Genetically engineered bacterium of candida and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium of candida and application of the genetically engineered bacterium. In the genetically engineered bacterium, the GOGAT gene is inactivated, and the GDH gene is over-expressed, so that the genetically engineered bacterium of the candida with remarkably improved binary acid yield is obtained.

Description

[0001] This patent application is a divisional application of the application with the application number: 202111478770.3, the application date: December 06, 2021, and the invention title: "A Genetic Engineering Bacterium for Producing Dicarboxylic Acid and Its Application". Technical Field

[0002] The present invention belongs to the field of microbial fermentation, and specifically relates to a genetic engineering bacterium of the genus Candida, a method for preparing a dicarboxylic acid, and the application of the genetic engineering bacterium in the preparation of a dicarboxylic acid. Background Art

[0003] During the microbial culture process, the absorption of inorganic nitrogen elements mainly includes nitrate nitrogen and ammonium nitrogen. As the ultimate precursor for converting ammonium nitrogen as an inorganic nitrogen source into organic nitrogen, its absorption is mainly achieved through two pathways: the GDH (glutamate dehydrogenase, EC: 1.4.1.4) pathway and the GS-GOGAT (glutamine synthetase - glutamine: α-ketoglutarate aminotransferase, E.C. 6.3.1.2 - E.C. 1.4.1.13) pathway.

[0004] As Figure 1 shown, the absorption of ammonium nitrogen by both pathways requires the consumption of reducing power: the GDH pathway is responsible for catalyzing the transfer of ammonium nitrogen element to α-ketoglutarate to generate glutamate, and at the same time requires the consumption of NADPH. Correspondingly, in the GS-GOGAT pathway, first under the catalysis of GS, one molecule of ammonium nitrogen is transferred to glutamate to generate glutamine, and then GOGAT is responsible for transferring ammonium nitrogen to α-ketoglutarate to convert it into glutamate. Compared with the GDH pathway, the GS-GOGAT pathway consumes NADH, but for each molecule of ammonium nitrogen absorbed, the GS-GOGAT pathway needs to consume one more molecule of ATP.

[0005] In different microorganisms, the GDH pathway and the GS-GOGAT pathway play different roles. For example, Macheda et al. found that when studying the nitrogen uptake pathway of Aspergillus Nidulans, compared with the wild type, the mutant lacking NADP-dependent GDH grew slowly on the medium with ammonium nitrogen as the sole nitrogen source. Further knocking out the gene encoding GOGAT, the cells could not grow on the medium with ammonium nitrogen as the sole nitrogen source at all. Schizosaccharomyces pombe lacking the GDH or GS-GOGAT pathway could still grow on the ammonium nitrogen medium. However, when both pathways were blocked, the growth ability of the mutant was completely lost (Perysinakis et al., 1995). It has also been found that in some microorganisms, only one of these pathways is involved in ammonium nitrogen uptake: for example, only the GS-GOGAT pathway exists in Agaricus bisporus, and in the heterocysts of the cyanobacterium PC7120, although GS and GDH are present, GOGAT is lacking, and it depends on the input of glutamate from adjacent vegetative cells to maintain the activity of GS. Holmes' research showed that the GS-GOGAT pathway is the main pathway for ammonium nitrogen uptake in Candida albicans, Candida tropicalis and other Candida yeasts (Holmes, 1989, 1991).

[0006] Glutamate dehydrogenase GDH plays a role in connecting carbon and nitrogen metabolism in most known species. Depending on the coenzyme it depends on, it is divided into two major categories: NADH-dependent glutamate dehydrogenase and NADPH-dependent glutamate dehydrogenase. In higher fungi (deuteromycetes, ascomycetes and basidiomycetes), there are two types of GDH, while most lower fungi only have NADH-dependent glutamate dehydrogenase. The former encodes a subunit with a size of 115 kDa, which usually functions as a tetramer in fungi and is involved in catabolism; while the NADPH-dependent glutamate dehydrogenase exists as a hexamer, and the encoded protein is relatively small, about 50 kDa, and is mainly involved in the uptake of ammonium nitrogen. In short, glutamate is at the core of the amino acid synthesis and metabolism pathway, and is involved in the uptake and utilization of nitrogen, having a systematic impact on cell growth and metabolism, but its impact on the production of dicarboxylic acids has been less studied.

[0007] In recent years, the production of long-chain dicarboxylic acids by microbial fermentation has gradually replaced the traditional chemical synthesis method due to its advantages of low carbon, green, high efficiency, and low by-product content, becoming the main method for the production of long-chain dicarboxylic acids. By genetic modification, changing the expression levels of the GS-GOGAT and GDH pathways in the ammonium nitrogen absorption pathway is of great significance for improving the nitrogen source utilization efficiency of microorganisms and increasing the yield of dicarboxylic acids during the biosynthesis of dicarboxylic acids. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a genetically engineered bacterium of the genus Candida and its application. The present invention uses genome editing methods to genetically modify the GS-GOGAT and GDH pathways in the ammonium nitrogen absorption pathway of dicarboxylic acid-producing strains such as Candida spp. By downregulating GOGAT to block / weaken the GS-GOGAT pathway and overexpressing the GDH pathway, the ammonium nitrogen absorption pathway is modified to obtain a genetically engineered bacterium of the genus Candida with a significantly increased yield of dicarboxylic acids.

[0009] The present invention solves the above technical problems through the following technical solutions.

[0010] The first aspect of the present invention provides a genetically engineered bacterium of the genus Candida, wherein the GOGAT gene in the genetically engineered bacterium is inactivated and the GDH gene is overexpressed; the amino acid sequence encoded by the GOGAT gene is as shown in SEQ ID NO: 27.

[0011] In a preferred embodiment, the amino acid sequence encoded by the GDH gene is as shown in SEQ ID NO: 28.

[0012] In a preferred embodiment, the nucleotide sequence of the GOGAT gene is as shown in SEQ ID NO: 25.

[0013] In a preferred embodiment, the nucleotide sequence of the GDH gene is as shown in SEQ ID NO: 26.

[0014] In a preferred embodiment, the genetically engineered bacterium lacks the amino acid sequence encoded by the GDH gene and overexpresses the amino acid sequence encoded by the GDH gene.

[0015] In a preferred embodiment, the genetically engineered bacterium is Candida viswanathii, Candida tropicalis, or Candida sake.

[0016] The second aspect of the present invention provides a genetically engineered bacterium for producing a dibasic acid, which is obtained by down-regulating the expression of the GOGAT gene and simultaneously over-expressing the GDH gene in a strain for producing a dibasic acid.

[0017] Among them, the method for down-regulating the expression of the GOGAT gene can be conventional in the art, and preferably includes:

[0018] i) Altering the nucleotide sequence encoding the GOGAT gene;

[0019] ii) Altering the regulatory sequence of the GOGAT gene;

[0020] iii) Using i) and ii) simultaneously;

[0021] iv) Reducing the expression of the GOGAT gene by RNA interference technology;

[0022] Among them, the method for over-expressing the GDH gene can be conventional in the art, and preferably includes:

[0023] i) Increasing the copy number of the nucleotide sequence encoding the GDH gene; for example, integrating a homologous or heterologous GDH gene into the genome of the strain or transforming the strain with an expression vector carrying a homologous or heterologous GDH gene;

[0024] ii) Altering the regulatory sequence of the GDH gene; for example, optimizing the nucleic acid sequence of the regulatory sequence, or increasing the number of promoters and / or enhancers;

[0025] iii) Using i) and ii) simultaneously.

[0026] In a preferred embodiment, the method for down-regulating the expression of the GOGAT gene is to delete the nucleotide sequence encoding the GOGAT gene; and, the method for over-expressing the GDH gene is to over-express the nucleotide sequence encoding the GDH gene.

[0027] The strain of the present invention is preferably selected from the genus Corynebacterium, Geotrichum, Candida, Pichia, Rhodotorula, Saccharomyces or Yarrowia.

[0028] The strain is more preferably Candida viswanathii, Candida tropicalis or Candida sake.

[0029] The dibasic acid of the present invention is preferably one or more selected from C9-C22 long-chain dibasic acids.

[0030] The dibasic acid of the present invention is more preferably one or more selected from C9-C18 long-chain dibasic acids.

[0031] The dibasic acid in the present invention is further preferably one or more of C10-C16 long-chain dibasic acids.

[0032] In a preferred embodiment, the dibasic acid is sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid or hexadecanedioic acid.

[0033] The third aspect of the present invention provides a method for preparing a dibasic acid, wherein the dibasic acid is obtained by fermenting and culturing the genetically engineered bacterium described in the first aspect or the second aspect of the present invention, and the dibasic acid is selected from one or more of C9-C22 long-chain dibasic acids.

[0034] In the preparation method, the temperature of the fermentation culture is preferably 25-35 °C, such as 30 °C, and the pH is preferably 7-9, such as 8.

[0035] The culture medium used for the fermentation culture preferably contains 200-400 mL / L of alkane, preferably one or more of C9-C22 long-chain alkanes.

[0036] The alkane is more preferably one or more of C10-C16 long-chain dibasic acids.

[0037] In a preferred embodiment, the alkane is decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane or n-hexadecane.

[0038] The fourth aspect of the present invention provides an application of the genetically engineered bacterium described in the first aspect or the second aspect of the present invention in the preparation of a dibasic acid, and the dibasic acid is selected from one or more of C9-C22 long-chain dibasic acids.

[0039] The GS-GOGAT pathway refers to the glutamine synthetase (E.C.6.3.1.2)-glutamine:α-ketoglutarate aminotransferase (E.C.1.4.1.13) pathway; the GDH gene refers to glutamate dehydrogenase (E.C.1.4.1.4).

[0040] In some embodiments, the amino acid sequence encoded by the GOGAT gene is as shown in SEQ ID NO:27 or has at least 70% sequence identity therewith, for example, a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95% or 99.96% identity. The amino acid sequence encoded by the GDH gene is as shown in SEQ ID NO:28 or has at least 70% sequence identity therewith, for example, a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95% or 99.96% identity.

[0041] The methods for down-regulating the expression of the GOGAT gene include: 1) obtaining a GOGAT protein with loss of function or reduced enzyme activity encoded thereby by altering the gene encoding GOGAT; 2) altering the upstream regulatory sequence of the encoding gene; 3) a combination of the above two methods. The ways for over-expressing the GDH pathway include: 1) increasing the copy number of the gene encoding NADPH-GDH; 2) altering the upstream regulatory sequence of the NADPH-GDH gene; 3) a combination of the above two methods.

[0042] The upstream regulatory sequence refers to the promoter that controls gene expression and may also contain an enhancer region, meaning that by altering the promoter and / or enhancer sequence that controls the expression of the GOGAT gene, the purpose of reducing the GOGAT transcription level is achieved. In a specific embodiment of the present invention, Candida viswanathii strain CAES2113 with the preservation number of CCTCC NO:2020048 is used. Among them: the nucleotide sequences of the GOGAT and GDH genes of Candida viswanathii are as shown in SEQ ID NO:25 and SEQ ID NO:26 respectively, and the amino acid sequences encoded by the GOGAT and GDH genes are as shown in SEQ ID NO:27 and SEQ ID NO:28 respectively.

[0043] The positive and progressive effects of the present invention are as follows:

[0044] The present invention uses a genome editing method to genetically modify the GS-GOGAT and GDH pathways in the ammonium nitrogen absorption pathway - by downregulating GOGAT to block / weaken the GS-GOGAT pathway and overexpressing the GDH pathway, the ammonium nitrogen absorption pathway is modified, and thus a genetically engineered strain of Candida with a significantly increased yield of dibasic acid is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 FIG. 6 is a schematic diagram of two ammonium nitrogen absorption pathways, where each abbreviation represents: α-KG: α-ketoglutarate; Glu: Glutamate; Gln: Glutamine. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0047] Example 1 Introduction to the culture medium, culture fermentation method and dibasic acid detection method

[0048] 1. YPD medium, the formula (w / v) is: 2% peptone, 2% glucose and 1% yeast extract (OXOID, LP0021). 1.5-2% agar powder is also required in the solid medium.

[0049] 2. Seed medium, the formula (w / v) is: 10-20 g / L of sucrose, 3-8 g / L of yeast extract, 2-4 g / L of corn steep liquor for industrial fermentation (referred to as corn steep liquor, total nitrogen content 2.5 wt%), KH 2 PO 4 4-12 g / L, 0.5-4 g / L of urea (sterilized separately at 115 °C for 20 min).

[0050] During cultivation, inoculate a glycerol tube of seeds into a 500 mL shake flask containing 30 mL of seed medium, and culture it on a shaker at 250 rpm and 30 °C until the OD 620 reaches 0.8 (after dilution 30 times).

[0051] 3. Fermentation medium (w / v): 10-40 g / L of sucrose, 1-5 g / L of corn steep liquor (total nitrogen content 2.5 wt%), 4-12 g / L of yeast extract, 0-3 g / L of NaCl, KNO 3 4-12 g / L, KH 2 PO 44 - 12 g / L, urea 0.5 - 3 g / L (sterilized alone at 115 °C for 20 min), and the fermentation substrate is alkane 200 - 400 mL / L (as shown below).

[0052] During fermentation, the seed liquid cultured in 2 was inoculated into a 500 mL shake flask containing 15 mL of fermentation medium, and the inoculation amount was 10 - 30%. Fermentation was carried out at a natural pH value under the conditions of 30 °C and 250 rpm on a shaker for 90 - 144 h.

[0053] 4. Steps for determining the yield of long-chain dibasic acid by acid-base titration

[0054] Adjust the pH of the fermentation broth to 3.0 with 1N hydrochloric acid solution, then add 100 mL of ether to extract the long-chain dibasic acid in the fermentation broth, and then evaporate to remove the ether to obtain long-chain dibasic acid powder; dissolve the obtained long-chain dibasic acid powder in ethanol and titrate it with 0.1 mol / L NaOH solution. Finally, obtain the titration amount of dibasic acid in the fermentation broth and calculate the yield of long-chain dibasic acid based on this.

[0055] Example 2 Cloning of GOGAT gene, construction of genome editing vector and preparation of knockout template

[0056] 1. Cloning of GOGAT gene

[0057] Extract the genomic DNA of Candida viswanathii CAES2113 yeast and use it as a template, and use HS high-fidelity DNA polymerase (Takara, R040A) to amplify the GOGAT gene.

[0058] Recover, purify and sequence verify the PCR product. The results show that the amplified sequence is the same as the known sequence of the GOGAT gene.

[0059] 2. Construction of genome editing vector, the steps are as follows:

[0060] Step 1: Prepare a mixed solution of the following two oligonucleotides with a final concentration of 10 mM, heat at 95 °C for 5 mins, slowly cool to 25 °C at 0.1 °C / S, and keep for 5 mins.

[0061] TGR_GOGAT-F: 5'-TACCTCCGCATTGCCAGTCGCTAA-3' (SEQ ID NO:1)

[0062] TGR_GOGAT-R: 5'-AAACTTAGCGACTGGCAATGCGGA-3' (SEQ ID NO:2)

[0063] Step 2: Digest the vector pC31 (whose sequence is shown in SEQ ID NO: 3) with the restriction endonuclease PaqCI, and after electrophoresis, recover and purify the vector backbone by gel extraction.

[0064] Step 3: Ligation and transformation

[0065] Vector backbone in Step 2: 100 ng

[0066] Annealed double-stranded DNA (diluted 1:200) in Step 2: 2 μl

[0067] 10×T4 DNA polymerase buffer: 2 μl

[0068] 10×T4 DNA polymerase: 1.5 μl

[0069] H 2 O to 20 μl

[0070] Ligate at room temperature for 30 mins, transform the above 6 - 8 μl reaction solution into TOP10 chemically competent cells (Tiangen Biochemical (Beijing) Co., Ltd.), and pick transformants for sequencing verification the next day. The constructed vector is named pC31 - GOGAT.

[0071] 3. Prepare the GOGAT gene knockout template

[0072] Prepare the following PCR reaction system:

[0073] 2×PrimeStarMax: 25 μl

[0074] Primer GOGAT_KO - F (10 mM): 2.5 μl

[0075] Primer GOGAT_KO - R (10 mM): 2.5 μl

[0076] Template: 20 ng of Candida viswanathii CAES2113 genomic DNA

[0077] H 2 O to 20 μl

[0078] The PCR reaction conditions are as follows: Step 1: 98℃ for 30 s, Step 2: 98℃ for 10 s, 52℃ for 30 s, 72℃ for 3 mins, for a total of 30 cycles, Step 3: 72℃ for 5 mins.

[0079] The primer sequences are GOGAT_KO - F (SEQ ID NO: 4) and GOGAT_KO - R (SEQ ID NO: 5).

[0080] After the reaction was completed, 5 μl of sodium acetate solution (3 M, pH 5.2) was added, precipitated with 2 volumes of absolute ethanol, washed twice with 75% ethanol, air-dried and dissolved in H 2 O. This fragment is GOGAT_KO.

[0081] Example 3 Cloning of GDH Gene

[0082] Using the genomic DNA described in Example 1 as a template, the GDH gene was amplified. The primers used were as follows:

[0083] GDH-F: 5'-ATGGTCTTGCCTTACGAACC-3' (SEQ ID NO: 6)

[0084] GDH-R: 5'-TTAGAAGACGTCACCTTGGTC-3' (SEQ ID NO: 7)

[0085] The PCR product was recovered, purified and verified by sequencing. The results showed that the amplified sequence was the same as the known sequence of the GDH gene.

[0086] Example 4 Preparation of Homologous Recombination Template

[0087] All the following steps were carried out using HS high-fidelity DNA polymerase for PCR amplification, (Takara, R040A) The template for amplifying the PCR fragment in Steps 1 to 5 was Candida genomic DNA.

[0088] Step 1: Amplification of the upstream homologous sequence of the integration site. The PCR product was recovered, purified and verified by sequencing, as shown in SEQ ID NO: 8.

[0089] Step 2: Amplification of the promoter sequence. The PCR product was recovered, purified and verified by sequencing, as shown in SEQ ID NO: 9.

[0090] Step 3: Amplification of the target gene GDH sequence. The PCR product was recovered, purified and verified by sequencing, as shown in SEQ ID NO: 10. This sequence contains the coding region of GDH, as well as the boundary sequences homologous to the upstream promoter and downstream terminator for fusion.

[0091] Step 4: Amplification of the terminator sequence Using primers TER-F (SEQ ID NO: 11) and TER-R (SEQ ID NO: 12) to amplify the terminator sequence 1. The PCR product was recovered, purified and verified by sequencing, as shown in SEQ ID NO: 13.

[0092] Using primer TER-2F (shown in SEQ ID NO:14) and TER-R, amplify the terminator sequence 2. The PCR product was confirmed by sequencing and is shown in SEQ ID NO:15.

[0093] Step 5: Amplify the downstream homologous sequence of the integration site, recover, purify the PCR product and perform sequencing verification, as shown in SEQ ID NO:16.

[0094] Step 6: PCR fusion

[0095] Mix the above SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16 in equimolar amounts to 0.05 pmol, and perform fusion PCR amplification with primers SEQ ID NO:6 and SEQ ID NO:12. Then recover, purify and perform sequencing verification, as shown in SEQ ID NO:17 (fusion sequence 1).

[0096] Mix the above SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16 in equimolar amounts to 0.05 pmol, perform fusion PCR amplification with primers SEQ ID NO:5 and SEQ ID NO:12, then recover, purify and perform sequencing verification, as shown in SEQ ID NO:18 (fusion sequence 2). Compared with SEQ ID NO:17, SEQ ID NO:18 does not contain the GDH gene.

[0097] Example 5 Construction of the integration site genome editing vector

[0098] The gRNA recognition site sequence is as follows

[0099] TGR_INTA-F: 5'-TACCACCCAAAAGATAGTTACAAA-3' (SEQ ID NO:19)

[0100] TGR_INTA-R: 5'-AAACTTTGTAACTATCTTTTGGGT-3' (SEQ ID NO:20)

[0101] The vector construction method refers to Example 2, and the constructed vector is called pC31-INTA.

[0102] Example 6 Construction of the Candida viswanathii genetic engineering strain

[0103] 1. The preparation method of yeast electrotransformation competent cells is as follows:

[0104] Pick a fresh streaked Candida versatilis monoclonal colony and inoculate it into 2 ml of YPD medium. Incubate at 30 °C and 200 rpm until the OD 620 reaches 1.3. Then centrifuge at 1500 g for 4 °C to collect the cells. Wash the cells twice with ice-cold sterile water, collect them, and resuspend the cells in 10 ml of 1 M sorbitol solution pre-cooled on ice. After centrifuging at 4 °C and 1500 g to collect the cells, resuspend them in 1 ml of the above sorbitol solution. Aliquot 100 μL of the cell suspension for genetic transformation.

[0105] 2. Yeast competent cell electroporation

[0106] Add 0.2 μg of plasmid DNA and 1 μg of recombinant DNA to the above competent cells, mix gently, place on ice for 5 min, then quickly transfer to a 0.2 cm electroporation cuvette. After electroporation (BioRad, MicropulserTM Electroporator, transformation program SC2), quickly add 1 mL of a mixture of YPD and 1 M sorbitol (1:1, v / v). Incubate at 30 °C and 200 rpm for 2 hours, then collect the bacterial solution and spread it on a YPD medium plate containing 100 mg / L hygromycin B. Incubate statically at 30 °C until single colonies grow.

[0107] 3. Colony PCR method

[0108] Pick a single colony and lyse it with 20 mM NaOH solution on a thermal cycler. The program is as follows:

[0109] 94 °C for 2 mins

[0110] 4 °C for 2 mins

[0111] 5 cycles

[0112] Use this lysate as a template for PCR amplification.

[0113] 1) Obtaining the GOGAT knockout strain

[0114] Add 0.2 μg of pC31-GOGAT plasmid DNA and 1 μg of DNA fragment GOGAT_KO to the competent cells. After electroporation, use the following primers for colony PCR.

[0115] GOGAT_SEQ-F: 5'-CCATCCGCATTGCCAGTCGCTAA-3' (SEQ ID NO:21)

[0116] GOGAT_SEQ-R: 5'-AACTTAGCGACTGGCAATGCGGA-3' (SEQ ID NO:22)

[0117] The amplified PCR fragment was confirmed by sequencing that the GOGAT locus was correctly edited.

[0118] 2) Plasmid deletion

[0119] Pick the positive clones verified by sequencing and inoculate them into 2 ml of YPD medium. Incubate at 30 °C and 200 rpm for 24 - 48 h. Pick a loop of bacteria and streak it on a YPD solid plate. After static incubation at 30 °C for 3 days, pick monoclonal colonies and inoculate them on YPD plates with and without antibiotics respectively. The clones that cannot grow on the plate containing antibiotics but can grow on the plate without antibiotics are the strains that have lost the plasmid. This strain is named gogat.

[0120] 4) Obtaining the overexpressing strain oxGDH

[0121] Using gogat as the host strain, further prepare electrocompetent cells. Add 0.2 μg of pC31 - INTA and 1 μg of the recovered and purified SEQ ID NO:17 to the freshly prepared competent cells. After electroporation, let it stand at 30 °C until monoclonal colonies grow. The primers for colony PCR identification are as follows:

[0122] INTA - F: 5'-AACAAGGAGCTGTAACGGGG-3' (SEQ ID NO:23)

[0123] INTA - R: 5'-AGTTGATGAGAAGCGGTGGG-3' (SEQ ID NO:24)

[0124] The PCR fragment was verified by sequencing, and the GDH expression cassette was correctly integrated into the genome. After the correctly identified clones were plasmid - deleted (by the method described in this example), they were named oxGDH.

[0125] 5) Obtaining the control strain inta

[0126] Using gogat as the host strain, further prepare electrocompetent cells. Add 0.2 μg of pC31 - INTA and 1 μg of the recovered and purified SEQ ID NO:18 to the freshly prepared competent cells. After electroporation, let it stand at 30 °C until monoclonal colonies grow. The primers for colony PCR identification are SEQ ID NO:23 and SEQ ID NO:24. The PCR fragment was verified by sequencing, and the fragment SEQ ID NO:18 was integrated into the genome. After the correctly identified clones were plasmid - deleted (by the method described in this example), they were named inta.

[0127] Example 7 Fermentation of the strain oxGDH to produce decanedioic acid

[0128] Inoculate the strain oxGDH into a 500 mL shake flask containing 30 mL of the seed medium of Example 1, and culture it on a shaker at 250 rpm and 30 °C for 36 - 48 h until the OD 620 reaches 0.8 (after dilution by 30 times). Inoculate the seed liquid into a shake flask containing 15 mL of the fermentation medium of Example 1, with an inoculation amount of 20%. The substrate in the fermentation medium is decane. Continue to culture on a shaker at 250 rpm and 30 °C until the fermentation ends, and use the method described in item 4 of Example 1 to measure the yield of the corresponding dicarboxylic acid with ten carbon atoms.

[0129] Comparative Example 1: Fermentation of long-chain dicarboxylic acid by strain CAES2113

[0130] The culture medium, culture method, and fermentation method are the same as those in Example 7, except that the inoculated strain is strain CAES2113. Use the method described in item 4 of Example 1 to measure the yield of the dicarboxylic acid.

[0131] Comparative Example 2: Fermentation of long-chain dicarboxylic acid by strain gogat

[0132] The culture medium, culture method, and fermentation method are the same as those in Example 7, except that the inoculated strain is strain gogat. Use the method described in item 4 of Example 1 to measure the yield of the dicarboxylic acid.

[0133] Comparative Example 3: Fermentation of long-chain dicarboxylic acid by strain inta

[0134] The culture medium, culture method, and fermentation method are the same as those in Example 7, except that the inoculated strain is inta. Use the method described in item 4 of Example 1 to measure the yield of the dicarboxylic acid.

[0135] Example 8: Fermentation of dodecanedioic acid by strain oxGDH

[0136] The culture medium, culture method, and fermentation method are the same as those in Example 7, except that the substrate is n-dodecane and the product is dodecanedioic acid.

[0137] Example 9: Fermentation of tridecanedioic acid by strain oxGDH

[0138] The culture medium, culture method, and fermentation method are the same as those in Example 7, except that the substrate is n-tridecane and the product is tridecanedioic acid.

[0139] Example 10: Fermentation of tetradecanedioic acid by strain oxGDH

[0140] The culture medium, culture method, and fermentation method are the same as those in Example 7, except that the substrate is n-tetradecane and the product is tetradecanedioic acid.

[0141] Example 11: Fermentation of pentadecanedioic acid by strain oxGDH

[0142] The culture medium, culture method, and fermentation method were the same as those in Example 7, except that the substrate was n-pentadecane and the product was pentadecanedioic acid.

[0143] Production of hexadecanedioic acid by strain oxGDH in Example 12

[0144] The culture medium, culture method, and fermentation method were the same as those in Example 7, except that the substrate was n-hexadecane and the product was hexadecanedioic acid.

[0145] Results of Examples

[0146] The yields of dicarboxylic acids of strain oxGDH and the control strains CAES2113, gogat, and inta in Examples 8 - 13 were statistically analyzed, and the results are shown in Table 1 below. It can be seen that the yields of dicarboxylic acids of strain oxGDH were significantly improved.

[0147] Table 1 Fermentation results of different strains using alkanes with different chain lengths as substrates

[0148]

[0149]

[0150] Among them, strain CAES2113 was the starting strain Candida viswanathii CAES2113;

[0151] Strain oxGDH was a strain overexpressing the GDH gene and knocking out the GOGAT gene;

[0152] Strain gogat was a strain only knocking out the GOGAT gene;

[0153] Strain inta was a strain knocking out the GOGAT gene, and compared with oxGDH, a DNA fragment not containing the GDH coding gene was integrated at the same integration site.

Claims

1. A genetically engineered bacterium of the genus Candida, characterized in that the GOGAT gene in the genetically engineered bacterium is inactivated, and the GDH gene is overexpressed; the amino acid sequence encoded by the GOGAT gene is as shown in SEQ ID NO:

27.

2. The genetically engineered bacterium according to claim 1, characterized in that the amino acid sequence encoded by the GDH gene is as shown in SEQ ID NO:

28.

3. The genetically engineered bacterium according to claim 1, characterized in that the nucleotide sequence of the GOGAT gene is as shown in SEQID NO:

25.

4. The genetically engineered bacterium according to claim 1, characterized in that the nucleotide sequence of the GDH gene is as shown in SEQ IDNO:

26.

5. The genetically engineered bacterium according to claim 1, characterized in that the genetically engineered bacterium lacks the amino acid sequence encoded by the GDH gene and overexpresses the amino acid sequence encoded by the GDH gene.

6. The genetically engineered bacterium according to any one of claims 1-5, characterized in that the genetically engineered bacterium is Candida viswanathii, Candida tropicalis or Candida sake.

7. A method for preparing a dibasic acid, characterized in that the dibasic acid is obtained by fermenting and culturing the genetically engineered bacterium according to any one of claims 1-6, and the dibasic acid is selected from one or more of C9-C22 long-chain dibasic acids.

8. The preparation method according to claim 7, characterized in that the dibasic acid is sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid or hexadecanedioic acid.

9. The preparation method according to claim 7, characterized in that the temperature of the fermentation culture is 25-35 °C, and / or the pH is 7-9.

10. Use of the genetically engineered bacterium according to any one of claims 1-6 in the preparation of a dibasic acid, and the dibasic acid is selected from one or more of C9-C22 long-chain dibasic acids.

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