CN111334445B

CN111334445B - Long-chain dicarboxylic acid producing strain and preparation method and application thereof

Info

Publication number: CN111334445B
Application number: CN201811555069.5A
Authority: CN
Inventors: 赖小勤; 晏礼明
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-12-19
Filing date: 2018-12-19
Publication date: 2021-08-03
Anticipated expiration: 2038-12-19
Also published as: CN111334445A

Abstract

The invention relates to a long-chain dicarboxylic acid production strain, a preparation method and application thereof, which solve the technical problem that the modification effect of the existing long-chain dicarboxylic acid strain is not obvious, are classified and named as Candida (Candida sp) TDTC019, and have the preservation number of: CGMCC No. 16660. The method can be widely applied to the field of preparation of long-chain dibasic acid.

Description

Long-chain dicarboxylic acid producing strain and preparation method and application thereof

Technical Field

The invention relates to a strain and a preparation method and application thereof, in particular to a strain for producing long-chain dicarboxylic acid and a preparation method and application thereof.

Background

The long-chain dibasic acid is an important chemical raw material, has extremely wide application, and can synthesize a series of chemicals with high added values, such as spices, special nylon, high-grade lubricating oil and the like. The long-chain dibasic acid can be applied to coatings of military fields, aerospace vehicles, pipelines, surface coatings of automobiles, oil pipes and the like; in the civil field, the method can be applied to more than ten high-tech industries such as automobiles, daily chemical spices, engineering plastics, nylon industries and the like, and can develop more downstream industries to form a new industrial chain.

In the past, long-chain dibasic acid is produced by a chemical synthesis method, and the patent technology is owned by foreign countries. The chemical synthesis method for producing the long-chain dibasic acid has the advantages of single product type, complex synthesis process, high cost and great pollution. China is the only country in the world which can realize large-scale industrial production of various long-chain dicarboxylic acids by adopting a microbial fermentation technology. In the past, the improvement of the dibasic acid production strains in China is realized by the traditional breeding methods such as mutagenesis in different modes. The traditional breeding method has great randomness and complex screening. It has been difficult to further improve the performance of strains by conventional breeding methods. Currently, there are many bottleneck problems in the industrial production process of long-chain dicarboxylic acid, such as the substrate conversion rate needs to be improved, the production energy consumption is very large, and the like.

The metabolic engineering technology can carry out targeted strain molecule modification on the gene level to obtain a new strain with better performance. As shown in fig. 1 and 2, the dibasic acid metabolic pathway mainly includes an ω -oxidation pathway and a β -oxidation pathway, wherein the former is a dibasic acid synthesis pathway and the latter is involved in a dibasic acid degradation pathway. The goal of metabolic engineering is to increase omega-oxidation activity and decrease beta-oxidation activity by molecular engineering means. Internationally, Henkel (the latter Cognis) has a patent report (US005254466A) that optimizes a dibasic acid-producing strain by gene knockout and sequentially knocks out 4 POX genes to completely block β -oxidation and increase the substrate conversion rate to 100%. On the basis, the company further co-expresses CYP monooxygenase and reductase by means of metabolic engineering so as to achieve the aim of enhancing omega-oxidation, and the yield is improved by 30 percent (World patent WO/91/06660).

However, the process of the bacterial strain for carrying out batch fermentation experiments cannot compete with other diacid production processes at that time, and finally, large-scale production is not carried out. The screening marker used by Henkel company for molecular modification of strains is uracil auxotrophy. The disadvantages of the Henkel invention are: 1. the starting strain is not a high-yield strain used for industrial production; 2. the candida used for producing long-chain dicarboxylic acid by the fermentation method is diploid, namely each cell has two sets of chromosomes, each gene has corresponding allele, and enzymes catalyzing biochemical reactions in each step are usually coded by a plurality of genes. Therefore, the enhancement or attenuation of the activity of a biochemical reaction in a body by means of metabolic engineering requires molecular modification of a key gene encoding the enzyme to have a significant effect. Otherwise, the effect after modification is not significant.

Disclosure of Invention

The invention aims to solve the technical problem that the effect of producing long-chain dibasic acid by using the existing strain is not obvious, and provides a long-chain dibasic acid producing strain with high production efficiency and a preparation method and application thereof.

Therefore, the invention provides a long-chain dicarboxylic acid producing strain, which is classified and named as Candida (Candida sp) TDTC019, and the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, CGMCC for short, 10 and 31 days in 2018, and the address is Beijing City Kogyao Xilu No.1 Homeh No. 3, China academy of sciences microorganism research institute; the preservation number is as follows: CGMCC No. 16660.

One of alleles Candidaa05236 and Candidaa04467 of the long-chain dibasic acid-producing strain of the present invention, the base sequences of which are shown in SEQ ID Nos. 5 and 6, was deleted. The gene is the most critical one for long-chain dibasic acid production in a plurality of genes for coding the enzyme in the genome of the strain.

The invention also provides a preparation method of the long-chain dicarboxylic acid production strain, which comprises the following steps: (1) preparing primers HCD-F and HCD-R; (2) preparing competent cells of the long-chain dicarboxylic acid production strain; (3) amplifying a clonNAT resistance gene by using the primer in the step (1), and knocking out one of alleles Candidaa05236 and Candidaa04467 in the strain by using an amplified product, wherein the base sequences of the alleles are shown as a sequence 5 and a sequence 6 in a sequence table; (4) the long-chain dicarboxylic acid producing strain is obtained through PCR amplification, purification, electrotransformation, screening and identification.

Preferably, in the method for preparing the long-chain dicarboxylic acid-producing strain according to the present invention, the screening marker used in the screening step is clonNAT.

Preferably, the long-chain dicarboxylic acid-producing strain used in step (2) is Candida (Candida sp.) DC 12.

The invention also provides the application of the long-chain dicarboxylic acid production strain in the production of long-chain dicarboxylic acid.

Preferably, after fermentation is finished, heating the fermentation liquor to 70-80 ℃; adjusting the pH value to 9-9.5, removing thalli precipitates, and keeping a supernatant; decoloring, keeping the temperature at 70-90 ℃ to obtain a filtrate, acidifying to pH2.5 with acid, keeping the temperature at 70-90 ℃, cooling, centrifuging or filter pressing, washing with water, taking out the cleaned precipitate, and drying in vacuum to obtain the long-chain dicarboxylic acid.

The long-chain dicarboxylic acid in the invention refers to a straight-chain dicarboxylic acid containing more than ten carbon atoms, and is an important fine chemical intermediate raw material, in particular to dodecanedioic acid (DC12), tetradecanedioic acid (DC14), hexadecanedioic acid (DC16) and octadecanedioic acid (DC 18).

The invention has the beneficial effects that in order to break through the bottleneck of genetic modification of production strains, the invention analyzes omega-oxidation and beta-oxidation metabolic pathways by analyzing the characteristics of genomics and transcriptomics of the production strains, establishes some key target sites related to dibasic acid metabolism on the global genome level, carries out molecular modification on the sites by means of metabolic engineering, and obtains strains with better performance through fermentation experiment verification. Compared with the DC12 strain, the TDTC019 strain (the preservation number is CGMCC No.16660) provided by the invention has the advantages that the conversion rate is obviously improved, and the beneficial effect is brought to large-scale production.

Drawings

FIG. 1 is a diagram of the omega-oxidative metabolic pathway involved in the synthesis of long chain diacids according to the present invention;

FIG. 2 is a diagram of the beta-oxidative metabolic pathway involved in the degradation of long chain diacids in accordance with the present invention;

FIG. 3 is a flow chart of gene knockout according to the present invention;

FIG. 4 is an HPLC analysis of long chain dibasic acid produced by fermentation of DC 12;

FIG. 5 shows HPLC analysis of long-chain dibasic acid produced by fermentation of TDTC 023.

The long-chain dicarboxylic acid production strain is classified and named as Candida (Candida sp.) TDTC 019; the preservation organization is China general microbiological culture Collection center (CGMCC) with the address of No. 3 of Xilu No.1 of Beijing university facing Yang district, China academy of sciences; the preservation date is 2018, 10 and 31, and the accession numbers are: CGMCC No. 16660.

Detailed Description

Sequence names in the sequence listing are as follows: sequence 1: HCD-F; sequence 2: HCD-R; and (3) sequence: HCD-U; and (3) sequence 4: HCD-D; and (5) sequence: candidaa 05236; and (3) sequence 6: candidaa04467

The colony and cell morphology observed in the following examples are summarized as follows:

on a solid culture medium flat plate, colonies are cheese-shaped, the surface is smooth, milk white, plump and convex, and the diameter of the colonies is about 2 mm. Yeast-like single cells, approximately 10X6 μm in size. In most cases, in the form of yeast-like single cells, there is a simultaneous pseudohyphal formation, e.g., the pseudohyphal proportion increases significantly during certain growth stages or under certain external conditions.

The following media were used in the following examples:

1. the YPD culture medium comprises the following components: 1% of yeast extract, 2% of peptone and 2% of glucose, and if a solid culture medium is prepared, 2% of agar powder is added. The above percentages are mass volume percentages, i.e. the grams of the component required per 100 ml of culture medium. If antibiotics are to be added, the liquid medium is added at the respective final concentration at the time of use. And (3) cooling the solid culture medium to about 50 ℃ after autoclaving, adding antibiotics to the corresponding final concentration, uniformly mixing, immediately pouring into a sterile culture dish, standing upside down after solidification, and placing in a refrigerator at 4 ℃ for use within two weeks.

2. The formula of the seed culture medium is as follows: 1-8 g/L of yeast extract, 1-8 g/L of corn steep liquor, 5-25 g/L of sucrose and KH₂PO₄4-12 g/L, 0.5-4 g/L of urea, 40-70 g/L of heavy wax, and sterilizing for 30 minutes at 121 ℃. Wherein, the sucrose and the urea are separately sterilized at 110 ℃ for 20 minutes, and then are combined and mixed evenly after sterilization.

3. The fermentation tank culture medium formula is as follows: 1-8 g/L of yeast extract, 1-8 g/L of corn steep liquor, 5-30 g/L of sucrose and KH₂PO₄4-15 g/L, 0.5-4 g/L urea, KNO₃5-15 g/L, NaCl 0.5-2.5 g/L, sterilizing at 121 ℃ for 30 minutes. Wherein, the sucrose and the urea are separately sterilized, sterilized for 20 minutes at 110 ℃, and then merged and mixed evenly after sterilization. A75% glucose solution was prepared, sterilized at 105 ℃ for 20 minutes, and fed-batch at the initial stage of fermentation.

The following cell culture and fermentation broth treatment protocols were used in the following examples:

1. plate culture: after streaking or spreading on YPD plates, the plates were placed in 30 ℃ incubator and large, full, milky colonies were observed for 2-3 days.

2. Shake cultivation: the plates were inoculated with single colonies into liquid media or transferred from liquid media and shake-cultured at 30 ℃ and maintained at 220 rpm.

3. Culturing in a fermentation tank: can accurately control the fermentation conditions in real time, such as feeding control, pH control, dissolved oxygen control, aeration stirring intensity control and the like. In the first stage, the pH value of fermentation liquor is controlled to be 5-6.8, and a glucose solution is fed at the same time to form a thallus growth stage; in the second stage, the pH value of the fermentation liquor is controlled to be 7.0-7.8, and simultaneously substrates (alkane, fatty acid or fatty acid derivatives such as methyl ester or ethyl ester and the like) are fed in, so that acid production through fermentation is mainly performed, and partial thalli also grow; in the third stage, only acid is produced, no bacteria are produced, and substrate (alkane, fatty acid or fatty acid derivative, such as methyl ester or ethyl ester, etc.) is continuously fed according to the fermentation condition. The pH value is controlled by using 10M NaOH solution to automatically feed in the whole fermentation process, and the dissolved oxygen is kept at 20-40% by adjusting the rotating speed.

4. And (3) treating fermentation liquor: after fermentation is finished, heating the fermentation liquor to 70-80 ℃, and maintaining for 60 minutes; adding 10M NaOH to adjust the pH value to 9-9.5, removing thallus precipitates by using a tubular centrifuge or filter pressing, and keeping a supernatant; adding a proper amount of activated carbon for decolorization, keeping the temperature at 70-90 ℃, and keeping the temperature for 60 minutes; removing the activated carbon to obtain a filtrate, continuously acidifying the filtrate to pH2.5 with concentrated hydrochloric acid or concentrated sulfuric acid, preserving the heat at 70-90 ℃ for 2 hours, cooling to 30 ℃, centrifuging or press-filtering, washing with clear water once, taking out the washed precipitate, and drying in vacuum to obtain the white dibasic acid.

Example 1: metabolic engineering of strains

1) Genome sequencing

The genome DNA of the long-chain dibasic acid producing strain Candida is prepared by utilizing a genome extraction Kit (Yeast DNAiso Kit) of Bao bioengineering Co., Ltd, and the specific operation is carried out according to the Kit instruction. And constructing the genomic DNA through a library, sequencing and analyzing by a new generation sequencing technology, assembling data and the like. Through genome annotation and data analysis, gene information related to the production of the dibasic acid by the production strain is obtained, and coding sequences of related genes of the strain in omega-oxidation and beta-oxidation metabolic pathways are excavated. Research has shown that (Eur.J.Lipid Sci.Technol.113: 548-. In the genome of the long-chain dibasic acid producing strain, Candida05236(2664bp) and Candida04467 (2721bp) open reading frames code HCD, the sequence similarity is up to 97 percent, and the sequences of the upstream region and the downstream region are also highly similar, so that the long-chain dibasic acid producing strain can be judged to be a pair of alleles.

2) Transcriptome sequencing

The total RNA of the long-chain dibasic acid producing strain Candida was prepared using an RNA extraction Kit (RNeasy Mini Kit) of Qiagen, Germany, and the specific procedures were performed according to the Kit instructions. And performing library construction, transcriptome sequencing and expression quantity analysis on the obtained total RNA. By transcriptome data analysis, transcript levels of the alleles were found to be 4336.8 and 3731.5RPKM for Candida05236 and Candida04467, respectively. RPKM is a method for calculating the gene expression level and is expressed as Reads Per Kb Per Million Reads. The calculation formula is

If RPKM (A) is the expression quantity of the gene A, C is the number of reads which are uniquely compared to the gene A, N is the total number of reads which are uniquely compared to a reference gene, and L is the number of bases of a coding region of the gene A. The RPKM method can eliminate the influence of the difference of gene length and sequencing quantity on the calculation of gene expression, and the calculated gene expression quantity can be directly used for comparing the gene expression difference.

Because beta-oxidation is a metabolic pathway related to degradation of long-chain dibasic acid, the aim of strain modification is to avoid the degradation of the dibasic acid as much as possible. On the other hand, however, beta-oxidation is essential for normal physiological metabolism and energy supply of the bacterial cells. Therefore, it is hoped that the beta-oxidation is inhibited but not completely blocked by means of genetic engineering, so as to achieve the purposes of not affecting the normal growth of thalli and the synthesis of long-chain dibasic acid, but also avoiding the degradation of the synthesized long-chain dibasic acid. Candida05236 and Candida04467 are pairs of alleles of diploid DC12 encoding hydroxyester acyl-CoA dehydrogenase (HCD) that plays a role in the beta-oxidative metabolism of diacid degradation processes. Therefore, one copy of the pair of alleles is knocked out by using a metabolic engineering means, and the beta-oxidative metabolic activity is reduced, so that the aim of more effectively accumulating the dibasic acid is fulfilled.

3) The gene knockout experiment flow is shown in FIG. 3. Primer design principles, homology arms and detection primers were designed to select regions that were identical in the alignment of Candida05236 and Candida04467, to achieve equal probability of both allele knockouts and detections. The 5' ends of the long primers HCD-F and HCD-R are homologous arm sequences corresponding to the upstream and downstream sequences of the gene knockout site. The 3' ends of these two long primers were paired with the upstream and downstream of the knock-out module (deletion cassette), respectively. The two ends of the DNA fragment obtained after PCR amplification are respectively provided with a downstream homology arm, and the middle part is a resistance screening marker.

The amplified DNA fragment is purified and then is introduced into a thallus cell by electrotransformation, and the upstream and downstream homologous arms of the DNA fragment and a target site on a thallus chromosome are subjected to double exchange to achieve the purpose of gene knockout. Since this double exchange is a small probability event, a method needs to be established for screening. The resistance gene used here was Sat1, encoding a nourseothricin acetyltransferase. The dibasic acid producing strain is sensitive to nourseothricin (called clonNAT or NTC), and can not grow on the plate containing nourseothricin, and the thallus can decompose nourseothricin after expressing the enzyme, so as to avoid lethal effect. The resistance selection marker can be expressed only by integrating into the chromosome, and plays a role in enabling the thalli to grow on a culture medium plate containing resistance and form colonies. At the same time, the resistant colonies were purified and verified.

In the verification, a pair of detection primers HCD-U and HCD-D are used to pair with the upstream and downstream regions of the target site, respectively. If no homologous recombination occurs, the fragments amplified by templating the pair of alleles are identical in length and only one band is observed in agarose electrophoresis. If one allele is replaced by a fragment of the resistance gene due to the double crossover event, the amplified fragment will vary in length and two bands will be observed in agarose electrophoresis. And (3) verifying whether the new strain has the advantage of performance in the aspect of binary acid production by the verified strain and the metabolic engineering modification through a fermentation experiment.

4) PCR amplification

Wherein, the plasmid pSFS2 is referenced as Gene (2004)341: 119-127, and the accession number of the GeneBank database is as follows: AY524979, available from institute of microbiology, academy of sciences of China.

The primers used for PCR amplification are HCD-F and HCD-R. The DNA polymerase was Pyrobest DNA polymerase from Takara Bio Inc., or Phusion DNA polymerase from NEB Inc., all having an activity of 5U/. mu.l.

The PCR cycling conditions were:

94 degree 2 min (pre-denaturation stage)

94 ℃ 20 seconds, 58 ℃ 20 seconds, 72 ℃ 1-6 minutes (30 cycles amplification stage)

72 degree 10 minutes (final extension phase)

5) DNA purification

After the reaction was completed, 5. mu.l of the above PCR sample was subjected to agarose electrophoresis, and it was confirmed that the amplified DNA fragment was the same in size as expected and free from a band, and two-step purification and concentration were carried out to prepare a DNA sample for the subsequent transformation.

The first step was performed using a PCR purification kit (purchased from Omega) according to the instructions. Typically, a 50. mu.l system was prepared in 4 tubes with a total volume of 200. mu.l, and the final step of PCR purification was performed by eluting the column with 50 or 100. mu.l TE buffer. The concentration of the purified DNA was measured by a NanoDrop apparatus, and the total amount of the purified DNA was calculated to be about 20. mu.g.

The second step is to precipitate the purified DNA by further treatment with ethanol/sodium acetate/glycogen. The specific method comprises the following steps: to the above DNA solution in TE buffer was added 1. mu.l of glycogen (20mg/ml), 100. mu.l of sodium acetate (3M, pH5.2) and 1ml of pre-cooled absolute ethanol; cooling the mixture for 30 minutes in a refrigerator at the temperature of-80 ℃; centrifuging for 10 minutes at 14000 rpm of a 4-DEG centrifuge and leaving a precipitate; washing the precipitate twice with 75% precooled ethanol; air drying in a super clean bench; the cells were stored in a refrigerator at 4 ℃ and resuspended in 10. mu.l of ultrapure water for further use before electroporation.

6) Competent preparation and electrotransfer

Selecting single colony of starting strain (Candida sp. DC12, see microbiology report 20 (1): 88-93,1980, n-alkane fermentation for producing long chain mixed dicarboxylic acid, available from microorganism of Chinese academy of sciences) from fresh activated plate, and shake culturing at 30 deg.C overnight at 220 r/min in 3ml liquid YPD medium; 2% of the cells were transferred to 20ml YPD, and shaking cultured at 30 ℃ for 220 rpm until OD600 reached 1.8; standing the bacterial liquid on ice for 15 minutes to stop the growth, centrifuging the bacterial liquid at 4000 revolutions per minute at 4 ℃ for 3 minutes, and leaving thalli to precipitate; washing with 4ml of precooled sterile water once; centrifuging at 4000 rpm and 4 ℃ for 3 minutes, and leaving thalli to precipitate; adding 4ml TE/0.1M LiOAc, rotating at 150 r/min, and oscillating in a shaker at 30 ℃ for 90 min; adding 0.1ml of 1M DTT, and continuing to oscillate in a shaking table box at 150 rpm and 30 ℃ for 30 minutes; centrifuging at 4000 rpm and 4 ℃ for 3 minutes, and leaving thalli to precipitate; adding 4ml of precooled sterile water, and washing for 3 times; adding 2ml of 1M sorbitol, washing for 1 time, rotating at 4000 rpm, and centrifuging for 3 minutes at 4 ℃; discard the supernatant and add 120. mu.l sorbitol to suspend the cells; taking out 40ul of the cell suspension, placing the cell suspension in a 1.5ml centrifuge tube, adding 5 ul of the purified PCR amplification product (about 10 ug) resuspended in sterile water, mixing well, and placing on ice for 5 minutes; transferring the electric rotating cup into a pre-cooled electric rotating cup, wiping the electric rotating cup dry, and performing electric rotating (the electric rotating condition is that the electric rotating cup with a 2mm slit is used, the voltage is 1800V, and the electric shock time is 5 milliseconds); after electrotransfer, immediately adding 1ml of sorbitol, uniformly mixing, sucking out and placing into a 1.5ml centrifuge tube; centrifuging at 4000 rpm for 3 min, removing supernatant, adding 1ml YPD medium, and culturing in a shaker at 37 deg.C for 2 hr; after centrifugation at 4000 rpm for 3 minutes, the supernatant was discarded, 100. mu.l of YPD was added to resuspend the cells, plates (YPD + clonNAT) were spread, and the cells were cultured in a 30 ℃ incubator until single colonies appeared.

7) Screening for resistant colonies

The single colonies growing on the resistant plates were inoculated into 1ml YPD medium centrifuge tubes, and clonNAT was added to a final concentration of 100. mu.g/ml, 220 rpm, and shake-cultured at 30 ℃. Growth occurs, which indicates resistance, and the resistance gene is integrated into the genome of the strain and plays a role, and this part of the colony is used for the next verification. No growing colonies appeared, indicating false positives, which equated to the starting strain, and treatment was stopped.

8) Identification

The resistant colonies growing on the surface are inoculated into YPD liquid culture medium for culture overnight, 500 mul of bacterial liquid is absorbed, genome DNA is prepared by utilizing a genome extraction kit of Bao bioengineering Co., Ltd, and the specific operation is carried out according to the kit instruction. PCR amplification was performed using the obtained genomic DNA as a template and HCD-U and HCD-D as primers, and the detailed reaction system and reaction conditions were as in item 4) of example 1. If the allele at the target site is not knocked out, the DNA fragments amplified from both alleles are the same size and appear as a single band on agarose electrophoresis. If one allele at the target site is knocked out, the amplified DNA fragments from the two alleles are different in size and appear as two bands on agarose electrophoresis (see FIG. 3). The bacterial liquid which is identified as resistance positive and the target site is knocked out is further purified by bacterial colonies and identified by the same method to obtain the long-chain dicarboxylic acid production strain TDTC 019.

Example 2: fermentative production of long-chain dicarboxylic acids

After the strain is cultured by a conventional slant, 50ml of first-class seeds are inoculated for 16 hours of culture, and then the first-class seeds are inoculated with 500ml of second-class seeds for 16 hours of culture.

The formula of the seed culture medium is as follows: 1-8 g/L of yeast extract, 1-8 g/L of corn steep liquor, 5-25 g/L of sucrose and KH₂PO₄4-12 g/L, 0.5-4 g/L of urea, 40-70 g/L of heavy wax, and sterilizing for 30 minutes at 121 ℃. Wherein, the sucrose and the urea are separately sterilized at 110 ℃ for 20 minutes, and then are combined and mixed evenly after sterilization.

After the secondary seed fermentation is completed, the second seed is transferred into a 5L fermentation tank. The fermentation tank culture medium formula is as follows: 1-8 g/L of yeast extract, 1-8 g/L of corn steep liquor, 5-30 g/L of sucrose and KH₂PO₄4-15 g/L, 0.5-4 g/L urea, KNO ₃ 5～15g/L，NaCl 0.5～2.5g/L，12Sterilizing at1 deg.C for 30 min. Wherein, the sucrose and the urea are separately sterilized, sterilized for 20 minutes at 110 ℃, and then merged and mixed evenly after sterilization. A75% glucose solution was prepared, sterilized at 105 ℃ for 20 minutes, and fed-batch at the initial stage of fermentation. The basal medium was 4L. 1 at 30 ℃ in the presence of a diluent: the aeration volume of 0.5, the pH value is controlled to be 5.5-6.5, the twelve-carbon straight-chain alkane is fed at the speed of 50ml/h after the 16 th hour, and the fermentation time is 144-156 hours. The pH value is controlled by the automatic feeding of 10M NaOH solution in the whole fermentation process, and the dissolved oxygen is kept at 30 percent by adjusting the rotating speed.

As shown in FIG. 4, HPLC analysis shows that the long-chain dibasic acid products obtained by fermenting the starting strain DC12 and the modified strain TDTC019 are consistent, and the purity is more than 98%.

As can be seen from the following table, the transformed strains also have improved conversion rate.

Example 3

The procedure of example 2 was followed except that the substrate was changed from a twelve-carbon linear alkane to methyl laurate.

Example 4

The procedure of example 2 was followed except that the substrate was changed from a twelve carbon linear alkane to ethyl laurate.

Sequence listing

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gttggtggtg acgacgatga tgatgacgaa gacgaagaag aagaagacga aggtgatgaa 1800

gaagaggaag aagaagacga agaagaagac gacccagtct ggagattcga cgacagagat 1860

gttatcttgt acaacattgc ccttggtgcc accaccaagc aattgaagta cgtctacgaa 1920

aacgactctg acttccaagt cattccaacc tttggtcact tgattacctt caactctggt 1980

aagtcacaaa actcctttgc caagttgttg cgtaacttca acccaatgtt gttgttgcac 2040

ggtgaacact acttgaaggt gcacagctgg ccaccaccaa ccgaaggtga aatcaagacc 2100

actttcgaac caattgccac tactccaaag ggtaccaacg ttgttatcgt gcacggttcc 2160

aagtctgttg acaacaagtc tggtgaattg atttactcca atgaagccac ttatttcatc 2220

agaaactgtc aggccgacaa caaggtctac gctgaccgtc cagcattcgc caccaaccaa 2280

ttcttggcac caaagagagc cccagactac caagttgatg ttccagtcgg tgaagacttg 2340

gctgctttgt accgtttgtc tggtgacaga aacccattgc acattgatcc aaactttgct 2400

aaaggtgcca agttccctaa gccaatctta cacggtatgt gcacttatgg tttgagtgct 2460

aaggctttga ttgacaagtt tggtatgttc aacgaaatca aggccagatt caccggtatt 2520

gtcttcccag gtgaaacctt gagagtcttg gcatggaagg aaagcgatga cactattatc 2580

ttccaaactc atgttgttga tagaggtact attgccatta acaacgctgc tattaagtta 2640

gttggtgaca aagcaaagat ctaa 2664

<210> 6

<211> 2721

<212> DNA

<213> Candida sp.

<400> 6

atgtctccag ttgattttaa agataaagtt gtgatcatta ccggtgccgg tggtggtttg 60

ggtaaatact actccctcga atttgccaag ttgggcgcca aagtcgtcgt taacgacttg 120

ggtggtgcct tgaacggtca aggtggaaac tccaaggccg ccgacgttgt cgttgacgaa 180

attgtcaaga acggtggtgt tgccgttgcc gattacaaca acgtcttgga cggtgacaag 240

attgtcgaaa ccgccgtcaa gaactttggt actgtccacg ttatcatcaa caatgccggt 300

atcttgagag atgcctccat gaagaagatg actgaaaaag actacaaatt ggtcattgac 360

gtgcacttga acggtgcctt tgccgtcacc aaggctgctt ggccatactt ccaaaagcaa 420

aaatacggta gaattgtcaa cacatcctcc ccagctggtt tgtacggtaa ctttggtcaa 480

gccaactacg cctccgccaa gtctgctttg ttgggattcg ctgaaacctt ggccaaggaa 540

ggtgccaaat acaacatcaa ggccaacgcc attgctccgt tggccagatc aagaatgact 600

gaatctatct tgccacctcc aatgttggaa aaattgggcc ctgaaaaggt tgccccattg 660

gtcttgtatt tgtcgtcagc tgaaaacgaa ttgactggtc aattctttga agttgctgct 720

ggcttttacg ctcagatcag atgggaaaga tccggtggtg tcttgttcaa gccagatcaa 780

tccttcaccg ctgaggttgt tgctaagaga ttctctgaaa tccttgatta tgacgactct 840

aggaagccag aatacttgaa gaaccaatac ccattcatgt tgaacgacta cgccactttg 900

accaacgaag ctagaaagtt gccagctaac gatgcttctg gtgctccaac tgtctccttg 960

aaggacaagg ttgttttgat caccggtgcc ggtgctggtt tgggtaaaga atacgccaag 1020

tggttcgcca agtacggtgc caaggttgtt gttaacgact tcaaggatgc taccaagacc 1080

gttgacgaaa tcaaagccgc tggtggtgaa gcttggccag atcaacacga tgttgccaag 1140

gactccgaag ctatcatcaa gaatgtcatt gacaagtacg gtaccattga tatcttggtc 1200

aacaacgccg gtatcttgag agacagatcc tttgccaaga tgtccaagca agaatgggac 1260

tctgtccaac aagtccactt gattggtact ttcaacttga gcagattggc atggccatac 1320

tttgttgaaa aacaatttgg tagaatcatc aacattacct ccaccagtgg tatctacggt 1380

aactttggtc aagccaacta ctcgtcttct aaggctggta tcttgggttt gtccaagacc 1440

atggccattg aaggtgctaa gaataacatt aaggtcaaca ttgttgctcc acacgctgaa 1500

actgccatga ccttgaccat cttcagagaa caagacaaga acttgtacca cgctgaccaa 1560

gttgctccat tgttggtcta cttgggtact gacgatgtcc cagtcaccgg tgaaactttc 1620

gaaatcggtg gtggttggat cggtaacacc agatggcaaa gagccaaggg tgctgtctcc 1680

cacgacgaac acaccactgt tgaattcatc aaggagcact tgaacgaaat cactgacttc 1740

accactgaca ctgaaaatcc aaaatctacc accgaatcct ccatggctat cttgtctgcc 1800

gttggtggtg atgacgatga tgatgacgaa gacgaagaag aagacgaagg tgatgaagaa 1860

gaagacgaag aagacgaaga agaagacgat ccagtctgga gattcgacga cagagatgtt 1920

atcttgtaca acattgccct tggtgccacc accaagcaat tgaagtacgt ctacgaaaac 1980

gactctgact tccaagtcat tccaaccttt ggtcacttga ttaccttcaa ctctggtaag 2040

tcacaaaact cctttgccaa gttgttgcgt aacttcaacc caatgttgtt gttgcacggt 2100

gaacactact tgaaggtgca cagctggcca ccaccaaccg aaggtgaaat caagaccact 2160

ttcgaaccaa ttgccactac tccaaagggt accaacgttg ttattgttca cggttccaaa 2220

tctgttgaca acaagtctgg tgaattgatt tactccaacg aagccactta cttcatcaga 2280

aactgtcaag ccgacaacaa ggtctacgct gaccgtccag cattcgccac caaccaattc 2340

ttggcaccaa agagagcccc agactaccaa gttgatgttc cagtcggtga agacttggct 2400

gctttgtacc gtttgtctgg tgacagaaac ccattgcaca ttgatccaaa ctttgctaaa 2460

ggtgccaagt tccctaagcc aatcttacac ggtatgtgca cttatggttt gagtgctaag 2520

gctttgattg acaagtttgg tatgttcaac gaaatcaagg ccagattcac cggtattgtc 2580

ttcccaggtg aaaccttgag agtcttggca tggaaggaaa gcgatgacac tattgtcttc 2640

caaactcatg ttgttgatag aggtactatt gccattaaca acgctgctat taagttagtc 2700

ggtgacaaag caaagatcta a 2721

Claims

1. The application of the long-chain dicarboxylic acid production strain in the production of long-chain dicarboxylic acid is characterized by comprising the following steps: after the strain is cultured by a conventional slant, 50ml of first-level seeds are inoculated for 16 hours of culture, and then the first-level seed culture solution is inoculated into 500ml of second-level seeds for 16 hours of culture; the formula of the seed culture medium is as follows: 1-8 g/L of yeast extract, 1-8 g/L of corn steep liquor, 5-25 g/L of sucrose, 0.5-4 g/L of KH2PO 44, 40-70 g/L of heavy wax, and sterilizing for 30 minutes at 121 ℃; wherein, the sucrose and the urea are separately sterilized for 20 minutes at 110 ℃, and then are combined and mixed evenly after sterilization; after the secondary seed fermentation is finished, transferring into a 5L fermentation tank; the fermentation tank culture medium formula is as follows: 1-8 g/L of yeast extract, 1-8 g/L of corn steep liquor, 5-30 g/L of sucrose, 0.5-4 g/L of KH2PO 44, 0.32-15 g/L of urea, 0.5-2.5 g/L of KNO 35 and 0.5-2.5 g/L of NaCl, and sterilizing for 30 minutes at 121 ℃; wherein, the sucrose and the urea are separately sterilized, sterilized for 20 minutes at 110 ℃, and then merged and mixed evenly after sterilization; preparing 75% glucose solution, sterilizing at 105 deg.C for 20 min, and fermenting at the initial stageAdding in a row flow manner; 4L of basic culture medium; 1 at 30 ℃ in the presence of a diluent: the aeration volume of 0.5, the pH value is controlled to be 5.5-6.5, the twelve-carbon straight-chain alkane is fed at the speed of 50ml/h after the 16 th hour, and the fermentation time is 144-156 hours; the pH value is controlled by the automatic feeding of 10M NaOH solution in the whole fermentation process, and the dissolved oxygen is kept at 30 percent by adjusting the rotating speed; after fermentation is finished, heating the fermentation liquor to 70-80 ℃; adjusting the pH value to 9-9.5, removing thalli precipitates, and keeping a supernatant; decolorizing, keeping the temperature at 70-90 ℃ to obtain a filtrate, acidifying to pH2.5 with acid, keeping the temperature at 70-90 ℃, cooling, centrifuging or filter pressing, washing with water, taking out the cleaned precipitate, and drying in vacuum to obtain long-chain dicarboxylic acid; the long-chain dicarboxylic acid producing strain is classified and named as candida (Candida sp.) TDTC019 with a preservation number of: CGMCC number 16660.