is produced using a microbial fermentation process, and the fermentation broth contains fatty acid impurities in an amount reduced to less than 1.5%, such as 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3% or less, by mass of the fermentation broth fatty acid impurities relative to the long-chain dibasic acid, preferably reduced to less than 1.1%, more preferably reduced to less than 1.0%, more preferably reduced to less than 0.9%.

In some embodiments of the invention, the long chain dibasic acids produced by the microbial fermentation process of the invention contain fatty acid impurities in an amount of 4000ppm or less, preferably 3000ppm or less, 2000ppm or less, 1000ppm or less, 290ppm, 270ppm, 250ppm, 200ppm, 150ppm, 100ppm or less.

The unit ppm of the impurity content of the invention is the mass ratio of the impurity to the long-chain dibasic acid, and 100ppm is 100 x 10^-60.01%. In some embodiments, the impurities of DC16 (hexadecanedioic acid) are collectively higher than DC12 (hexadecanedioic acid) and DC10 (decadioic acid), such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, 60%, at least 80%, at least 100%, or higher, where DC refers to long-chain dibasic acids.

In some embodiments of the invention, when the long chain dibasic acid with twelve carbons is produced using a microbial fermentation process, the fatty acid impurity is predominantly lauric acid, and the lauric acid impurity is present in an amount of less than 3000ppm, preferably less than 500ppm, 400ppm, 300ppm, 200ppm or less.

In some embodiments of the invention, when a microbial fermentation process is used to produce a long chain dibasic acid of ten carbons, the fatty acid impurity is predominantly decanoic acid, the decanoic acid impurity being present in an amount less than 2000ppm, preferably less than 500ppm, 400ppm, 300ppm, 200ppm or less.

In some embodiments of the invention, when using microbial fermentation to produce long chain dibasic hexadecanoic acids, the fatty acid impurities are predominantly palmitic acid, and the palmitic acid impurities are present in an amount of less than 4000ppm, preferably less than 500ppm, 400ppm, 300ppm or less.

The content of the dibasic acid and impurities can be measured by a method known to those skilled in the art, such as internal standard method or normalization method of gas chromatography.

The CPR-b gene (GenBank accession No. AY823228) encodes NADPH-dependent cytochrome reductase, binds to the endoplasmic reticulum membrane in a complex with P450 cytochrome oxidase in ω -oxidation, and supplies electrons to P450 as an electron donor. The skilled person will appreciate that the CPR-b gene or its homologous gene is also present in other long-chain dicarboxylic acid-producing microorganisms, and that the sequences may differ, but fall within the scope of the present invention.

The term "isolated" when used with respect to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components to which it is bound in its native state. It may be, for example, in a homogeneous state, and may be dry or in aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

As used herein, the expression "relative to GenBank accession number AY 823228" refers to a pair having the described mutation at the corresponding position as compared to the sequence set forth in GenBank accession number AY823228 (SEQ ID NO: 22). The corresponding position refers to the residue numbering of the reference sequence when the given polynucleotide sequence (e.g., mutated CPR-b gene sequence) is compared to the reference sequence (e.g., SEQ ID NO: 22). A base in a nucleic acid "corresponds to" a given base when it occupies the same basic structural position within the nucleic acid as the given base. In general, to identify the corresponding positions, the nucleic acid sequences are arranged so as to obtain the highest level of matching (see, for example, comparative Molecular Biology, Lesk, A.M., ed., Oxford university Press, New York, 1988; Biocomputing: information and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffing, A.M., and Griffing, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis, Prime, development in Molecular Biology, City health, G., Academic Press, 1988; sample J.1998; sample Analysis, plant J.S. and sample J., growth Press, New York, 1988; Mat.8; Mat.M. D.S. 3, plant J.S. App.S. M. D. Tokup.. Nucleotide sequence alignments may also take into account conservative differences in nucleotides and/or the frequency of substitutions. Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignments of sequences over the full length and including all residues) or local (alignments of a portion of a sequence, including only the most similar region or regions).

As used herein, a base mutation "XXX N0> N1" refers to the mutation of the base N0 at position XXX to N1. For example, "the base mutation of-322G > A" based on-1 being the first base upstream of the ATG of the initiation codon means that-1 is the first base upstream of the base "A" immediately adjacent to the ATG of the initiation codon, and A is the base G at the-322 th position; and "mutation of 3' UTR.19C > T with the first base downstream of the stop codon TAG as 1; 3' UTR.76_77insT "means that the first base immediately downstream of the base" G "of the stop codon TAG is 1, the base C at position 19 is mutated to T, and a T is inserted between the bases 76 and 77.

In one embodiment, the CPR-b gene of the invention has the sequence set forth in SEQ ID NO: 22, wherein the protein coding sequence is nucleotide 764-2803. Accordingly, the mutation "-322G > a" corresponds to SEQ ID NO: 22 nucleotide G at position 442 is mutated to A; the mutation "3' utr.19c > T" corresponds to SEQ ID NO: the 2822 th nucleotide C is mutated into T; the mutation "3' utr.76 — 77 insT" corresponds to the amino acid sequence shown in SEQ ID NO: a T is inserted between nucleotides 2879 and 2880 of 22.

In this context, when referring to bases, G refers to guanine, T refers to thymine, A refers to adenine, C refers to cytosine, and U refers to uracil.

As used herein, "unmutated CPR-b gene" refers to a CPR-b gene that does not contain a mutation-322G > A, 3'UTR.19C > T or 3' UTR.76_77insT as described herein, e.g., a naturally occurring, wild-type allele, e.g., the CPR-b gene having accession number AY823228 in GenBank. Exemplary unmodified CPR-b genes are set forth in SEQ ID NO: 22, respectively. The CPR-b gene may contain other mutations, such as silent mutations in the coding region that result in no change in the coding amino acid.

As used herein, "non-mutated microorganism" refers to a microorganism that does not contain a mutated CPR-b gene or homologous gene of the present invention, e.g., contains only the CPR-b gene with accession No. AY823228 in GenBank. In one embodiment, the non-mutated microorganism contains the unmutated CPR-b gene of the invention.

The invention screens a bacterial strain with mutated CPR-b gene, relative to GenBank accession number AY823228, the promoter region of the bacterial strain has a base mutation-322G > A by taking the first base at the upstream of the ATG of the initiation codon as-1; the mutation of the terminator region is as follows according to the first base in the downstream of the termination codon TAG as 1: 3'UTR.19C > T and 3' UTR.76_77 insT.

As used herein, a homologous gene refers to two or more gene sequences with sequence similarity of up to 80%, including orthologous genes (also referred to as vertically homologous genes, orthologous genes, or orthologous genes), transversely homologous genes (also referred to as paralogs, or paralogs), and/or heterologous homologous genes. The homologous gene of the CPR-b gene referred to in the present invention may be an orthologous gene of the CPR-b gene, or a transversely homologous gene or a heterologous homologous gene thereof.

Sequence identity refers to the percentage of residues of a variant polynucleotide sequence that are identical to a non-variant sequence after alignment of the sequences and the introduction of gaps. In particular embodiments, a polynucleotide variant has at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.1%, at least about 99.2%, at least about 99.3%, 99.4%, at least about 99.5%, at least about 99.6%, 99.7%, at least about 99.8%, at least about 99.9%, at least about 99.91%, at least about 99.92%, at least about 99.93%, at least about 99.94%, at least about 99.95%, or at least about 99.96% polynucleotide homology to a polynucleotide described herein.

As used herein, the terms "homology" and "identity" are used interchangeably to refer to the degree to which a nucleotide sequence does not vary, as can be detected by aligning the number of identical nucleotide bases between a polynucleotide and a reference polynucleotide. Sequence identity can be determined by standard alignment algorithm programs using default gap penalties established by each supplier. A homologous nucleic acid molecule refers to a predetermined number of identical or homologous nucleotides. Homology includes substitutions that do not alter the encoded amino acid (silent substitutions) as well as identical residues. Substantially homologous nucleic acid molecules typically hybridize to a full length nucleic acid or at least about 70%, 80%, or 90% of a full length nucleic acid molecule of interest under moderately stringent conditions or under highly stringent conditions. Nucleic acid molecules containing degenerate codons instead of codons in the hybrid nucleic acid molecule are also encompassed by the present invention. Whether any two nucleic acid molecules have a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical" can be determined using known computer algorithms, such as BLASTN, FASTA, DNAStar, and Gap (University of Wisconsin genetics computer Group (UWG), Madison Wis, USA). For example, the percent homology or identity of nucleic acid molecules can be determined, for example, by comparing sequence information using the GAP computer program (e.g., Needleman et al.J.mol.biol.48:443(1970), revised by Smith and Waterman (adv.appl.Math.2:482 (1981)). briefly, the GAP program defines similarity based on the number of symbols (i.e., nucleotides) of similar alignment divided by the total number of symbols of the shorter of the two sequences.

Directed evolution refers to the process of simulating natural selection by means of technical means. Through artificial mutation and specific screening pressure, protein or nucleic acid is mutated in a specific direction, so that the evolution process which can be completed in nature can be realized in thousands of years at a molecular level in a short time. A variety of methods for performing directed evolution are known in the art, including, for example, error-prone PCR and the like (see, e.g., Technique,1,11-15,1989; Genome Research,2,28-33,1992).

In some embodiments, in the error-prone PCR of the present invention, Mg²⁺In a concentration range of 1 to 10mM, preferably 2 to 8mM, more preferably 5 to 6mM, and/or a concentration of dNTPs of 0.1 to 5mM, preferably 0.2 to 3mM, more preferably 0.5 to 2mM, more preferably 0.8 to 1.5mM, e.g. 1mM, and/or the addition of freshly prepared MnCl₂To a final concentration of 0.1-5 mM, preferably 0.2-2 mM, more preferably 0.3-1 mM, more preferably 0.4-0.7 mM, e.g., 0.5 mM. In some embodiments, the chance of mutation is increased by decreasing the amount of template and increasing to 40 or more cycles of PCR as appropriate, e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 or more cycles.

PCR overlap extension is also called SOE (gene splicing by overlap extension) PCR, and refers to a method of splicing different DNA fragments together by PCR amplification by designing primers with complementary ends.

Homologous recombination refers to recombination between DNA molecules that rely on sequence similarity, most commonly found in cells for the repair of mutations that occur during mitosis. Homologous recombination techniques have been widely used for genome editing, including gene knock-out, gene repair, and the introduction of new genes into specific sites. The microorganism represented by saccharomyces cerevisiae has very high probability of homologous recombination in cells, does not depend on sequence specificity, and has obvious advantages in the aspect of genome editing. And site-specific recombination only occurs between specific sites, such as Cre/loxP, FLP/FRT and the like, depending on the participation of specific sites and site-specific recombinases. The homologous recombination technique used in this patent does not belong to site-specific recombination, which relies on intracellular DNA repair systems.

A resistance marker is one of the selectable markers, which often carries a marker conferring to the transformant the ability to survive in the presence of an antibiotic. The resistance marker genes comprise NPT, HPT, HYG, BLA, CAT and the like, and can resist kanamycin, hygromycin, ampicillin/carbenicillin, chloramphenicol and the like. Preferably, the resistance marker gene is the hygromycin B resistance gene HYG.

In the fermentation production process, the fermentation medium comprises: carbon source, nitrogen source, inorganic salts and nutrient salts.

In some embodiments, the carbon source comprises one or more selected from the group consisting of glucose, sucrose, and maltose; and/or the amount of the carbon source added is 1% to 10% (w/v), for example, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%.

In some embodiments, the nitrogen source comprises one or more selected from the group consisting of peptone, yeast extract, corn steep liquor, ammonium sulfate, urea, and potassium nitrate; and/or the total amount of nitrogen sources added is 0.1% to 3% (w/v), for example 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.5%.

In some embodiments, the inorganic salt comprises one or more selected from the group consisting of potassium dihydrogen phosphate, potassium chloride, magnesium sulfate, calcium chloride, ferric chloride, copper sulfate; and/or the total amount of inorganic salts added is 0.1% to 1.5% (w/v), e.g. 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%.

In some embodiments, the trophic factors include one or more selected from the group consisting of vitamin B1, vitamin B2, vitamin C, biotin; and/or the total addition amount of the nutritional factors is 0-1% (w/v), such as 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%. According to the common knowledge in the field of fermentation, the percentage is mass-volume ratio, namely: w/v; % means g/100 mL.

The amount of the above-mentioned substances to be added can be easily determined by those skilled in the art.

In one embodiment of the invention, the amount of inoculum of the fermentation strain is 10% to 30%, such as 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, 27%, 29%. The strain is cultured to the thallus Optical Density (OD)₆₂₀) When the concentration is more than 0.5 (diluted by 30 times), adding substrate for fermentation conversion.

Extracting and purifying long-chain dicarboxylic acid: and extracting and purifying the fermentation liquor obtained by fermentation to obtain a long-chain dicarboxylic acid finished product. The steps of extracting and purifying comprise: sterilizing and acidifying the fermentation liquor, and acidifying, solid-liquid separating and/or solvent crystallizing the obtained clear liquid.

The extraction and purification of the invention can be repeated more than once, and multiple extraction and purification steps are performed to help further reduce the impurity content in the dibasic acid product, for example, in one embodiment of the invention, the twelve carbon long-chain dibasic acid product obtained by the invention is further treated by referring to the refining process in example 1 of chinese patent CN 101985416a, and the impurity content of lauric acid in the obtained twelve carbon long-chain dibasic acid can be reduced from 5000ppm or more before treatment to 4000ppm or less, such as 3000ppm or less, 2000ppm or less, 1000ppm or less, 500ppm or less, 400ppm or less, 300ppm or less, even 250ppm, 200ppm or less, and 150ppm or less.

The fermentation liquor comprises fermentation liquor containing long-chain dibasic acid salt generated in the process of biologically fermenting long-chain dibasic acid, and the fermentation liquor containing the long-chain dibasic acid salt may contain long-chain dibasic acid sodium salt, long-chain dibasic acid potassium salt or long-chain dibasic acid ammonium salt and the like.

The sterilization is preferably membrane filtration: residual bacteria, large protein and other impurities are separated by using a filtering membrane and are effectively separated from the fermentation liquor containing the long-chain dibasic acid salt. Further, a ceramic membrane filtration process is preferable. When the ceramic membrane is used for membrane filtration, the pressure before the membrane is preferably 0.2-0.4 MPa; the preferred filtration membrane pore size is 0.05-0.2 microns.

And the acidification is to carry out acidification treatment on the obtained membrane clear liquid containing the long-chain dibasic acid salt after membrane filtration, and to convert the long-chain dibasic acid salt into long-chain dibasic acid precipitate by adding acid. It is preferable to use an inorganic acid such as sulfuric acid, hydrochloric acid, nitric acid, or a mixed acid thereof for the acidification. The addition amount of the inorganic acid in the acidification treatment needs to fully precipitate the long-chain dibasic acid in the solution, mainly based on the end-point pH of the solution, preferably the end-point pH of the acidification is lower than 5, and more preferably the end-point pH is lower than 4.0. When inorganic acid is added for acidification treatment, long-chain diacid precipitate and corresponding inorganic salt solution can be obtained.

The solid-liquid separation is to separate the obtained long-chain dicarboxylic acid precipitate from the acidified mother liquor, and the solid-liquid separation comprises filtration or/and centrifugal separation, and common solid-liquid separation equipment can be used.

Preferably, the step of extracting and purifying further comprises decoloring the fermentation liquor containing the long-chain dibasic acid salt, adding activated carbon into the fermentation liquor or the membrane clear liquid containing the long-chain dibasic acid salt for decoloring, filtering to remove the activated carbon after decoloring, wherein the decoloring step can further remove impurities in the long-chain dibasic acid solution. Preferably, the amount of the activated carbon added is 0.1 to 5 wt%, and more preferably 1 to 3 wt% (relative to the amount of the long-chain dibasic acid contained in the solution).

And (3) crystallizing the solvent, namely dissolving the long-chain dicarboxylic acid precipitate in an organic solvent, crystallizing the long-chain dicarboxylic acid by cooling, evaporating and dissolving, and separating crystals to obtain the purified long-chain dicarboxylic acid. The organic solvent comprises one or more of alcohol, acid, ketone and ester; wherein the alcohol comprises one or more of methanol, ethanol, isopropanol, n-propanol, and n-butanol; the acid comprises acetic acid; the ketone comprises acetone; the esters include ethyl acetate and/or butyl acetate.

In another preferred embodiment, the long-chain dicarboxylic acid precipitate is dissolved in an organic solvent, then decolorized, and then separated to obtain a clear solution, wherein the decolorization temperature is 85-100 ℃ and the decolorization time is 15-165 min when activated carbon is used for decolorization; in another preferred embodiment, after separation of the supernatant, crystallization is carried out at reduced temperature, which may include the following steps: firstly, cooling to 65-80 ℃, preserving heat for 1-2 hours, then cooling to 25-35 ℃, and crystallizing. In another preferred embodiment, after the crystallization, the resulting crystals are separated, thereby obtaining the long-chain dibasic acid, and the manner of separating the crystals may be centrifugation.

In some embodiments, the invention relates to the use of the dibasic acid product obtained in the above way to produce nylon filaments, engineering plastics, synthetic perfumes, cold-resistant plasticizers, high-grade lubricating oils, polyamide hot melt adhesives and the like.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally included step refers to the presence or absence of that step.

As used herein, the term "about" refers to a range of values that includes the particular value, which one of skill in the art can reasonably consider similar to the particular value. In some embodiments, the term "about" means within standard error using measurements commonly accepted in the art. In some embodiments, about refers to +/-10% of the specified value.

The invention will now be further illustrated by the following non-limiting examples, and it will be apparent to those skilled in the art that many modifications can be made without departing from the spirit of the invention, such modifications also falling within the scope of the invention.

The following experimental methods are all conventional methods unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.