CN111394400B - Application of SCT1 gene in production of long-chain dicarboxylic acid - Google Patents

Abstract

The invention provides an application of a SCT1 gene in long-chain dicarboxylic acid production, which comprises knocking out a copy of SCT1 gene or homologous gene thereof in a genome of a long-chain dicarboxylic acid production strain by a genetic engineering means, and carrying out fermentation production of long-chain dicarboxylic acid by utilizing the modified engineering bacteria. Compared with the long-chain dicarboxylic acid production strain before modification, the modified engineering bacteria have the advantage that the mass ratio of the impurities of the acylglyceride in the fermentation liquid after the fermentation is finished is obviously reduced. The long-chain dibasic acid with low content of impurities in the acylglyceride greatly reduces the difficulty of the post-extraction and purification of the dibasic acid and the wastewater treatment process, simplifies the process and saves the energy consumption; meanwhile, the quality of downstream products is improved.

Description

Application of SCT1 gene in production of long-chain dicarboxylic acid

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of an SCT1 gene in production of long-chain dicarboxylic acid.

Background

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

The microbial fermentation method for producing the long-chain dibasic acid has the characteristics of low pollution, environmental friendliness, capability of synthesizing products which are difficult to synthesize by a chemical synthesis method, such as long-chain dibasic acid with more than 12 carbon atoms, relatively high product purity, reduction of extraction and purification cost and the like, and gradually replaces the chemical synthesis method in recent years and becomes a main way for producing the long-chain dibasic acid.

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 long-chain binary acid is produced through microbial fermentation process with alkane as main component and through omega-oxidation. Which in turn can be degraded via the beta-oxidation pathway. Previous studies have mostly focused on acyl-coa oxidase knock-out and acyl-coa oxidase POX gene knock-out to block the β -oxidation pathway for product accumulation and overexpression of the P450 oxidase and CPR cytochrome oxidoreductase genes for improved ω -oxidation efficiency (mol. cell. biol.,11(9), 4333-.

Although the microbial fermentation method for producing long-chain dibasic acid has higher purity than the dibasic acid produced by the traditional chemical method, the excessive accumulation of metabolic byproducts, such as diacyl glyceride (DAG) and triacyl glyceride (TAG) which can be produced by the excessive accumulation of fatty acid, can still be caused in the fermentation process due to the oxidation rate of intermediate metabolites and the coordination of transportation and accumulation. DAG and TAG are formed by esterifying glycerol and two or three molecules of fatty acid, and are the main existing forms of neutral fat in cells; usually generated when the energy is excessive and stored as an energy substance. The presence of such impurities can present a number of challenges to the subsequent extractive purification process, resulting in increased costs and decreased yields from the purification steps. Meanwhile, the product quality problem caused by the impurities in the long-chain dicarboxylic acid product or crude product causes great troubles to downstream processes, and the development of the biological long-chain dicarboxylic acid industry is influenced to a certain extent.

At present, no report is found on the research of modifying a dibasic acid production strain by adopting a genetic engineering means to reduce the content of acylglycerol.

Disclosure of Invention

The invention aims to provide application of an SCT1 gene in production of long-chain dicarboxylic acid.

In order to achieve the purpose, the invention provides an application of the SCT1 gene in the production of long-chain dicarboxylic acid, wherein the application comprises knocking out a copy of the SCT1 gene or a homologous gene thereof in the genome of a long-chain dicarboxylic acid producing strain by genetic engineering means, and carrying out fermentation production of the long-chain dicarboxylic acid by utilizing the modified engineering bacteria.

Wherein, the genetic engineering means includes but is not limited to a homologous recombination mode.

In yeast cells, the first step in the assembly of DAG with TAG synthesis is catalyzed by the 3-phosphoglycerate acyltransferase SCT1(EC2.3.1.15), a key step in the entry of glycerol triphosphate into acylglycerol synthesis.

The results of amino acid sequence alignment of 3-phosphoglyceryl acyltransferase from different microbial species are shown in FIG. 2.

In a second aspect, the invention provides an engineered bacterium for producing long-chain dicarboxylic acid, which is a modified engineered bacterium obtained by knocking out a copy of SCT1 gene or its homologous gene in the genome of a long-chain dicarboxylic acid producing strain by using a genetic engineering means.

The long-chain dicarboxylic acid producing strain of the present invention is selected from the group consisting of strains of Corynebacterium (Corynebacterium), Geotrichum (Geotrichum), Candida (Candida), Pichia (Pichia), Rhodotorula (Rhodotorula), Saccharomyces (Saccharomyces), Yarrowia (Yarrowia); preferably a Candida species; more preferably Candida tropicalis (Candida tropicalis) or Candida sake (Candida sake).

The long-chain dibasic acid is selected from C9-C22 long-chain dibasic acids, preferably C9-C18 long-chain dibasic acids, and comprises sebacic acid or at least one of dodecanedioic acid (DC10), undecanedioic acid (DC11), dodecanedioic acid (DC12), tridecanedioic acid (DC13), tetradecanedioic acid (DC14), pentadecanedioic acid (DC15), hexadecanedioic acid (DC16), heptadecanedioic acid (DC17) and octadecanedioic acid (DC 18).

In a specific embodiment of the invention, a strain used for fermentation is an engineering bacterium 630 for producing long-chain dicarboxylic acid, and the engineering bacterium 630 is obtained by knocking out a strain with a preservation number of CCTCC NO: candida tropicalis (Candida tropicalis) CAT N145 genome of M2011192 was derived from a copy of the SCT1 gene. The candida tropicalis CAT N145 is biologically preserved in 2011 at 6, 9 months, and the preservation unit is as follows: china center for type culture Collection (Address: Wuhan, Wuhan university, China, zip code: 430072).

In another embodiment of the invention, the strain used for fermentation is an engineering bacterium 631 producing long-chain dicarboxylic acid, and the engineering bacterium 631 is obtained by knocking out a strain with a preservation number of CCTCC NO: m203052 was obtained as a copy of the SCT1 gene in the Candida tropicalis genome. The preservation number is CCTCC NO: m203052 Candida tropicalis has been biologically deposited at 6.6.2003, depository: china center for type culture Collection (Address: Wuhan, Wuhan university, China, zip code: 430072).

The Candida tropicalis with the preservation number of CCTCC M2011192 can be seen in CN201110168672. X. The preservation number is CCTCC NO: candida tropicalis of M203052 is described in CN 200710170899.1. The preservation number is CCTCC NO: m2011192 or CCTCC NO: candida tropicalis (Candida tropicalis) M203052 is available through the China Center for Type Culture Collection (CCTCC).

The preservation number is CCTCC NO: m2011192 Candida tropicalis has a SCT1 gene part CDS sequence shown as SEQ ID NO: 16.

The preparation method of the dibasic acid production strain comprises the following steps: (1) preparing a homologous recombination template which comprises a recombination template at the upper and lower reaches of a target site and a resistance selection marker gene HYG (hygromycin resistance gene), and then obtaining a complete recombination template by a PCR (polymerase chain reaction) overlap extension method; (2) the complete recombinant template is transformed into competent cells, and strains containing the resistance marker are obtained by screening on a resistance medium containing hygromycin.

In a third aspect, the invention provides a method for reducing the content of acylglycerol esters in the fermentation production of long-chain dibasic acids, which utilizes the engineering bacteria to perform the fermentation production of the long-chain dibasic acids, and the obtained long-chain dibasic acids have low content of acylglycerol esters (neutral fats) impurities.

Wherein the acylglycerides include diacylglycerides (i.e., DAG) and triacylglycerides (i.e., TAG). The diacylglycerides are compounds consisting of one molecule of glycerol and two molecules of fatty acid, and the triacylglycerides are compounds consisting of one molecule of glycerol and three molecules of fatty acid.

Preferably, the diacylglycerides are C ₃₉ H ₇₂ O ₅ Wherein said triacylglycerol is C ₅₇ H ₁₀₄ O ₆ 。

Preferably, the fatty acid is oleic acid.

Further, the engineering bacteria are utilized to produce the long-chain dicarboxylic acid through fermentation, and the content of impurities of diacyl glyceride and triacylglycerol in the fermentation liquor after the fermentation is finished is reduced by at least 10%, preferably at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher compared with the content of the microorganism (namely the long-chain dicarboxylic acid production strain before modification) with the SCT1 gene not knocked out.

Further, the total content of diacylglyceride and triacylglycerol in the obtained long-chain dibasic acid is 0.3ppm or less.

Preferably, when the long-chain dibasic acid produced by fermentation is a long-chain dibasic acid with twelve carbon atoms, the total content of the diacylglycerol ester and the triacylglycerol ester in the obtained long-chain dibasic acid is less than 0.3 ppm; wherein the content of DAG impurity is lower than 0.1ppm, and the content of TAG impurity is lower than 0.2 ppm.

Preferably, when the diacylglyceride is C ₃₉ H ₇₂ O ₅ The triacylglycerol is C ₅₇ H ₁₀₄ O ₆ When C is in contact with ₃₉ H ₇₂ O ₅ And C ₅₇ H ₁₀₄ O ₆ The total content of (B) is 0.3ppm or less, preferably 0.25ppm or less.

The feedstock for fermentative conversion comprises long-chain alkanes, fatty acids, fatty acid derivatives or mixtures thereof.

Preferably, the fermentation substrate of the present invention comprises long-chain alkane, which belongs to saturated chain hydrocarbon, is a saturated hydrocarbon under hydrocarbon, and the whole structure of the long-chain alkane is mostly only composed of carbon, hydrogen, carbon-carbon single bond and carbon-carbon single bond, and comprises the chemical formula CH ₃ (CH ₂ ) _n CH ₃ Wherein n.gtoreq.7. The n-alkanes are preferably C9-C22, and more preferably C9-C18, i.e., C9, C10, C11, C12, C13, C14, C15, C16, C17 or C18. Suitable fermentation substrates for the engineered bacteria 630 of the present invention include n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-hexadecane, n-heptadecane, or n-octadecane.

The foregoing method comprises the step of culturing the long-chain dicarboxylic acid-producing microorganism under conditions suitable for growth of the long-chain dicarboxylic acid-producing microorganism. The concentration of the fermentation substrate is controlled to be 0.1-17%, preferably 0.1-15%, more preferably 0.5-15% during fermentation, and the addition speed of the fermentation substrate can be adjusted during the fermentation process. The concentration of the fermentation substrate is controlled in a preferred range, so that the microorganisms are helped to fully utilize the fermentation substrate to produce the long-chain dibasic acid by fermentation, and the impurity content of the diacylglycerol and the triacylglycerol is further reduced.

In a fourth aspect, the present invention provides a long chain dibasic acid product having a substantially reduced content of DAG and TAG impurities, obtainable according to the above process.

By means of the technical scheme, the invention at least has the following advantages and beneficial effects:

compared with the long-chain dicarboxylic acid production strain before modification, the modified engineering bacteria have the advantages that the mass ratio of DAG and TAG impurities in the finally obtained long-chain dicarboxylic acid product is remarkably reduced, and the total content of DAG and TAG impurities can be reduced to be below 0.3 ppm. In addition, during the fermentation process, the concentration of the fermentation substrate is preferably controlled to be 0.1-17%. The reduction of DAG and TAG impurity content further improves the purity of the fermentation product long-chain dibasic acid, so that the dibasic acid product can be used as an important raw material of products such as engineering plastics, synthetic perfumes, cold-resistant plasticizers, high-grade lubricating oil, polyamide hot melt adhesives and the like, is more favorable for the production and manufacture of downstream products, and improves the quality of the downstream products. More importantly, the long-chain dibasic acid with low DAG and TAG impurity content greatly reduces the difficulty of the extraction and purification of the dibasic acid in the later period and the wastewater treatment process, simplifies the process and saves the energy consumption.

Drawings

FIG. 1 is a schematic flow chart of the method for knocking out a copy of SCT1 by homologous recombination in example 2 of the present invention.

FIG. 2 shows the alignment of amino acid sequences of 3-phosphoglycerate acyltransferase from different microbial species.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning Laboratory Manual, Sambrook, et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or following the conditions recommended by the manufacturer's instructions.

In the invention, the method for testing the contents of the dibasic acid, the alkane and the impurities can adopt a liquid chromatography-mass spectrometry combined technology.

Homologous genes refer to two or more gene sequences with 70% sequence similarity, including orthologous genes (also referred to as orthologous, or orthologous), transversely homologous genes (also referred to as paralogous, or paralogous), and/or heterologous homologous genes. The homologous gene of SCT1 in the invention can be the orthologous gene of SCT1 gene, and can also be the horizontal homologous gene or the heterologous homologous gene.

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 or polypeptide homology to a polynucleotide described herein.

PCR overlap extension is also called SOE (gene splicing by overlap extension) PCR, and refers to a method for 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 the present invention does not belong to site-specific recombination, and recombination relies on an intracellular DNA repair system.

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, 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 resistance gene, HYG.

In the process of fermentation production of the long-chain dicarboxylic acid, the fermentation medium at least comprises a carbon source, a nitrogen source, inorganic salts and optional nutrient salts. According to the common knowledge in the field of fermentation, the percentage of the added amount of the raw materials of the fermentation medium is the mass-to-volume ratio, namely w/v; % means g/100 mL.

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

Optionally, the nitrogen source comprises one or more selected from peptone, yeast extract, corn steep liquor, ammonium sulfate, urea and potassium nitrate; and/or the total amount of the nitrogen source added is 0.1-3% (w/v), such as 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.5%.

Optionally, the inorganic salt comprises one or more selected from 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), such as 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%.

Optionally, the trophic factors include one or more selected from 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%.

The OD value in the present invention is a value measured when the optical density of the bacterial cells is diluted 30-fold in each of the following embodiments and examples, which are not particularly described.

The production method of the long-chain dibasic acid comprises a thallus growth period and a fermentation conversion period (acid production period).

The invention provides a method for producing long-chain dicarboxylic acid by fermentation, which is preferably carried out under the following conditions when long-chain alkane is taken as a substrate: the fermentation temperature is 28-32 ℃, the air volume is 0.3-0.7 vvm, the pressure is 0.05-0.14 MPa, the pH value of the thallus in the growth period is 3.5-6.5, and the pH value of the thallus in the conversion period is 5.0-8.0.

In a preferred embodiment of the invention, the Dissolved Oxygen (DO) during the fermentative conversion is not less than 15%.

In a preferred embodiment of the invention, the concentration of the fermentation substrate is controlled between 0.1% and 17%, preferably between 0.1% and 15%, when the long-chain dicarboxylic acid is produced by fermentation. Preferably, the concentration of the fermentation substrate is controlled within any one of the ranges of 0.1% to 0.5%, 0.5% to 1%, 0.5% to 2%, 1% to 3%, 2% to 4%, 3% to 5%, 4% to 6%, 5% to 7%, 6% to 8%, 7% to 9%, 8% to 10%, 9% to 11%, 10% to 12%, 11% to 13%, 12% to 14%, 13% to 15%, 14% to 16%, or 15% to 17%, or within any combination of the endpoints of these ranges, such as within the range of 1% to 10% of the concentration of 1% and 10% of the endpoint, or within the range of 2% to 12% of the concentration of 2% and 12% of the endpoint. The substrate concentration refers to the volume concentration of the fermentation substrate in the fermentation broth. By controlling the substrate concentration is meant controlling the volume concentration of the fermentation substrate in the fermentation broth throughout the fermentation process. The concentration of the substrate can be controlled by adjusting the adding speed of the fermentation substrate in the fermentation process and detected by gas chromatography-mass spectrometry.

In a preferred embodiment of the invention, the fermentation substrate can be added in a batch or continuous feed.

In a preferred embodiment of the present invention, the process for producing the long-chain dicarboxylic acid may comprise the following processes:

the seed bottle culture process comprises the following steps:

inoculating the glycerol tube strain of the candida tropicalis into a seed bottle filled with a YPD liquid culture medium, and performing shake culture at 200-250 rpm for 1-2 days at the temperature of 28-32 ℃ under natural pH.

YPD medium, formula (w/v) is: 1-3% of peptone, 1-3% of glucose and 1-3% of yeast extract.

The seeding tank culture process comprises the following steps:

inoculating seeds obtained from a seed bottle into a seed tank filled with a seed culture medium, wherein the inoculation amount is 10-30% (v/v, relative to the initial volume of seed culture), the initial pH value of a fermentation system after inoculation is 6.0-6.8, the temperature is 28-32 ℃, the ventilation amount is 0.3-0.7 vvm, the tank pressure is 0.05-0.14 MPa, a certain stirring speed is kept to control the dissolved oxygen in the seed culture process to be not less than 10%, the culture is finished when the seeds are mature, and the standard of mature seed culture is OD after 30 times of dilution ₆₂₀ ≥0.5，OD ₆₂₀ Can be about 0.5 to 1.0, and can be about 0.8.

The formulation (w/v) of the seed culture medium is preferably: 10-20 g/L of sucrose, 3-8 g/L of yeast extract, 2-4 g/L of corn steep liquor for industrial fermentation and KH ₂ PO ₄ 4-12 g/L and 0.5-4 g/L urea (preferably sterilized separately at 115 ℃ for 20 min).

In some preferred embodiments of the invention, a fermentation substrate, preferably n-alkanes, including any of n-decane to n-nonadecane, may be added to the initial seed medium.

Fermentation process of the fermentation tank:

inoculating the seed liquid obtained by culturing in the seed tank into a fermentation tank containing a fermentation culture medium, wherein the inoculation amount is 10-30% (v/v, relative to the fermentation initial volume), the temperature in the fermentation process is controlled to be 28-32 ℃, the ventilation rate is about 0.3-0.7 vvm, the tank pressure (gauge pressure) is about 0.05-0.14 MPa, a certain stirring speed is kept, and the dissolved oxygen is controlled to be not less than 10%. Controlling the pH of the thallus in the growth period to be 3.5-6.5 until the optical density OD of the thallus is obtained ₆₂₀ When the pH value is more than 0.5, controlling the pH value in the conversion period to be 5.0-8.0 until the fermentation is finished. When the fermentation is carried out for 10-20 h, adding a fermentation substrate, and controllingThe concentration of the fermentation substrate in the fermentation liquid in the whole fermentation process is 0.1-17%, the total addition amount of the fermentation substrate is 300-500 mL/L (v/v, relative to the initial volume of fermentation), and the fermentation is finished when the fermentation substrate detects 0. And in the fermentation process, 1N HCl and 1NNaOH are used for adjusting the pH value to 5.0-8.0.

The formulation (w/v) of the fermentation medium is preferably: 10-40 g/L of sucrose, 1-5 g/L of corn steep liquor, 4-12 g/L of yeast extract, 0-3 g/L of NaCl and KNO ₃ 4～12g/L，KH ₂ PO ₄ 4-12 g/L and 0.5-3 g/L urea (preferably sterilized separately at 115 ℃ for 20 min).

In a preferred embodiment of the invention, a fermentation substrate, preferably n-alkanes, including any of n-decane to n-nonadecane, may be added to the fermentation medium. Fermentation substrates may also be added during the fermentation process.

In some preferred embodiments of the invention, the strain may be inoculated in an amount of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, 27%, 29%.

Extraction of long-chain dibasic acid: and (4) separating and extracting fermentation liquor obtained after fermentation to obtain a long-chain dicarboxylic acid finished product. The extraction step comprises acidifying the fermentation liquor or fermentation treatment liquid, and separating to obtain long-chain dicarboxylic acid.

The fermentation treatment liquid can be a mixed solution obtained by alkalization, decoloration, solid-liquid separation and the like of fermentation liquid. The fermentation treatment liquid may or may not include bacterial cells.

The acidification is to perform acidification treatment on the fermentation liquid or the fermentation treatment liquid, adjust the pH value of the fermentation liquid or the fermentation treatment liquid to 2-6.5, and preferably use inorganic acid such as sulfuric acid, hydrochloric acid, nitric acid or mixed acid thereof during acidification. Preferably the end point pH of the acidification is below 5.

In some embodiments of the present invention, the extracting step includes adjusting the pH of the long-chain dibasic acid fermentation solution to be 7 or more after the end of fermentation by adding alkali before acidification to obtain an alkali solution containing a long-chain dibasic acid salt or a fermentation treatment solution, and then performing solid-liquid separation. The solid-liquid separation includes filtration and/or centrifugal separation, and a commonly used solid-liquid separation apparatus may be used. For the alkalization treatment, sodium hydroxide, ammonia water or potassium hydroxide may be used.

In some embodiments of the present invention, the alkaline solution containing the long-chain dibasic acid salt is centrifuged by using a centrifugal device, and preferably, after centrifugation, the impurities containing the bacterial cells are continuously separated by using a filter membrane. Or treating the alkali solution containing the long-chain dibasic acid salt directly by using a filter membrane to separate impurities such as residual bacteria, large protein and the like from the solution. The filter membrane is preferably a ceramic membrane. When ceramic membrane is used for membrane filtration, the pressure before membrane is preferably 0.2-0.4 MPa; the pore diameter of the membrane core of the filtering membrane is preferably 0.05-0.2 micron.

Preferably, the step of extracting and purifying further comprises decolorizing the fermentation broth or fermentation treatment solution containing the long-chain dibasic acid salt, adding activated carbon into the fermentation broth or membrane clear solution containing the long-chain dibasic acid salt for decolorization, filtering to remove the activated carbon after the decolorization, wherein the decolorization step can further remove impurities in the long-chain dibasic acid solution. Preferably, the amount of activated carbon added is 0.1 to 10 wt%, preferably 1 to 8 wt% (relative to the amount of long chain dibasic acid contained in the solution). When the activated carbon is used for decoloring, the decoloring temperature is 60-100 ℃, and the decoloring time is 15-165 min.

Preferably, the obtained long-chain dicarboxylic acid precipitate is further purified, that is, the long-chain dicarboxylic acid precipitate is dissolved in an organic solvent, the long-chain dicarboxylic acid is crystallized by cooling, evaporation and dissolution, and then the crystal is separated, so that the purified long-chain dicarboxylic acid is obtained. 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 or formic acid; the ketone comprises acetone; the esters include ethyl acetate and/or butyl acetate. Preferably, the purification conditions are: preserving the heat at 70-100 ℃ for 30-180 minutes, cooling for crystallization, and then carrying out solid-liquid separation.

In a preferred embodiment of the present invention, the long-chain dibasic acid product obtained by the present invention can be further subjected to extraction treatment by referring to the refining process in example 1 of CN 101985416 a.

Example 1 culture Medium, culture fermentation method, and Long-chain dicarboxylic acid detection method

1. YPD medium, formula (w/v) is: 2% peptone, 2% glucose and 1% yeast extract (OXOID, LP 0021).

2. Seed culture medium, formula (w/v) is: sucrose (10 g/L), yeast extract (total nitrogen content 6.5 wt%, the same applies hereinafter) 3g/L, corn steep liquor for industrial fermentation (corn steep liquor for short, total nitrogen content 2.5 wt%), KH ₂ PO ₄ 4g/L, urea 0.5g/L (sterilized separately at 115 ℃ C. for 20 min).

3. The fermentation medium has the following formula (w/v): 10g/L of sucrose, 1g/L of corn steep liquor, 4g/L of yeast extract and KNO ₃ 4g/L，KH ₂ PO ₄ 4g/L, urea 0.5g/L (115 ℃,20 min single sterilization).

4. Extraction of long-chain dibasic acid:

(1) firstly, adjusting the pH value of the fermentation liquor to 8.5 by using a sodium hydroxide solution with the mass concentration of 30%, adding water to adjust the concentration of the long-chain dibasic acid to 8.5 wt%, heating to 50 ℃, and filtering the fermentation liquor by using a ceramic membrane with the pore diameter of 0.1 micrometer. The membrane area of the ceramic membrane used was 0.84m ² And setting the pressure before the membrane to be 0.3MPa, and collecting membrane clear liquid. (2) 7 wt% of powdered activated carbon is added into the collected membrane clear liquid for decolorization for 1h at 60 ℃, and a clear liquid is obtained by filtration. (3) And adding sulfuric acid into the clear liquid, adjusting the pH value to 3.5, cooling to room temperature, filtering to obtain a wet solid, washing a filter cake with purified water with the weight of 3 times that of the wet solid, filtering, and drying to obtain a corresponding long-chain dicarboxylic acid primary product. (4) Adding 95% acetic acid into the corresponding long-chain dicarboxylic acid product, heating to dissolve, adding 1 wt% macroporous powder activated carbon for decolorization, decolorizing at 85 deg.C for 1h, filtering to obtain clear solution, cooling to 30 deg.C, washing with water, and oven drying to obtain the long-chain dicarboxylic acid product.

5. Liquid chromatography-mass spectrometry combined technology for detecting DAG (C) ₃₉ H ₇₂ O ₅ ) And TAG (C) ₅₇ H ₁₀₄ O ₆ ) Method for impurity content and acid yield

Liquid chromatograph: agilent 1290 Infinity II LC (Cat No. 54983)

And (3) chromatographic column: agilent Elipse Plus-C18 (2.1X 50mm, 1.8. mu.M)

The method comprises the following steps: the initial temperature is 100 ℃, the temperature is raised to 230 ℃ at the speed of 15 ℃/min, and the temperature is kept for 2 min. The carrier gas is hydrogen, the injection port temperature is 280 ℃, the FID temperature is 280 ℃, and the injection amount is 4 mu L.

And (4) calculating the yield of the dibasic acid according to an internal standard method, and calculating the content of the impurities according to the peak area of the dibasic acid product and the peak area of the impurities.

The mass spectrum parameters were as follows: the ion mode is APCI, Negative, gas temperature 325 ℃, drying gas flow 10L/min, atomizing gas 40psig, sheath gas temperature 350 ℃, sheath gas flow 12L/min, capillary voltage 4000V, nozzle voltage 150V, scanning mode: 3 times/s.

And calculating the purity and the impurity content of the product according to the peak area of the dibasic acid product and the peak area of the impurity.

Example 2 cloning of the SCT1 Gene

1. Total RNA extraction and transcriptome sequencing

Mixing CCTCC NO: single colonies of M2011192 Candida tropicalis were inoculated into 2ml centrifuge tubes containing 1ml of YPD medium (containing 100mg/L hygromycin B) described in example 1 and shake-cultured at 250rpm at 30 ℃ for 1 day. The bacterial solution was inoculated into a 500mL shake flask containing 30mL of the seed medium of example 1 (containing 100mg/L hygromycin B), the inoculum size was 3%, and the mixture was cultured at 250rpm and 30 ℃ to OD ₆₂₀ Up to 0.8. The seed solution was inoculated into a 500mL shake flask containing 15mL of the fermentation medium described in example 1, at an inoculum size of 20%. After culturing at 250rpm and 30 ℃ for 36h, the fermentation was terminated, and the mixture was centrifuged at 5000rpm for 5min (Jouman BR4i, rotor AB50.10A) to collect the bacterial solution. The fermentation substrate in the fermentation medium was 400mL/L n-dodecane. And in the culture process, the pH value is adjusted to 7.5-7.6 by intermittently supplementing 1N HCl and 1N NaOH.

RNA extraction was performed using TRNzol univarial Reagent (Tiangen) kit, and grinding the disrupted cells with liquid nitrogen. Transcriptome sequencing adopts Miseq (illumina) platform and double-end sequencing method to obtain 20M Reads with length of 2 × 251 bp. After the measured Reads had removed the linker and filtered the low quality bases and Reads with CutAdpt (v1.1.6), Unigene was assembled with Trinity software (http:// trinityareq. sf. net) and functionally annotated with the Non-Redundant protein database at NCBI.

2. Bioinformatic analysis

The resulting Unigene was pooled using the local Blast (Blast +2.7.1) method and the candidate genes were searched for alignments by tblastn using known glycerol triphosphate acyltransferase genes from Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida tropicalis (Candida tropicalis MYA3404), Yarrowia lipolytica (Yarrowia lipolytica) and Candida albicans (Candida albicans) as the query. Finally, a candidate CtSCT1 gene is screened by analysis, and the partial CDS sequence is shown as SEQ ID NO. 16. The accession numbers of glycerol-3-phosphate acyltransferase genes in Genbank in different microorganisms are Saccharomyces cerevisiae (EWG87580), Candida tropicalis MYA3404 (XP-002548333), Yarrowia lipolytica (XP-501275) and Candida albicans (KGU30921), respectively.

EXAMPLE 3 preparation of homologous recombination templates

In this example, Takara was used for all DNA fragments

HS high fidelity DNA polymerase (Takara, R040A). The purified DNA fragment was recovered by an Axygen gel recovery kit (Axygen, AP-GX-250G) after electrophoresis in 1% agarose gel.

1. Amplification of upstream and downstream homology arms of SCT1 gene

Extraction of yeast (strain preservation number: CCTCC NO: M2011192) genome DNA adopts an Ezup yeast genome DNA rapid extraction kit (product number 518257, manufactured and biological engineering (Shanghai) GmbH) and is assisted with a liquid nitrogen grinding method to improve the wall breaking efficiency. Mu.g of genome was added per 50. mu.L reaction as a template for PCR amplification. The primers used for the upstream homology arm amplification are as follows:

SCT_UP-F：

5’-GAGCGACCAAAGCGATTCAG-3’(SEQ ID NO:1)

SCT_UP-R:

5’-TTTGCCATCGTTCCAACAGC-3’(SEQ ID NO:2)

the primers used for the downstream homology arm amplification are as follows:

SCT_DOWN-F：

5’-ATGAGTGACGGGGTCTCCTT-3’(SEQ ID NO:3)

SCT_DOWN-R:

5’-ATGAGTGACGGGGTCTCCTT-3’(SEQ ID NO:4)

the PCR reaction conditions were as follows: 30s at 98 ℃; 30 cycles of 98 ℃ for 10s,55 ℃ for 10s, and 72 ℃ for 30 s; 5min at 72 ℃.

The obtained products are amplified into SCT _ UP and SCT _ DOWN which are proved to be error-free by sequencing, and the sequences are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6.

2. Amplification of the resistance selection marker (HYG, i.e., the hygromycin resistance gene), the amplification template being the vector pCIB2(SEQ ID NO:7), the primer sequences and PCR reaction conditions were as follows:

SCT_HYG-F：

5’-GCTGTTGGAACGATGGCAAAGCATGCGAACCCGAAAATGG-3’(SEQ ID NO:8)

SCT_HYG-R：

5’-AAGGAGACCCCGTCACTCATGCTAGCAGCTGGATTTCACT-3’(SEQ ID NO:9)

the PCR reaction conditions were as follows: 30s at 98 ℃; 10s at 98 ℃, 10s at 55 ℃, 1min at 72 ℃ for 50s, and 5 cycles; 10s at 98 ℃, 2min at 72 ℃ and 25 cycles; 5min at 72 ℃.

The obtained amplification product is called HYG, and the sequence of the HYG is shown in SEQ ID NO. 10.

3. PCR overlap extension to obtain complete recombinant template

And (3) performing overlapping extension on 3 recovered and purified PCR fragments of SEQ ID NO 5, 6 and 10 to obtain a homologous recombination template, and recovering and purifying. The specific method comprises the following steps:

adding equimolar amount of SCT _ UP, SCT _ DOWN and HYG fragments as template, primers SCT _ UP-F and SCT _ Down-R, and using

Performing PCR overlap extension by using HS high-fidelity DNA polymerase, wherein the PCR reaction conditions are as follows: 30s at 98 ℃; 10s at 98 ℃, 10s at 55 ℃, 2min at 72 ℃ for 30s, and 20 cycles; and 8min at 72 ℃.

The gel electrophoresis was followed by recovery and purification of a recombinant fragment of about 2.2Kb in size, the sequence of which is shown in SEQ ID NO 11.

FIG. 1 is a schematic flow chart of the process of knocking out a copy of SCT1 by homologous recombination.

Example 4 transformation of recombinant transformants

1. Preparation of Yeast electrotransformation competent cells

The yeast cells cultured overnight at 30 ℃ and 250rpm in a shaking table have the CCTCC NO: m2011192 was inoculated into 100mL YPD medium of example 1 to OD ₆₂₀ Is 0.1. Cultured to OD under the same conditions ₆₂₀ When the temperature reached 1.3, cells were collected by centrifugation at 3000g and 4 ℃. After washing the cells twice with ice-cold sterile water and harvesting, the cells were resuspended in 10ml of 1M sorbitol solution pre-cooled on ice, after centrifugation at 1500g at 4 ℃ and harvesting, the cells were resuspended in 1ml of the above sorbitol solution, and 100. mu.L of the cell suspension was aliquoted for genetic transformation.

2. Yeast competent electroporation transformation

Mu.g of the recovered and purified DNA fragment SEQ ID NO:11 was added to the competent cells, and after 5min on ice, the cells were quickly transferred to a 0.2cm cuvette and transformed by electric shock (BioRad, Micropulser. TM. electroporation, transformation program SC2, 1.5kV, 25uFD, 200 ohms). Quickly adding 1mL of mixed solution of YPD and 1M sorbitol (1:1, v/v), culturing at 30 ℃ and 200rpm for 2h, collecting bacterial solution, coating a YPD medium plate containing 100mg/L hygromycin B, and performing static culture at 30 ℃ for 2-3 days until a single colony grows out.

Example 5 screening of recombinant transformants

The single colony obtained in example 3 was inoculated into 1ml YPD medium (containing 100mg/L hygromycin B) of example 1 in a 2ml centrifuge tube, and cultured overnight at 30 ℃ and 250 rpm. The colony PCR identification is carried out the next day, and the primer sequences and the PCR reaction conditions are as follows:

the primer sequences for identifying the SCT1 gene are as follows:

SCT-1F:5’-AAGATCCCCAGAGAGGGTCC-3’(SEQ ID NO:12)

SCT-1R:5’-ATGCACCCATCATCAGCCAA-3’(SEQ ID NO:13)

the primer sequences for identifying homologous recombination are as follows:

HYG-2F:5’-TGTGCTACCCACGCTTACT-3’(SEQ ID NO:14)

SCT1-Down-2R:5’-ACTGACTGTCCATTCTGTTCTCTC-3’(SEQ ID NO:15)

the PCR reaction conditions were as follows: 30s at 98 ℃; 30 cycles of 98 ℃ for 10s,55 ℃ for 10s, and 72 ℃ for 40 s; 5min at 72 ℃.

The two groups of PCR primers are amplified to respectively obtain strains with fragments of about 500bp and 600bp, namely the strains with one copy of the SCT1 gene knocked out, and the strains are named as 630.

EXAMPLE 6 production of Dodecarbyl Long chain dibasic acid by recombinant Strain in Shake flask fermentation (DC12)

Single colonies of strain 630 were picked and inoculated into 2ml centrifuge tubes containing 1ml YPD medium (containing 100mg/L hygromycin B) as described in example 1 and shake-cultured at 30 ℃ and 250rpm for 1 day. The bacterial solution was inoculated into a 500mL shake flask containing 30mL of the seed medium (containing 100mg/L hygromycin B) described in example 1, the inoculum size was 3%, and shake cultivation was carried out at 250rpm and 30 ℃ until OD was reached ₆₂₀ Up to 0.8. The seed solution was inoculated into a 500mL shake flask containing 15mL of the fermentation medium described in example 1 at an inoculum size of 20% and continued shake cultivation at 250rpm and 30 ℃ until the end of the fermentation. The fermentation substrate in the fermentation medium was 400mL/L n-dodecane. And in the culture process, the pH value is adjusted to 7.5-7.6 by intermittently supplementing 1N HCl and 1N NaOH.

Meanwhile, the strain CCTCC NO: m2011192 as a control: the cultivation and fermentation process was the same as above except that the medium did not contain hygromycin B. The results of the acid yield of DC12 in the fermentation broth are shown in table 1.

The method for extracting the dibasic acid product after the fermentation is finished is as described in step 4 of example 1. The content of DAG and TAG impurities in the finished diacid product is detected as described in example step 5, and the results are shown in Table 1.

TABLE 1

As can be seen from table 1, the total amount of impurities DAG and TAG in the strain in which one copy of SCT1 gene was knocked out was reduced by about 18.5% and the amount of acid produced was slightly increased, as compared to the control group.

EXAMPLE 7 production of undecanoic Long-chain dibasic acid by strain 630 shake flask fermentation (DC11)

The fermentation conditions, extraction and purification, and DAG and TAG content detection methods were all the same as those in example 5 except that the fermentation substrate in the fermentation medium was 300mL/L n-undecane.

The detection results of the acid yield of DC11 in the fermentation liquid and the contents of DAG and TAG in the impurities of the finished product of the dibasic acid are shown in Table 2.

TABLE 2

As can be seen from table 2, the total amount of impurities DAG and TAG in the strain in which one copy of SCT1 gene was knocked out was reduced by about 18.0% and the amount of acid produced was slightly increased, as compared to the control group.

EXAMPLE 8 production of hexadecane long chain dicarboxylic acid (DC16) by Strain 630 Shake flask fermentation

The fermentation conditions, extraction and purification, and DAG and TAG content detection methods were all the same as those in example 5 except that the fermentation substrate in the fermentation medium was 500mL/L n-hexadecane.

The acid yield of DC16 in the fermentation liquid and the detection results of the contents of DAG and TAG as impurities in the finished product of the dibasic acid are shown in Table 3.

TABLE 3

As can be seen from table 3, the total amount of impurities DAG and TAG in the strain in which one copy of SCT1 gene was knocked out was reduced by about 19.4% and the amount of acid produced was slightly increased, as compared to the control group.

From the above experimental results of the fermentation production of long-chain dibasic acids in examples 6, 7 and 8 in the shake flask stage for different fermentation substrates, it can be seen that the acid yield for different fermentation substrates such as n-dodecane, n-undecane and n-hexadecane is slightly increased under the same fermentation conditions, and the content of DAG as an impurity and TAG in the product after the extraction of the finished product of dibasic acid is reduced by at least 18% compared to the parent strain.

EXAMPLE 9 Strain 630 production of dodecanedioic acid (DC12) by fermentation at 10L tank scale

The glycerol tube strain of strain 630 was inoculated into a seed flask containing YPD liquid medium described in example 1, and shake-cultured at 28 ℃ and 200rpm for 1 day under a natural pH.

Inoculating the shake flask seeds into a seed tank filled with the seed culture medium (containing 100mg/L hygromycin B) described in example 1, wherein the inoculation amount is 10%, the initial pH value of a fermentation system after inoculation is 6.0, the ventilation rate is 0.3vvm at 28 ℃, the tank pressure is 0.14MPa, a certain stirring speed is kept to control the dissolved oxygen in the seed culture process to be not less than 10%, the seeds are cultured for 15h until the seeds are mature, and the OD is diluted by 30 times ₆₂₀ Is 0.8.

The seed solution obtained by the seed tank culture was inoculated into a fermenter containing the fermentation medium described in example 1, the initial volume of fermentation after inoculation was 4L, the inoculum amount was 10% (v/v, relative to the initial volume of fermentation), 30ml (v/v, relative to the initial volume of fermentation) of n-dodecyl alkane was added at the beginning of fermentation, the temperature of fermentation was controlled at 29 ℃, the aeration rate was about 0.6vvm, the pressure (gauge pressure) of the tank was about 0.1MPa, and the stirring speed was maintained at a constant rate so as to control the dissolved oxygen content to be not less than 10%. Controlling the pH of a fermentation system of the thallus in the growth period to be 4.4-4.6 until the optical density OD of the thallus ₆₂₀ And (4) controlling the pH value of the fermentation system in the conversion period to be 7.5-7.6 until the fermentation is finished, wherein the pH value is more than 0.5. When the fermentation is carried out for 15 hours, adding n-dodecane in batches, controlling the volume concentration of the n-dodecane in the fermentation liquid in the whole fermentation process within four concentration ranges of 0.1-0.5%, 1-3%, 8-10% and 15-17%, wherein the total alkane addition is 400mL/L, and finishing the fermentation when the alkane is detected to be 0. The results of the acid production by DC12 in the fermentation broth are shown in Table 4.

The extraction of the product after fermentation was as described in step 4 of example 1. The content of DAG and TAG as impurities in the finished diacid product is detected as described in step 5 of example 1, and the results are shown in Table 4.

TABLE 4

Substrate concentration (%)	0.1～0.5	1～3	8～10	15～17
					Acid yield (mg/g) of DC12 in fermentation broth	145.5	157.8	158.0	125.6
DAG content (C) 39 H 72 O 5 ，ppm)	0.053	0.079	0.088	0.109
					TAG content (C) 57 H 104 O 6 ，ppm)	0.084	0.118	0.135	0.184
Total (DAG + TAG, ppm)	0.137	0.197	0.223	0.293
					Fermentation time (h)	188	164	167	177

As can be seen from Table 4, the lower the concentration of the fermentation substrate is controlled, the lower the DAG and TAG impurities are.

Example 10 fermentation of Strain 630 on a 10L Scale to produce undecanedioic acid (DC11)

The glycerol tube strain of strain 630 was inoculated into a seed flask containing YPD liquid medium described in example 1, and shake-cultured at 220rpm at 30 ℃ for 2 days with natural pH.

Inoculating shake flask seeds into a seed tank filled with the seed culture medium (containing 100mg/L hygromycin B) described in example 1, wherein the inoculation amount is 30%, the initial pH value of a fermentation system after inoculation is 6.5, the ventilation rate is 0.3vvm at 30 ℃, the tank pressure is 0.14MPa, a certain stirring speed is kept, the DO in the seed culture process is controlled to be not less than 10%, the seeds are cultured for 20h, and the standard for culturing mature seeds is OD after 30 times of dilution ₆₂₀ Is 1.0.

The seed solution obtained by the seed tank culture was inoculated into a fermentation tank containing the fermentation medium described in example 1, the initial fermentation volume after inoculation was 5L, the inoculum amount was 20% (v/v, relative to the initial fermentation volume), the fermentation process was controlled at 30 ℃ with the aeration rate of about 0.3vvm and the tank pressure (gauge pressure) of about 0.14MPa, and the agitation speed was maintained with the dissolved oxygen being not less than 10%. Controlling the pH of the fermentation system of the thallus in the growth period to be 5.5 until the optical density OD of the thallus ₆₂₀ And when the pH value is more than 0.5, controlling the pH value of the fermentation system in the conversion period to be 7.4-7.5 until the fermentation is finished. When sending outAnd (3) starting to add n-undecane when the fermentation time is 10 hours, controlling the volume concentration of the n-undecane in the fermentation liquor in the whole fermentation process to be in four concentration ranges of 0.1-0.5%, 1-3%, 8-10% and 15-17%, wherein the total alkane addition is 300mL/L, and ending the fermentation when the alkane is detected to be 0. The results of the acid production of DC11 in the fermentation broth are shown in table 5.

The extraction of the product after fermentation was as described in step 4 of example 1. The content of DAG and TAG as impurities in the finished diacid product is detected as described in step 5 of example 1, and the results are shown in Table 5.

TABLE 5

As can be seen from Table 5, the lower the concentration of the fermentation substrate is controlled, the lower the DAG and TAG impurities are.

EXAMPLE 11 fermentation of Strain 630 on a 10L Scale to produce hexadecanedioic acid (DC16)

Inoculating the strain of Candida tropicalis into seed bottle containing YPD liquid culture medium, and shake culturing at 32 deg.C and 250rpm for 2 days under natural pH.

Inoculating shake flask seeds into a seed tank filled with the seed culture medium (containing 100mg/L hygromycin B) described in example 1, wherein the inoculation amount is 30%, the initial pH value of a fermentation system after inoculation is 6.5, the ventilation rate is 0.7vvm at 32 ℃, the tank pressure is 0.1MPa, a certain stirring speed is kept to control the dissolved oxygen in the seed culture process to be not less than 10%, the seeds are cultured for 30 hours until the seeds are mature, and the OD (optical density) is 30 times after dilution ₆₂₀ Is 0.9.

Inoculating the seed solution obtained by seed tank culture into a fermentation tank containing the fermentation medium described in example 1, wherein the initial fermentation volume after inoculation is 6L, the inoculation amount is 30% (v/v, relative to the initial fermentation volume), 10% (v/v, relative to the initial fermentation volume) of n-hexadecane is added at the beginning of fermentation, the temperature of the fermentation process is controlled at 32 ℃, and the ventilation rate is about0.7vvm, a tank pressure (gauge pressure) of about 0.05MPa, a constant stirring speed, and a dissolved oxygen content of not less than 10%. Controlling the pH of a fermentation system of the thallus in the growth period to be 6.4-6.5 until the optical density OD of the thallus ₆₂₀ And when the pH value is more than 0.5, controlling the pH value of the fermentation system in the conversion period to be 7.5-7.6 until the fermentation is finished. And when the fermentation is carried out for 20 hours, adding n-hexadecane, controlling the volume concentration of the n-hexadecane in the fermentation liquor in the whole fermentation process to be in four concentration ranges of 0.1-0.5%, 1-3%, 8-10% and 15-17%, wherein the total alkane addition is 500mL/L, and finishing the fermentation when the alkane is detected to be 0. The results of the acid production of DC16 in the fermentation broth are shown in table 6.

The extraction of the product after fermentation was as described in step 4 of example 1. The content of DAG and TAG as impurities in the finished diacid product is detected as described in step 5 of example 1, and the results are shown in Table 6.

TABLE 6

Substrate concentration (%)	0.1～0.5	1～3	8～10	15～17
					Acid yield (mg/g) of DC16 in fermentation broth	129.7	141.5	143.0	129.5
DAG impurity content (C) 39 H 72 O 5 ，ppm)	0.045	0.077	0.084	0.108
					TAG impurity content (C) 57 H 104 O 6 ，ppm)	0.085	0.113	0.124	0.169
Total (DAG + TAG, ppm)	0.130	0.190	0.208	0.277
					Fermentation time (h)	190	168	170	178

As can be seen from Table 6, the lower the concentration of the fermentation substrate is controlled, the lower the DAG and TAG impurities are.

From the experimental results in tables 4-6, it can be known that when the concentration of the fermentation substrate is controlled to be too low, such as 0.1% -0.5%, the content of impurities is low, but under the condition of the same total alkane addition, the fermentation time is relatively long, and the fermentation efficiency is reduced; when the concentration of the fermentation substrate is controlled within the range of 0.5-15%, the effects in the aspects of reducing the impurity content, ensuring the fermentation efficiency and acid yield are better; when the concentration of the fermentation substrate is controlled within the range of 15-17%, the acid production amount is reduced, and the concentration may be too high, for example, more than 15% can inhibit the acid production of cells.

EXAMPLE 12 construction of other recombinant strains and production of Dodecanedioic acid by Shake flask fermentation (DC12)

The 2.2Kb recombinant DNA fragment (SEQ ID NO:11) described in example 3 was constructed into Candida tropicalis (CCTCC: M203052) using the homologous recombination method, which was the same as in example 3. The method for transforming and screening the recombinant was the same as in examples 4 and 5, and the recombinant obtained by screening was named 631.

The method for producing the twelve-carbon long-chain dicarboxylic acid (DC12) by the shake flask fermentation of the recombinant strain 631 is the same as that in example 6, and the control strain is CCTCC NO: m203052. The results showed that the amounts of impurities DAG and TAG in the dibasic acid product were measured as described in step 5 of example 1 in strain 631 compared with the parental strain, and the results are shown in Table 7. The total content of DAG and TAG impurities was reduced by about 23.6% compared to the control group.

TABLE 7

Strain of bacillus	CCTCC NO：M 203052	631
			Acid yield (mg/g) of DC12 in fermentation broth	136.4	137.7
DAG content (C) 39 H 72 O 5 ，ppm)	0.118	0.091
			TAG content (C) 57 H 104 O 6 ，ppm)	0.187	0.142
Total (DAG + TAG, ppm)	0.305	0.233

Example 12 shows that an engineered strain obtained by knocking out a copy of the SCT1 gene in the genome of another Candida tropicalis (Candida tropicalis) by homologous recombination can significantly reduce the mass ratio of DAG to TAG impurities in a fermentation broth after completion of fermentation, as compared with a long-chain dibasic acid-producing strain before the engineering.

Example 13 fermentation of Pre-engineered strains to produce Dodecadioic acid on a 10L tank Scale

The glycerol tube strain of the original strain (CCTCC NO: M2011192) before transformation is inoculated into a seed bottle filled with YPD liquid culture medium described in example 1, the pH is natural, and the strain is subjected to shake culture at the temperature of 28 ℃ and the rpm of 200 for 1 day.

The cultivation method of the seed tank and the fermenter was the same as in example 9. In the fermentation process, when the fermentation is started for 15 hours, n-dodecane is added in batch, the volume concentration of the n-dodecane in the fermentation liquid in the whole fermentation process is controlled to be in four concentration ranges of 0.1-0.5%, 1-3%, 8-10% and 15-17%, the total alkane addition is 400mL/L, and the fermentation is finished when the alkane is detected to be 0. The results of the acid yield of DC12 in the fermentation broth are shown in table 8.

The extraction of the product after fermentation was as described in step 4 of example 1. The content of DAG and TAG as impurities in the finished diacid product is detected as described in step 5 of example 1, and the results are shown in Table 8.

TABLE 8

Substrate concentration (%)	0.1～0.5	1～3	8～10	15～17
					Acid yield (mg/g) of DC12 in fermentation broth	141.3	154.8	155.0	123.3
DAG content (C) 39 H 72 O 5 ，ppm)	0.064	0.099	0.112	0.124
					TAG content (C) 57 H 104 O 6 ，ppm)	0.099	0.136	0.183	0.215
Total (DAG + TAG, ppm)	0.163	0.235	0.295	0.339
					Fermentation time (h)	190	168	170	179

As can be seen from Table 8, the lower the concentration of the fermentation substrate is, the lower the contents of DAG and TAG impurities are, but the fermentation time is relatively long, while the concentration of the fermentation substrate is controlled to be 0.5% -15%, so that the effect is relatively better in the aspects of reducing the impurity content, ensuring the fermentation efficiency and acid yield. Comparing the results of example 13 with those of example 9, it is clear that the modified strain obtained lower levels of impurities. The reduction of the impurity content reduces the difficulty and the production cost of the later extraction and purification process on one hand, and is more beneficial to the production and the manufacture of downstream nylon, plastic, polyamide hot melt adhesive and other products, and the product quality is improved on the other hand.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Shanghai Kaiser Biotechnology research and development center, Inc

Kaisai Biological Industry Co.,Ltd.

Application of <120> SCT1 gene in production of long-chain dicarboxylic acid

<130> KHP181116613.8

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gagcgaccaa agcgattcag 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tttgccatcg ttccaacagc 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgagtgacg gggtctcctt 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgagtgacg gggtctcctt 20

<210> 5

<211> 271

<212> DNA

<213> Candida tropicalis (Candida tropicalis)

<400> 5

gagcgaccaa agcgattcag gttggtcatt tacgactgct tgctctggct cttgtcagtg 60

atatttgatt gttttttcag agagatcaga ccgagagggg cgtttaagat ccccagagag 120

ggtccggtga tctttgttgc cgccccccat cataatcagt ttgttgatcc catcgtgttg 180

atgaaccagg tgaaaagaga agcaaaccga agaatatcat tcttgattgc ggccaagtca 240

tatcaattga aagctgttgg aacgatggca a 271

<210> 6

<211> 267

<212> DNA

<213> Candida tropicalis (Candida tropicalis)

<400> 6

atgagtgacg gggtctcctt attaaactca gataattctt tgacaaatct cccaatgttt 60

tcggattacg tgttgcacaa gaacgcaaag aacccggact tggagcttga tccccaatct 120

ttggcggcat cgagagtcaa ttcgatggtc aacatacctc atgcttctca tggcgttaat 180

acgccatcac cgtcaacttc aagacgccca ccattgaccg aaaccgcctc gtcggagcat 240

atggaattga actttgggtc tggagcc 267

<210> 7

<211> 5873

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120

cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180

tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcggtct 240

agtatgattg tcaataatga tgggtcatcg tttcctgatt cgacgttccc tgtggtgtcg 300

ttaaatagcc tgtctgaaat ctcctccatg attgtgttgg tgtgtgttgt ttgactttcc 360

caattgctta catttttttc ttcaaggatt cgctccaaaa tagacagaaa ttatcgcgac 420

aagtcagacg aacgtcgcac gaggcgaacc aaattcttta gaagcatacg aaaactcact 480

ttatttccat tagaagtatt aaattaacaa atatataata tacaggatac aaagtaaaag 540

cacgcttaag caaccaaagc ggaagcggta gcggattcgt atttccagtt aggtggcaag 600

acagcgacgg ttctgtagta tctggccaat ctgtggattc tagattcaat caaaatcaat 660

ctgaacttgg agtccttgtc ctttctgttt ctttccaagt gctttctgac agagacagcc 720

ttcttgatca agtagtacaa gtcttctggg atttctggag ccaaaccgtt ggatttcaag 780

attctcaaga tcttgttacc agtgacaacc ttggcttggg aaacaccgtg agcatctctc 840

aagataacac caatttgaga tggagtcaaa ccctttctgg cgtacttgat gacttgttca 900

acaacttcgt cagaagacaa cttgaaccaa gatggagcgt ttcttgagta tggaagagcg 960

gaggaggaaa tacctttacc ctaaaataac aagagctaat gttagtaatt tgaaaaaaaa 1020

gacgttgagc acgcacaccc catccacccc acaggtgaaa cacatcaaac gtagcaagaa 1080

caatagttgg ccctcccgtc aagggggcag gtaattgtcc aagtacttta gaaaagtatg 1140

tttttaccca taagatgaac acacacaaac cagcaaaagt atcaccttct gcttttcttg 1200

gttgaggttc aaattatgtt tggcaataat gcagcgacaa tttcaagtac ctaaagcgta 1260

tatagtaaca attctaggtc tgtatagtcg accgtaggtg aatcgtttac tttaggcaag 1320

accttgtccc tgataaagcc aggttgtact ttctattcat tgagtgtcgt ggtggtggta 1380

gtggtggttg attgggctgt tgtggtagta gtagtggttg tgatttggaa catacagatg 1440

aatgcatacg acccatgatg actgatttgt ttctttattg agttgatggt aagaaagaga 1500

agaagaggag gtaaaaaggt ggtagagtga aaaatttttt tctcttaaaa gtgagagaga 1560

gaaagagaaa aatttcactg cgaaacaaat ggttggggac acgacttttt tcaggaattt 1620

ttactcgaag cgtatatgca ggaaagttgt tgttagggaa tatggagcca caagagagct 1680

gcgaattcga gctcggtacc cggggatcct ctagagtcga cctgcaggca tgcgaacccg 1740

aaaatggagc aatcttcccc ggggcctcca aataccaact cacccgagag agagaaagag 1800

acaccaccca ccacgagacg gagtatatcc accaaggtaa gtaactcagg gttaatgata 1860

caggtgtaca cagctccttc cctagccatt gagtgggtat cacatgacac tggtaggtta 1920

caaccacgtt tagtagttat tttgtgcaat tccatgggga tcaggaagtt tggtttggtg 1980

ggtgcgtcta ctgattcccc tttgtctctg aaaatctttt ccctagtgga acactttggc 2040

tgaatgatat aaattcacct tgattcccac cctcccttct ttctctctct ctctgttaca 2100

cccaattgaa ttttcttttt ttttttactt tccctccttc tttatcatca aagataagta 2160

agtttatcaa ttgcctattc agaatgaaaa agcctgaact caccgcgacg tctgtcgaga 2220

agtttctcat cgaaaagttc gacagcgtct ccgacctcat gcagctctcg gagggcgaag 2280

aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctccgg gtaaatagct 2340

gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc 2400

cgattccgga agtgcttgac attggggaat tcagcgagag cctcacctat tgcatctccc 2460

gccgtgcaca gggtgtcacg ttgcaagacc tccctgaaac cgaactcccc gctgttctcc 2520

agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt 2580

tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg 2640

cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt 2700

ccgtcgcgca ggctctcgat gagctcatgc tttgggccga ggactgcccc gaagtccggc 2760

acctcgtgca cgcggatttc ggctccaaca atgtcctcac ggacaatggc cgcataacag 2820

cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct 2880

tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc 2940

atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc 3000

aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat 3060

gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa 3120

gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc 3180

ccagcactcg tccgagggca aaggaatagt gtgctaccca cgcttactcc accagagcta 3240

ttaacatcag aaatatttat tctaataaat aggatgcaaa aaaaaaaccc cccttaataa 3300

aaaaaaaaga aacgattttt tatctaatga agtctatgta tctaacaaat gtatgtatca 3360

atgtttattc cgttaaacaa aaatcagtct gtaaaaaagg ttctaaataa atattctgtc 3420

tagtgtacac attctcccaa aatagtgaaa tccagctgct agcgtgtaag cttggcactg 3480

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 3540

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 3600

tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg 3660

catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc 3720

gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 3780

ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 3840

aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 3900

ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 3960

aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 4020

atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 4080

caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 4140

cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 4200

tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 4260

tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 4320

gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 4380

tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 4440

gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 4500

aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 4560

gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 4620

atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 4680

caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 4740

ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 4800

attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 4860

agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 4920

aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 4980

catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 5040

ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 5100

tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 5160

ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 5220

ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 5280

ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 5340

gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 5400

aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 5460

acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 5520

gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 5580

gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 5640

cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 5700

aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 5760

gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 5820

cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga aga 5873

<210> 8

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gctgttggaa cgatggcaaa gcatgcgaac ccgaaaatgg 40

<210> 9

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aaggagaccc cgtcactcat gctagcagct ggatttcact 40

<210> 10

<211> 1776

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gctgttggaa cgatggcaaa gcatgcgaac ccgaaaatgg agcaatcttc cccggggcct 60

ccaaatacca actcacccga gagagataaa gagacaccac ccaccacgag acggagtata 120

tccaccaagg taagtaactc agagttaatg atacaggtgt acacagctcc ttccctagcc 180

attgagtggg tatcacatga cactggtagg ttacaaccac gtttagtagt tattttgtgc 240

aattccatgg ggatcaggaa gtttggtttg gtgggtgcgt ctactgattc ccctttgtct 300

ctgaaaatct tttccctagt ggaacacttt ggctgaatga tataaattca ccttgattcc 360

caccctccct tctttctctc tctctctgtt acacccaatt gaattttctt ttttttttta 420

ctttccctcc ttctttatca tcaaagataa gtaagtttat caattgccta ttcagaatga 480

aaaagcctga actcaccgcg acgtctgtcg agaagtttct catcgaaaag ttcgacagcg 540

tctccgacct catgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag 600

gagggcgtgg atatgtcctc cgggtaaata gctgcgccga tggtttctac aaagatcgtt 660

atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg 720

aattcagcga gagcctcacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag 780

acctccctga aaccgaactc cccgctgttc tccagccggt cgcggaggcc atggatgcga 840

tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg 900

gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact 960

ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctca 1020

tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca 1080

acaatgtcct cacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt 1140

tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta 1200

tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc 1260

tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca 1320

atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg 1380

ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg 1440

tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat 1500

agtgtgctac ccacgcttac tccaccagag ctattaacat cagaaatatt tattctaata 1560

aataggatgc aaaaaaaaaa ccccccttaa taaaaaaaaa agaaacgatt ttttatctaa 1620

tgaagtctat gtatctaaca aatgtatgta tcaatgttta ttccgttaaa caaaaatcag 1680

tctgtaaaaa aggttctaaa taaatattct gtctagtgta cacattctcc caaaatagtg 1740

aaatccagct gctagcatga gtgacggggt ctcctt 1776

<210> 11

<211> 2274

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gagcgaccaa agcgattcag gttggtcatt tacgactgct tgctctggct cttgtcagtg 60

atatttgatt gttttttcag agagatcaga ccgagagggg cgtttaagat ccccagagag 120

ggtccggtga tctttgttgc cgccccccat cataatcagt ttgttgatcc catcgtgttg 180

atgaaccagg tgaaaagaga agcaaaccga agaatatcat tcttgattgc ggccaagtca 240

tatcaattga aagctgttgg aacgatggca agcatgcgaa cccgaaaatg gagcaatctt 300

ccccggggcc tccaaatacc aactcacccg agagagataa agagacacca cccaccacga 360

gacggagtat atccaccaag gtaagtaact cagagttaat gatacaggtg tacacagctc 420

cttccctagc cattgagtgg gtatcacatg acactggtag gttacaacca cgtttagtag 480

ttattttgtg caattccatg gggatcagga agtttggttt ggtgggtgcg tctactgatt 540

cccctttgtc tctgaaaatc ttttccctag tggaacactt tggctgaatg atataaattc 600

accttgattc ccaccctccc ttctttctct ctctctctgt tacacccaat tgaattttct 660

tttttttttt actttccctc cttctttatc atcaaagata agtaagttta tcaattgcct 720

attcagaatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tcatcgaaaa 780

gttcgacagc gtctccgacc tcatgcagct ctcggagggc gaagaatctc gtgctttcag 840

cttcgatgta ggagggcgtg gatatgtcct ccgggtaaat agctgcgccg atggtttcta 900

caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct 960

tgacattggg gaattcagcg agagcctcac ctattgcatc tcccgccgtg cacagggtgt 1020

cacgttgcaa gacctccctg aaaccgaact ccccgctgtt ctccagccgg tcgcggaggc 1080

catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc 1140

gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca 1200

tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct 1260

cgatgagctc atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga 1320

tttcggctcc aacaatgtcc tcacggacaa tggccgcata acagcggtca ttgactggag 1380

cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg 1440

gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg 1500

atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt 1560

ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg 1620

atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac 1680

cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag 1740

ggcaaaggaa tagtgtgcta cccacgctta ctccaccaga gctattaaca tcagaaatat 1800

ttattctaat aaataggatg caaaaaaaaa acccccctta ataaaaaaaa aagaaacgat 1860

tttttatcta atgaagtcta tgtatctaac aaatgtatgt atcaatgttt attccgttaa 1920

acaaaaatca gtctgtaaaa aaggttctaa ataaatattc tgtctagtgt acacattctc 1980

ccaaaatagt gaaatccagc tgctagcatg agtgacgggg tctccttatt aaactcagat 2040

aattctttga caaatctccc aatgttttcg gattacgtgt tgcacaagaa cgcaaagaac 2100

ccggacttgg agcttgatcc ccaatctttg gcggcatcga gagtcaattc gatggtcaac 2160

atacctcatg cttctcatgg cgttaatacg ccatcaccgt caacttcaag acgcccacca 2220

ttgaccgaaa ccgcctcgtc ggagcatatg gaattgaact ttgggtctgg agcc 2274

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aagatcccca gagagggtcc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgcacccat catcagccaa 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gtgtgctacc cacgcttact 20

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

actgactgtc cattctgttc tctc 24

<210> 16

<211> 2217

<212> DNA

<213> Candida tropicalis (Candida tropicalis)

<400> 16

cttaagtacg agcgaccaaa gcgattcagg ttggtcattt acgactgctt gctctggctc 60

ttgtcagtga tatttgattg ttttttcaga gagatcagac cgagaggggc gtttaagatc 120

cccagagagg gtccggtgat ctttgttgcc gccccccatc ataatcagtt tgttgatccc 180

atcgtgttga tgaaccaggt gaaaagagaa gcaaaccgaa gaatatcatt cttgattgcg 240

gccaagtcat atcaattgaa agctgttgga acgatggcaa aatgtcagtt gtcaattcct 300

gttatccgcc cacaagactg tttgaccaaa ggtagtggta agatatttgt ggactttgac 360

aaagatccgt taaaagttat cggcaagaat accaaattca cgaccgaatg tatggtcaag 420

ggcttaattg cattgccaca atctttgggt gcctctgagg tggctgaaat cgtctcggat 480

accgagttgc ttatccgtaa agagttcaaa cccctggaga aagttaagga attgttgagc 540

caaggaacct ccttcaagcg tgctgataag gtcgaccaaa agcaagtcta tcaaatggtc 600

tttgaccact tggctgatga tgggtgcatt gggatctttc ccgaaggcgg gtctcacgat 660

cggccagact tattgccctt gaaagcaggc gttgctgtga tggctcttgg cgccatggac 720

aaaaatccaa attgcaatat caagattgtt ccatgtggta tgaactattt cagtgctcac 780

aagtttagaa gcagagccgt tgttgagttt ggcgatccaa ttgaaattcc aaaggagcta 840

gtgaagaaat acgcgaaccc ggagaccaac cgtgaagcgg ttaaggattt actagacacc 900

atcactactg gtttgaaagc agttaccgtt acctgcgagg actacgagac gttaatgtta 960

atacaggcgg caagacgttt gtatgctggt aacttcgcac agcaattgcc cttgccgttg 1020

attgtggaaa tgaacaggag attggtcatc ggctacgaac acttcaaaaa cgtgccaaaa 1080

gtgcaagagc tcaaggaaaa agtgttggcg tataatgagt ttctcaaaac tttgcattta 1140

ccagatcacc acgtcgagag ttgcaacgac aagacacaca agatagcttt gattccaaca 1200

tttttcataa gactttttga ggttatcttt ttgtttattt tggctttgcc aggagctgtg 1260

ttgttttcgc ccgtctttat ttcaagcaaa ttgatatccc gcaggaaggc aaaggaagcg 1320

ttagcgaact ccgtagtcaa gatccaagca aacgatgtga tcgcgacgtg gaaaatatta 1380

gttgctatgg gtatcgcgcc ggtcgtttat tctttctatg caagtattgg aacgtattac 1440

tgttctgcac ataaatattt cctgcattgg cgattgtttt gggtttgggt gtttttgtac 1500

atctgtgggg tcttggttac ttattcggca ttggtgactg gggaacaagg catggacttg 1560

ttgaaatcga ttcggccgtt gtacttgtcc atcacttcag ggtcgtcgat taaggagttg 1620

aagcgaatgc gagcggagct tagtgaagaa attaccgact tggtcaacac atatggccca 1680

cagttgtatc caaaagactt taatttgtta gagttgcaaa aatcgttgaa catcaatggc 1740

gatgttaatt atgttgacag tgacgaggaa gaagaattga aaaccgagga gttgcgtcaa 1800

agacgtttgg caaggcgcag ggcagagaag aaggccaccg atgcagatga cgaatctgaa 1860

ttgaaacatt catcttctat gagtgacggg gtctccttat taaactcaga taattctttg 1920

acaaatctcc caatgttttc ggattacgtg ttgcacaaga acgcaaagaa cccggacttg 1980

gagcttgatc cccaatcttt ggcggcatcg agagtcaatt cgatggtcaa catacctcat 2040

gcttctcatg gcgttaatac gccatcaccg tcaacttcaa gacgcccacc attgaccgaa 2100

accgcctcgt cggagcatat ggaattgaac tttgggtctg gagccaggac aaggaaatcg 2160

aatttgaggg acaaaatcaa actgaaactc agagagaaca gaatggacag tcagtct 2217

Claims (9)

1. The application of the SCT1 gene in the production of long-chain dicarboxylic acid is characterized in that the application comprises knocking out a copy of SCT1 gene in a genome of a long-chain dicarboxylic acid production strain by a genetic engineering means, and performing fermentation production of the long-chain dicarboxylic acid by utilizing the transformed engineering bacteria;

   the long-chain dibasic acid-producing strain is Candida tropicalis (Candida tropicalis).
2. 2. The engineering bacteria for producing the long-chain dicarboxylic acid are characterized in that the engineering bacteria are obtained by knocking out a copy of SCT1 gene in the genome of a long-chain dicarboxylic acid production strain by using a genetic engineering means;

   the long-chain dibasic acid-producing strain is Candida tropicalis (Candida tropicalis).
3. 3. The engineered bacterium of claim 2, wherein the long-chain dibasic acid is at least one selected from long-chain dibasic acids having a carbon chain length of C9-C22.
4. 4. The engineering bacteria of claim 3, wherein the long-chain dibasic acid comprises one or more of dodecanedioic acid, undedecane dicarboxylic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecane dicarboxylic acid, or octadecanedioic acid.
5. 5. The engineering bacteria for producing the long-chain dicarboxylic acid are characterized in that the engineering bacteria knock out the long-chain dicarboxylic acid with the preservation number of CCTCC NO: m2011192 or CCTCC NO: m203052 was obtained as a copy of the SCT1 gene in the Candida tropicalis genome.
6. 6. The method for reducing the content of acylglyceride in the fermentation production of long-chain dibasic acid is characterized in that the engineering bacteria of any one of claims 2 to 5 are used for carrying out the fermentation production of the long-chain dibasic acid, and the obtained long-chain dibasic acid has low content of acylglyceride impurities;

   wherein the acyl glycerides comprise diacyl glycerides and triacylglycerides;

   the raw material for fermentation comprises long-chain alkane, fatty acid derivative or mixture thereof;

   during fermentation, the concentration of fermentation substrate in the fermentation liquid is controlled to be 0.1-10%.
7. 7. The method of claim 6, wherein the diacylglyceride is C ₃₉ H ₇₂ O ₅ The triacylglycerol is C ₅₇ H ₁₀₄ O ₆ 。
8. 8. The method according to claim 6 or 7, wherein the total content of diacylglycerol esters and triacylglycerides in the obtained long-chain dibasic acid is 0.3ppm or less.
9. 9. The method of claim 8, wherein when the diacylglyceride is C ₃₉ H ₇₂ O ₅ Wherein said triacylglycerol is C ₅₇ H ₁₀₄ O ₆ When C is in contact with ₃₉ H ₇₂ O ₅ And C ₅₇ H ₁₀₄ O ₆ The total content of (B) is 0.3ppm or less.

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