Genetically engineered bacteria for producing dibasic acid and its application

Abstract

The present invention discloses a genetically engineered bacterium for producing dibasic acid and its application. The genetically engineered bacterium is obtained by down-regulating the expression of GOGAT gene and over-expressing GDH gene in a strain producing dibasic acid. Specifically, the existing Candida visweiss is used as a starting strain, and the GS-GOGAT and GDH pathways in the ammonium nitrogen absorption pathway are genetically modified by a genome editing method. The ammonium nitrogen absorption pathway is transformed by down-regulating GOGAT to block / weaken the GS-GOGAT pathway and over-expressing the GDH pathway, thereby obtaining a strain for producing long-chain dibasic acid with significantly improved dibasic acid yield.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and specifically relates to a genetic engineering bacterium for producing dibasic acid, a method for preparing dibasic acid, and application of the genetic engineering bacterium in preparing dibasic acid. Background Art

[0002] The absorption of inorganic nitrogen elements during microbial cultivation mainly includes nitrate nitrogen and ammonium nitrogen. Ammonium nitrogen is the final precursor of inorganic nitrogen source converted into organic nitrogen, and its absorption is mainly achieved through two pathways: GDH (glutamate dehydrogenase, EC:1.4.1.4) pathway and GS-GOGAT (glutamine synthetase-glutamine: α-ketoglutarate aminotransferase, EC6.3.1.2-EC1.4.1.13) pathway.

[0003] like Figure 1 As shown in the figure, both pathways require the consumption of reducing power to absorb ammonium nitrogen: the GDH pathway is responsible for catalyzing the transfer of ammonium nitrogen elements to α-ketoglutarate to generate glutamate, and it also consumes NADPH. The corresponding GS-GOGAT pathway first transfers one molecule of ammonium nitrogen to glutamate under the catalysis of GS to generate glutamine, and then GOGAT is responsible for transferring ammonium nitrogen to α-ketoglutarate and converting 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.

[0004] In different microorganisms, the roles of the GDH pathway and the GS-GOGAT pathway are not the same. For example, when studying the nitrogen absorption pathway of Aspergillus Nidulans, Macheda et al. found that compared with the wild type, mutants lacking NADP-dependent GDH grew slowly on a medium with ammonium nitrogen as the only nitrogen source. Further knocking out the gene encoding GOGAT made the cells completely unable to grow on a medium with ammonium nitrogen as the only nitrogen source. Pombe Schizosaccharomyces pombe lacking the GDH or GS-GOGAT pathway can still grow on ammonium nitrogen medium, but when both pathways are blocked, the mutant's growth ability is completely lost (Perysinakis et al., 1995). Some studies have also found that in some microorganisms, only one of the pathways is involved in ammonium nitrogen absorption: for example, only the GS-GOGAT pathway exists in Agaricus bisporus, and in the heterocysts of cyanobacteria PC7120, although GS and GDH exist, GOGAT is lacking, and it relies on the import of glutamate from adjacent nutrient cells to maintain the activity of GS. Holmes' research showed that the GS-GOGAT pathway is the main pathway for ammonium nitrogen absorption by Candida albicans, Candida tropicalis and other Candida (Holmes, 1989, 1991).

[0005] Glutamate dehydrogenase GDH plays a role in connecting carbon and nitrogen metabolism in most known species. According to the different coenzymes it depends on, it is divided into two categories: NADH-dependent glutamate dehydrogenase and NADPH-dependent glutamate dehydrogenase. There are two types of GDH in higher fungi (deuteromycetes, ascomycetes and basidiomycetes), while most lower fungi only have NADH-dependent glutamate dehydrogenase. The former encodes a subunit of 115kDa in size, which usually functions as a tetramer in fungi and participates in catabolism; while NADPH-dependent glutamate dehydrogenase exists in a hexamer form, and the protein it encodes is relatively small, about 50kDa, and is mainly involved in the absorption of ammonium nitrogen. In short, glutamate is at the core of the amino acid anabolic pathway, and involves the absorption and utilization of nitrogen elements, which has a systemic effect on the growth and metabolism of cells, but its effect on the production of dibasic acids has been less studied.

[0006] In recent years, the production of long-chain dibasic acids by microbial fermentation has gradually replaced the traditional chemical synthesis method and become the main method for the production of long-chain dibasic acids due to its advantages of low carbon, green, high efficiency and low by-product content. By genetic modification, changing the expression level of GS-GOGAT and GDH pathways in the ammonium nitrogen absorption pathway is of great significance for improving the efficiency of microbial nitrogen source utilization and increasing the production of dibasic acids in the process of biosynthesis of dibasic acids. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a genetically engineered bacterium for producing dibasic acids and its application. The present invention adopts a genome editing method to genetically modify the GS-GOGAT and GDH pathways in the ammonium nitrogen absorption pathway of dibasic acid-producing strains such as Candida, and transforms the ammonium nitrogen absorption pathway by down-regulating GOGAT to block / weaken the GS-GOGAT pathway and over-expressing the GDH pathway, thereby obtaining a strain for producing long-chain dibasic acids with significantly improved dibasic acid yield.

[0008] The first 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.

[0009] Wherein, the method of down-regulating the expression of GOGAT gene can be conventional in the art, preferably including:

[0010] i) changing the nucleotide sequence encoding the GOGAT gene;

[0011] ii) altering the regulatory sequence of the GOGAT gene;

[0012] iii) i) and ii) are used simultaneously;

[0013] iv) RNA interference technology reduces the expression of GOGAT gene;

[0014] The method of overexpressing the GDH gene may be conventional in the art, and preferably includes:

[0015] i) increasing the copy number of the gene encoding GDH; for example, integrating a homologous or heterologous GDH gene into the genome of the strain or transforming an expression vector carrying a homologous or heterologous GDH gene into the strain;

[0016] ii) changing 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;

[0017] iii) i) and ii) are used simultaneously.

[0018] In a preferred embodiment, the method of down-regulating the expression of the GOGAT gene is to delete the nucleotide sequence encoding the GOGAT gene; and the method of over-expressing the GDH gene is to over-express the nucleotide sequence encoding the GDH gene.

[0019] The strain of the present invention is preferably selected from the genus Corynebacterium, Geotrichum, Candida, Pichia, Rhodotorula, Saccharomyces or Yarrowia.

[0020] More preferably, the strain is Candida viswanathii, Candida tropicalis or Candida sake.

[0021] The dibasic acid of the present invention is preferably selected from one or more of C9-C22 long-chain dibasic acids.

[0022] The dibasic acid of the present invention is preferably one or more of C9-C18 long-chain dibasic acids.

[0023] The dibasic acid of the present invention is further preferably one or more of C10-C16 long-chain dibasic acids.

[0024] In a preferred embodiment, the dibasic acid is sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedicarboxylic acid or hexadecanedioic acid.

[0025] The second 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 bacteria described in the first aspect of the present invention.

[0026] 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.

[0027] The culture medium used in the fermentation culture preferably contains 200 to 400 mL / L of alkanes, preferably one or more of C9 to C22 long-chain alkanes.

[0028] The alkane is more preferably one or more of C10-C16 long-chain dibasic acids.

[0029] In a preferred embodiment, the alkane is decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, or n-hexadecane.

[0030] The third aspect of the present invention provides a use of the genetically engineered bacteria as described in the first aspect of the present invention in the preparation of dibasic acids.

[0031] The GS-GOGAT pathway refers to the glutamine synthetase (EC6.3.1.2)-glutamine:α-ketoglutarate aminotransferase (EC1.4.1.13) pathway; the GDH gene refers to glutamate dehydrogenase (EC1.4.1.4).

[0032] 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 thereto, such as 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 thereto, such as 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.

[0033] The method of down-regulating the expression of the GOGAT gene includes: 1) obtaining a GOGAT protein with loss of function or reduced enzyme activity by changing the gene encoding GOGAT; 2) changing the upstream regulatory sequence of the encoding gene; 3) a combination of the above two methods. The method of overexpressing the GDH pathway includes: 1) increasing the copy number of the gene encoding NADPH-GDH; 2) changing the upstream regulatory sequence of the NADPH-GDH gene; 3) a combination of the above two methods.

[0034] The upstream regulatory sequence refers to a promoter that controls gene expression and may also include an enhancer region, which means that by changing the promoter and / or enhancer sequence that controls GOGAT gene expression, the purpose of reducing the transcription level of GOGAT is achieved. In a specific embodiment of the present invention, the Candida visweiss CAES2113 strain with a deposit number of CCTCC NO: 2020048 is used. Wherein: the nucleotide sequences of the Candida visweiss GOGAT and GDH genes are 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 shown in SEQ ID NO: 27 and SEQ ID NO: 28.

[0035] The positive and progressive effects of the present invention are:

[0036] The present invention adopts a genome editing method to genetically modify the GS-GOGAT and GDH pathways in the ammonium nitrogen absorption pathway - by down-regulating GOGAT to block / weaken the GS-GOGAT pathway and over-expressing the GDH pathway, the ammonium nitrogen absorption pathway is transformed, thereby obtaining a strain for producing long-chain dicarboxylic acids with significantly improved dicarboxylic acid production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of two ammonium nitrogen absorption pathways, where the abbreviations represent: α-KG: α-ketoglutarate (α-ketoglutarate); Glu: Glutamate (glutamate); Gln: Glutamine (glutamine). DETAILED DESCRIPTION

[0038] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0039] Example 1 Introduction to culture medium, culture and fermentation method, and dibasic acid detection method

[0040] 1. YPD medium, formula (w / v): 2% peptone, 2% glucose and 1% yeast extract (OXOID, LP0021). 1.5-2% agar powder should also be added to the solid medium.

[0041] 2. Seed culture medium, formula (w / v): sucrose 10-20 g / L, yeast extract 3-8 g / L, corn syrup for industrial fermentation (referred to as corn syrup, total nitrogen content 2.5 wt%) 2-4 g / L, KH2PO4 4-12 g / L, urea 0.5-4 g / L (115°C, sterilized separately for 20 min).

[0042] During the culture, inoculate one glycerol tube seed into a 500 mL shake flask containing 30 mL of seed medium and culture at 250 rpm and 30 °C in a shaker until the OD 620 Reach 0.8 (after dilution 30 times).

[0043] 3. Fermentation medium (w / v): sucrose 10-40 g / L, corn steep liquor (total nitrogen content 2.5 wt%) 1-5 g / L, yeast extract 4-12 g / L, NaCl 0-3 g / L, KNO3 4-12 g / L, KH2PO4 4-12 g / L, urea 0.5-3 g / L (115°C, 20 min separate sterilization), fermentation substrate is alkane 200-400 mL / L (as shown below).

[0044] During fermentation, the seed liquid of 2 cultures was inoculated into a 500 mL shaking flask containing 15 mL fermentation medium, with an inoculation amount of 10-30%, and fermented at natural pH at 30°C and 250 rpm, and cultured in a shaking table for 90-144 hours.

[0045] 4. Steps for determining the yield of long-chain dibasic acids by acid-base titration

[0046] The pH of the fermentation broth was adjusted to 3.0 with 1N hydrochloric acid solution, and then 100 mL of ether was added to extract the long-chain dibasic acid in the fermentation broth, and then the ether was removed by evaporation to obtain long-chain dibasic acid powder; the obtained long-chain dibasic acid powder was dissolved in ethanol and titrated with 0.1 mol / L NaOH solution to finally obtain the dibasic acid titration in the fermentation broth, and the long-chain dibasic acid yield was calculated based on this.

[0047] Example 2 Cloning of GOGAT gene, construction of genome editing vector and preparation of knockout template

[0048] 1. Cloning of GOGAT gene

[0049] The genomic DNA of Candida visweiss CAES2113 yeast was extracted and used as a template. HS high-fidelity DNA polymerase (Takara, R040A) was used to amplify the GOGAT gene.

[0050] The PCR product was recovered, purified and sequenced for verification, and the results showed that the amplified sequence was consistent with the known sequence of the GOGAT gene.

[0051] 2. Construct a genome editing vector. The steps are as follows:

[0052] Step 1: Prepare a mixed solution of the following two oligonucleotides with a final concentration of 10 mM, incubate at 95°C for 5 mins, then slowly cool to 25°C at 0.1°C / s and maintain for 5 mins.

[0053] TGR_GOGAT-F:5'-TACCTCCGCATTGCCAGTCGCTAA-3'(SEQ ID NO:1)

[0054] TGR_GOGAT-R:5'-AAACTTAGCGACTGGCAATGCGGA-3'(SEQ ID NO:2)

[0055] Step 2: The vector pC31 (whose sequence is shown in SEQ ID NO: 3) was digested with restriction endonuclease PaqCI, and the vector backbone was purified by gel recovery after electrophoresis.

[0056] Step 3: Connect and convert

[0057] Step 2 Vector backbone: 100ng

[0058] Step 2 annealed double-stranded DNA (1:200 dilution) 2 μl

[0059] 10×T4 DNA polymerase buffer: 2 μl

[0060] 10×T4 DNA polymerase: 1.5 μl

[0061] H2O to 20 μl

[0062] Ligation was carried out at room temperature for 30 minutes, and 6-8 μl of the reaction solution was transformed into TOP10 chemically competent cells (Tiangen Biochemical (Beijing) Co., Ltd.), and the transformants were picked out for sequencing verification the next day. The constructed vector became pC31-GOGAT.

[0063] 3. Preparation of GOGAT gene knockout template

[0064] Prepare the following PCR reaction system:

[0065] 2× PrimeStarMax: 25 μl

[0066] Primer GOGAT_KO-F (10 mM): 2.5 μl

[0067] Primer GOGAT_KO-R (10 mM): 2.5 μl

[0068] Template: Candida visweiss CAES2113 genomic DNA 20ng

[0069] H2O to 20 μl

[0070] The PCR reaction conditions were as follows: step 1: 98°C for 30 s, step 2: 98°C for 10 s, 52°C for 30 s, 72°C for 3 mins, for a total of 30 cycles, step 3: 72°C for 5 mins.

[0071] Primer sequences: GOGAT_KO-F (SEQ ID NO: 4), GOGAT_KO-R (SEQ ID NO: 5).

[0072] After the reaction, 5 μl of sodium acetate solution (3 M, pH 5.2) was added, precipitated with 2 volumes of anhydrous ethanol and washed twice with 75% ethanol, dried and dissolved in H2O. This fragment is GOGAT_KO.

[0073] Example 3 Cloning of GDH gene

[0074] The GDH gene was amplified using the genomic DNA described in Example 1 as a template. The primers used were as follows:

[0075] GDH-F:5'-ATGGTCTTGCCTTACGAACC-3'(SEQ ID NO:6)

[0076] GDH-R:5'-TTAGAAGACGTCACCTTGGTC-3'(SEQ ID NO:7)

[0077] The PCR product was recovered, purified and sequenced for verification, and the results showed that the amplified sequence was consistent with the known sequence of the GDH gene.

[0078] Example 4 Preparation of homologous recombination template

[0079] All the following steps use HS high-fidelity DNA polymerase was used for PCR amplification (Takara, R040A). The template used for amplifying the PCR fragments from step 1 to step 5 was Candida genomic DNA.

[0080] Step 1: Amplify the upstream homologous sequence of the integration site, recover and purify the PCR product and perform sequencing verification, as shown in SEQ ID NO:8.

[0081] Step 2: Amplify the promoter sequence, recover and purify the PCR product and perform sequencing verification, as shown in SEQ ID NO:9.

[0082] Step 3: Amplify the target gene GDH sequence, recover and purify the PCR product and perform sequencing verification, as shown in SEQ ID NO: 10, which contains the coding region of GDH and boundary sequences homologous to the upstream promoter and downstream terminator used for fusion.

[0083] Step 4: Amplify the terminator sequence The terminator sequence 1 was amplified using primers TER-F (SEQ ID NO: 11) and TER-R (SEQ ID NO: 12). The PCR product was recovered and purified and sequenced for verification, as shown in SEQ ID NO: 13.

[0084] Primers TER-2F (as shown in SEQ ID NO: 14) and TER-R were used to amplify terminator sequence 2. The PCR product was confirmed by sequencing, as shown in SEQ ID NO: 15.

[0085] Step 5: Amplify the downstream homologous sequence of the integration site, recover and purify the PCR product and perform sequencing verification, as shown in SEQ ID NO:16.

[0086] Step 6: PCR fusion

[0087] The above SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16 were mixed in equal moles to 0.05 pmol, and fusion PCR amplification was performed with primers SEQ ID NO:6 and SEQ ID NO:12, and then recovered, purified and sequenced for verification, as shown in SEQ ID NO:17 (fusion sequence 1).

[0088] The above SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 16 were mixed to 0.05 pmol, and fusion PCR amplification was performed with primers SEQ ID NO: 5 and SEQ ID NO: 12, and then recovered and purified and sequenced for 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.

[0089] Example 5 Construction of integration site genome editing vector

[0090] The gRNA recognition site sequence is as follows

[0091] TGR_INTA-F:5'-TACCACCCAAAAGATAGTTACAAA-3'(SEQ ID NO:19)

[0092] TGR_INTA-R:5'-AAACTTTGTAACTATTCTTTTGGGT-3'(SEQ ID NO:20)

[0093] The vector construction method was similar to that in Example 2, and the constructed vector was called pC31-INTA.

[0094] Example 6 Construction of genetically engineered strains of Candida vesica

[0095] 1. The preparation method of yeast electroporation competent cells is as follows:

[0096] Pick a freshly streaked Candida visweiss single colony and inoculate it into 2 ml YPD medium, and culture it at 30°C and 200 rpm until OD 620 When the cell density reaches 1.3, the cells are collected by centrifugation at 1500 g and 4°C. The cells are washed twice with ice-cold sterile water and collected, and then resuspended in 10 ml of 1 M sorbitol solution precooled on ice. The cells are collected by centrifugation at 4°C and 1500 g, and then resuspended in 1 ml of the above sorbitol solution. 100 μL of the cell suspension is dispensed for genetic transformation.

[0097] 2. Electroporation of Competent Yeast

[0098] 0.2 μg of plasmid DNA and 1 μg of recombinant DNA were added to the competent cells, mixed gently, placed on ice for 5 min, and then quickly transferred to a 0.2 cm electroporation cup. After electroporation transformation (BioRad, MicropulserTM Electroporator, transformation program SC2), 1 mL of a mixture of YPD and 1 M sorbitol (1:1, v / v) was quickly added. After culturing at 30°C, 200 rpm for 2 hours, the bacterial solution was collected and coated on a YPD medium plate containing 100 mg / L hygromycin B, and cultured at 30°C until a single colony grew.

[0099] 3. Colony PCR method

[0100] Pick a single clone and lyse it with 20mM NaOH solution in a thermal cycler. The procedure is as follows:

[0101] 94℃ 2mins

[0102] 4℃ 2mins

[0103] 5 cycles

[0104] The lysate was used as a template for PCR amplification.

[0105] 1) Obtaining GOGAT knockout strain

[0106] 0.2 μg pC31-GOGAT plasmid DNA and 1 μg DNA fragment GOGAT_KO were added to the competent cells, and colony PCR was performed using the following primers after electroporation transformation.

[0107] GOGAT_SEQ-F:5'-CCATCCGCATTGCCAGTCGCTAA-3'(SEQ ID NO:21)

[0108] GOGAT_SEQ-R:5'-AACTTAGCGACTGGCAATGCGGA-3'(SEQ ID NO:22)

[0109] The amplified PCR fragment was sequenced to confirm that the GOGAT site was correctly edited.

[0110] 2) Plasmid deletion

[0111] The positive clones verified by sequencing were picked and inoculated into 2 ml YPD medium, and cultured at 30°C, 200 rpm for 24-48 h. A ring of bacteria was picked and streaked onto a YPD solid plate. After static culture at 30°C for 3 days, a single clone was picked and inoculated onto YPD plates with and without antibiotics. The clones that could not grow on the plates containing antibiotics but could grow on the plates without antibiotics were the strains that had lost the plasmid, and this strain was named gogat.

[0112] 4) Obtaining oxGDH overexpression strain

[0113] Using gogat as the host bacteria, further prepare electroporation competent cells, add 0.2 μg pC31-INTA and 1 μg recovered and purified SEQ ID NO: 17 to the freshly prepared competent cells, and after electroporation, stand at 30°C until a single clone grows. The primers for colony PCR identification are as follows:

[0114] INTA-F: 5'-AACAAGGAGCTGTAACGGGG-3' (SEQ ID NO: 23)

[0115] INTA-R: 5'-AGTTGATGAGAAGCGGTGGG-3' (SEQ ID NO: 24)

[0116] The PCR fragment was sequenced and verified that the GDH expression cassette was correctly integrated into the genome. The correct clone was deleted from the plasmid (the method is described in this example) and named oxGDH.

[0117] 5) Obtaining the control strain inta

[0118] Using gogat as the host bacteria, further prepare the electroporation competent cells, add 0.2 μg pC31-INTA and 1 μg recovered and purified SEQ ID NO: 18 to the freshly prepared competent cells, and after electroporation, stand at 30°C until a single clone grows. The primers for colony PCR identification are SEQ ID NO: 23 and SEQ ID NO: 24. The PCR fragment is verified by sequencing, and the fragment SEQ ID NO: 18 is integrated into the genome. The correctly identified clone is named inta after plasmid deletion (the method is described in this embodiment).

[0119] Example 7 Fermentation of oxGDH to produce deca-dicarboxylic acid

[0120] Inoculate the strain oxGDH into a 500 mL shake flask containing 30 ml of the seed culture medium of Example 1 and culture at 250 rpm and 30°C for 36 to 48 h until the OD 620 When the concentration reaches 0.8 (after dilution 30 times), the seed solution is inoculated into a shake flask containing 15 ml of the fermentation medium of Example 1, with an inoculation amount of 20%, and the substrate in the fermentation medium is decane. The shaking table is continued at 250 rpm and 30° C. until the fermentation is completed, and the yield of the corresponding deca-dicarboxylic acid is determined using the method described in 4 of Example 1.

[0121] Comparative Example 1 Fermentation of long-chain dibasic acid by strain CAES2113

[0122] The culture medium, culture method and fermentation method were the same as those in Example 7, except that the inoculated strain was strain CAES2113. The yield of dibasic acid was determined using the method described in Example 1, step 4.

[0123] Comparative Example 2: Production of long-chain dibasic acid by fermentation of strain gogat

[0124] The culture medium, culture method and fermentation method are the same as those in Example 7, except that the inoculated strain is strain gogat. The yield of dibasic acid is determined using the method described in Example 1, step 4.

[0125] Comparative Example 3: Production of long-chain dibasic acid by inta fermentation of strain

[0126] The culture medium, culture method and fermentation method are the same as those in Example 7, except that the inoculated strain is inta. The yield of dibasic acid is determined using the method described in Example 1, Part 4.

[0127] Example 8 Fermentation of dodecanedioic acid by strain oxGDH

[0128] 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 dodecane dibasic acid.

[0129] Example 9: Fermentation of tridecanedioic acid by strain oxGDH

[0130] 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.

[0131] Example 10 Fermentation of oxGDH to produce tetradecanedioic acid

[0132] 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.

[0133] Example 11 Fermentation of 15-carbon dicarboxylic acid by strain oxGDH

[0134] The culture medium, culture method and fermentation method are the same as those in Example 7, except that the substrate is n-pentadecane and the product is pentadecanedioic acid.

[0135] Example 12: Fermentation of oxGDH strain to produce hexadecane dibasic acid

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

[0137] Results Example

[0138] The dibasic acid production of the strain oxGDH in Examples 8-13 and the comparative examples CAES2113, gogat and inta was statistically analyzed, and the results are shown in Table 1. It can be seen that the dibasic acid production of the strain oxGDH was significantly improved.

[0139] Table 1 Fermentation results of different strains using alkanes of different chain lengths as substrates

[0140]

[0141] Among them, strain CAES2113 is the starting strain Candida visweiss CAES2113;

[0142] The strain oxGDH is a strain that overexpresses the GDH gene and knocks out the GOGAT gene;

[0143] Strain gogat is a strain in which only the GOGAT gene is knocked out;

[0144] The strain inta is a strain in which the GOGAT gene is knocked out, and compared with oxGDH, a DNA fragment that does not contain the GDH encoding gene is integrated at the same integration site. SEQUENCE LISTING <110> Shanghai Cathay Biotechnology Co., Ltd. CIBT USA Cathay Biotech (Taiyuan) Co., Ltd. <120> Genetically engineered bacteria for producing dibasic acid and its application <130> P21017751C <160> 28 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> TGR_GOGAT-F primer <400> 1 tacctccgca ttgccagtcg ctaa 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TGR_GOGAT-R primer <400> 2 aaacttagcg actggcaatg cgga 24 <210> 3 <211> 11738 <212> DNA <213> Artificial Sequence <220> <223> Vector pC31 <400> 3 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggtctagta tgattgtcaa 420 taatgatggg tcatcgtttc ctgattcgac gttccctgtg gtgtcgttaa atagcctgtc 480 tgaaatctcc tccatgattg tgttggtgtg tgttgtttga ctttcccaat tgcttacatt 540 tttttcttca aggattcgct ccaaaataga cagaaattat cgcgacaagt cagacgaacg 600 tcgcacgagg cgaaccaaat tctttagaag catacgaaaa ctcactttat ttccattaga 660 agtattaaat taacaaatat ataatataca ggatacaaag taaaagcacg cttaagcaac 720 caaagcggaa gcggtagcgg attcgtattt ccagttaggt ggcaagacag cgacggttct 780 gtagtatctg gccaatctgt ggattctaga ttcaatcaaa atcaatctga acttggagtc 840 cttgtccttt ctgtttcttt ccaagtgctt tctgacagag acagccttct tgatcaagta 900 gtacaagtct tctgggattt ctggagccaa accgttggat ttcaagattc tcaagatctt 960 gttaccagtg acaaccttgg cttgggaaac accgtgagca tctctcaaga taacaccaat 1020 ttgagatgga gtcaaaccct ttctggcgta cttgatgact tgttcaacaa cttcgtcaga 1080 agacaacttg aaccaagatg gagcgtttct tgagtatgga agagcggagg aggaaatacc 1140 tttaccctaa aataacaaga gctaatgtta gtaatttgaa aaaaaagacg ttgagcacgc 1200 acaccccatc caccccacag gtgaaacaca tcaaacgtag caagaacaat agttggccct 1260 cccgtcaagg gggcaggtaa ttgtccaagt actttagaaa agtatgtttt tacccataag 1320 atgaacacac acaaaccagc aaaagtatca ccttctgctt ttcttggttg aggttcaaat 1380 tatgtttggc aataatgcag cgacaatttc aagtacctaa agcgtatata gtaacaattc 1440 taggtctgta tagtcgaccg taggtgaatc gttacttta ggcaagacct tgtccctgat 1500 aaagccaggt tgtactttct attcattgag tgtcgtggtg gtggtagtgg tggttgattg 1560 ggctgttgtg gtagtagtag tggttgtgat ttggaacata cagatgaatg catacgaccc 1620 atgatgactg atttgtttct ttattgagtt gatggtaaga aagagaagaa gaggaggtaa 1680 aaaggtggta gagtgaaaaa ttttttctc ttaaaagtga gagagaaa gagaaaaatt 1740 tcactgcgaa acaatggtt ggggacacga cttttcag gatttttac tcgaagcgta 1800 Tatgcaggaa agttgttgtt agggaatg gagccacaag agagctgcga attcgagctc 1860 ggtaccttgc ccgctatgaa caatacagtt tggttgtgct gtttatcgcg taataaatat 1920 tgtttaccaa ttcattacta aaaactctaa gggatgttgg tttgataatg gattactaaa 1980 gcctgagcga agagactga agagcagtg agagcaaggg agcgcgaggc agaggaaaaa 2040 agagtatggg cgtgctgttc ctgcacgtag agcgcaccat tactttaatt ttcgacccag 2100 cttgattcac tcggcgaccc tcttgcttct aatgggatca ggaggaagc aatgggaggg 2160 gagcgaaaaa aacatgcaat tggtacatgg ggggacgcga tgtagggaca cctctaatgg 2220 agccaggtt ttgccggaag ttgaggag tccattggtt atttttacta attggggttga 2280 gtttttttt ttactgattt tcttattaca aaatttcagc aagtccggaa gttgagaat 2340 tttgtgagta gctgtttaat aatttgaca cacacaccca aaagttttt ctgtatcctg 2400 gacggaaatc gctcctggtg cgttggaagc ccaaaggta acgtgtgtag accggagcgt 2460 tctgtataaa tattcaaa ttctccttg aacgaattta aattctga aagaaccttt 2520 eat eat eat eat eat eat eat eat eat eat eat eat eat eat eat eat eat eat eat eat eat 2580 ttaatacttc attaacaac tacaactata atgtcttcag ctccaagta tgtacctchca 2640 ttcgtttta tcattggaa cagagagat aggacggcag attccagag attggacagg 2700 aacaagaaca attaggcaat gggcgagcgg aaatcaatg gatctactca attgatattt 2760 ttaaccagaa cccagccact tggcaata ctcacatgt ccgttccacc cccctcctag 2820 aaattttcac attggctgga attttttt aggaggagat gaatcgttg agaaccgagc 2880 gttcctcgcc cccaattttt tttctgttc gggctcacacg cccaggt caggataat 2940 cagctgcaag gaaaaaaaat aaagtataaa gagaatgccg ttttccccc acatgaaag 3000 3060 tcccaagtag ctataattta atcggtaaa taccgtag tggcatggtt tatctgagtt 3120 cavattctcag tagttacccc aagcaggtgt cgcgacacct gcgtaggtttt tagagctaga 3180 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 3240 gcttttggcc ggcatggtcc cagcctcctc gctggcgccg gctgggcaac atgcttcggc 3300 atggcgaatg ggacctcgag acatgctttg agttttgtt tcccttctct atcctatcat 3360 tctttagttt ttggttatat acttaatatt aatcgaattt tttacaaata aatacattta 3420 agatttgttt tacatctttc gtactcttcg aaatgaaaat cttactttag tctatacaaa 3480 tatcaagcta tgtaataat atgcaaaaaa attataaaat atcaatcttt ccttttcctg 3540 tttttgcccc tgcaaataaa taataataaa aagtatactc tgtgaactga aaccaccctt 3600 ttttcatcct ctggtttgat cacaacggca cgtcgtatcc aagaatcatg cttccgatca 3660 agtacaagtt ggatccttgc ccgctatgaa caatacagtt tggttgtgct gtttatcgcg 3720 taataaatat tgtttaccaa ttcattacta aaaactctaa gggatgttgg tttgataatg 3780 gattactaaa gcctgagcga acgagactga caaggcagtg agagcaaggg agcgcgaggc 3840 agaggaaaaa agagtatggg cgtgctgttc ctgcacgtag agcgcaccat tactttaatt 3900 ttcgacccag cttgattcac tcggcgaccc tcttgctct aatgggatca gggaggaagc 3960 aatgggagggg gagcgaaaa aacatgcaat tggtacatgg ggggacgcga tgtagggaca 4020 cctctaatgg agcccaggtt ttgccggag tttgaggac tccattggtt atttttacta 4080 attgggttga gtttttttt ttactgatt tcttattaca aaatttcagc aagtccggaa 4140 gttgagaaat ttgtgagta gctgtttaat aatttgaca cacacaccca aaagttttt 4200 ctgtatcctg gacggaaatc gctcctggtg cgttggaagc ccaaaggta acgtgtgtag 4260 accggagcgt tctgtataaa tattcacaa ttctccttg aacgaattta aattctga 4320 aagaaccttt tctttctt cttcttctt ctcctttca acatcattaa 4380 ttctttcaa ttaatacttc attaacaac tacaactata atgtctcag ctccaagta 4440 tgtacctca ttcgttttt tcattggaa caagagagat aggacggcag aattccagag 4500 attggacagg aacaagaaca attaggcaat gggcgagcgg aaatcaatg gatctactca 4560 attgatattt tcaccagaa cccagccact tggcaata ctcacatgt ccgttccacc 4620 cccctcctag aaatttcac attggctgga attttttt aggaggagat gaatcgttg 4680 agaaccgagc gttcctcgcc cccaattttt tttctgttc ggctcacacg cccaaggt 4740 caagtaat cagctgcaag gaaaaaaaat aaagtataaa gagaatgccg ttttccccc 4800 acattgaaag ttttcaga ttttgcaac caacgttta tcaagttcttt ttttttgg 4860 cacacgtct tccaacta gtatggacaa gaatacagt atcggattag atatcggtac 4920 taattcagtg ggatgggctg ttattactga tgaatacaag gtgcctagta aaagtttaa 4980 ggttttggtt atactgacc gtcactccat tagagaat ttaatcggtg ctttgttatt 5040 cgattcaggt gaaccgctg aagctaccag attaaagcgt actgctagac gtagatacac 5100 ccgtcgtaaa aaccgtattt gttacttaca ggaaatttc agtaacgaga tggctaggt 5160 ggatgattca ttctccaca gattggaaga gtcctttta gtcgaagagg ataaaaagca 5220 cgagcgtcat cctatctcg gaaacatcgt tgacgaggtt gcttaccatg agaagtatcc 5280 aaccatctac cacttgagaa agaagttagt ggattccacc gataagccg acttgcgttt 5340 gatctactta gctttggcac acatgatcaa gtttcgtgga cactttttga tcgagggcga 5400 cttgaaccca gataactccg acgttgacaa gttgtttatt cagttggtgc aaacatacaa 5460 tcagttattt gaggagaacc ctattaacgc ctccggtgtc gatgcaaagg ccatcttaag 5520 tgctagattg tccaaatcca gacgtttgga gaatttgatt gctcagttac caggagaaaa 5580 aaagaacggt ttatttggaa acttaatcgc tttatcattg ggcttgaccc caaactttaa 5640 atccaatttt gatttggccg aggatgccaa gttgcaatta tccaaagaca catacgatga 5700 cgatttagat aatttgttgg ctcaaatcgg agatcaatat gcagatttgt tcttggcagc 5760 taaaaactta tcagatgcca ttttgttgtc cgatattttg cgtgttaata ccgaaattac 5820 aaaggctcca ttgtcagcct ccatgattaa gagatatgat gagcatcatc aagacttaac 5880 cttattgaag gctttggtga gacagcaatt acctgaaaag tacaaagaaa tcttcttcga 5940 tcagtccaaa aacggatacg ctggttacat tgacggaggt gcatcccaag aggaatttta 6000 caagttcatt aaaccaatct tggaaaaaat ggacggaacc gaagagttat tagttaagtt 6060 aaatagagaa gacttgttaa gaagcaag aacctttgat aacggttcta ttccacca 6120 gatccacttg gtgaattac acgctatctt gcgtagacag gaggacttct acccattctt 6180 aaaagataac agagagaaaa tcgaaagat tttgacattc agaattccat actacgttgg 6240 tccattggct agaggtact cccgttcgc atggatgaca cgtaaatccg aggaccact 6300 cacccatgg aacttcgagg aggttgtgga tagggcgcc cacccagt ccttcattga 6360 gagaatgacc aatttcgata agaacttacc aaatgaaag gtcttgccta aacactcatt 6420 gttatacgag tattcactg tgtataacga attgactaa gtgaagtacg tcaccgaagg 6480 tatgcgtaag ccagcatttt tgtccggcga gcaaagaag gcaattgttg atttattgtt 6540 cagaccaat agaaaagtca ccgttaaaca atttaaggag gattacttca agaaaattga 6600 atgtttcgac tccgtcgaga ttccggagt ggaggataga ttcaatgcct ctttaggaac 6660 ctatcacgac ttgttgaaa tcattaagga caagacttc ttagacaacg aggaaacga 6720 agacatttg gaggacattg tcttacttt gacttgttc gagaccgtg agatgattga 6780 6840 tagacgttac actggttgg gacgtttaag tcgtaagttg attaacggca ttagagataa 6900 gcagtccggt aagaccattt tagatttttt gaagtccgat ggtttcgcta atcgtaactt 6960 tatgcagttg atccacgacg attctttgac ctttaaggaa gatatccaga aggcccaagt 7020 ctccggtcag ggagactctt tacacgagca catcgctaac ttggctggtt cccctgctat 7080 taagaagggc attttgcaga ccgtcaaggt ggtcgatgag ttagtgaaag ttatgggaag 7140 acacaagcca gaaaacatcg ttatcgagat ggcacgtgag aaccaaacaa cacaaaaggg 7200 tcagaagaat tccagagaga gaatgaagag aatcgagaa ggtattaagg agttgggctc 7260 ccaaatctta aaggaacatc ctgtggagaa cacccaattg caaaacgaga aattgtattt 7320 gtattactta cagaatggaa gagacatgta cgttgaccaa gaattagaca ttaacagatt 7380 gagtgattac gatgtcgatc atattgttcc tcaaagttt ttgaaagacg attccattga 7440 taacaaggtc ttgactcgtt ctgataagaa cagaggtaag tccgacaatg ttccatccga 7500 ggaggtcgtc aagaagatga agaactattg gcgtcagtta ttgaacgcta aattgatcac 7560 acaaagaaaa ttcgacaatt tgactaaagc agaaagaggc ggattgtccg aattggacaa 7620 agctggattt atcaaacgtc aattggtcga aaccagacag attaccaagc atgttgctca 7680 gattttagat agtcgtatga acaccaagta cgacgaaaac gacaagttaa tcagagaggt 7740 gaaggtcatt accttgaagt ctaaattagt ttccgatttc cgtaaagact tccagttcta 7800 taaggtccgt gaaattaaca actatcacca cgctcacgat gcctacttaa atgcagtggt 7860 tggtacagct ttaatcaaga aatacccaaa attagaatct gaattcgtgt atggcgacta 7920 caaggtttat gatgttcgta aaatgattgc taagtcagag caagagattg gcaaagctac 7980 cgcaaaatac ttcttcttatt caaacattat gaatttcttc aaaaccgaga ttacattggc 8040 taatggtgaa atccgtaaga gaccattaat tgaaacaaac ggtgaaactg gtgagattgt 8100 gtgggataaa ggtagagatt ttgctactgt cagaaaagtg ttatctatgc cacaggtcaa 8160 tatcgtgaaa aagactgaag ttcagaccgg aggtttcagt aaagagtcaa tcttgccaaa 8220 gagaaacagt gatagttga tcgctagaa gaggattgg gacctaaa agtacggcgg 8280 ctttgattct cctaccgtcg cttactcagt gttagtcgtt gctaagtcg aaaagggaaa 8340 gagtaagag ttaagtccg ttaggatt attgggaatt acattatgg aagatccctc 8400 cttcgagaag aaccctattg acttcttgga ggcaagggt tacaagagg tcaaaagga 8460 cttaatcatt aagttaccta agtattcttt attcgagtta gaaatggcc gtaaacgtat 8520 gttggcttcc gctggtgaat tgcagaaggg taacgagttg gccttgccta gtaagtagt 8580 gaacttttg tacttagctt cccactacga aaattgaag ggaagtccctg aagataatga 8640 gcaaaaacag tgttcgttg aaaacataa gcacttg gatgaatca tcgagcaaat 8700 ttctgagttt tccaagcgtg tgattttggc tgatgcaac ttagatagg tttaagtgc 8760 ctacaacaag cacagagata agccaattag agaacaagct gagaatatca ttcattgtt 8820 caccttaacc aacttgggtg ctccagctgc attcaagtat ttcgacaca ccatcgatcg 8880 taagcgttac acctccacca aggagtgtt ggacgctacc ttaatccatc agtcattac 8940 aggcttatat gaaacacgta tcgacttgtc acaattaggc ggtgatgagg gtgctgatcc 9000 aaaaaagaag cgtaaggttg atcctaagaa aaaacgtaaa gttgacccaa agaagaaaag 9060 aaaggtcgat tacaaggatc atgacggtga ttataaggac cacgacattg actataaaga 9120 tgacgatgat aagtagctgc agacatgctt tgagtttttg tttcccttct ctatcctatc 9180 attctttagt ttttggttat atacttaata ttaatcgaat tttttacaaa taaatacatt 9240 taagatttgt tttacatctt tcgtactctt cgaaatgaaa atcttacttt agtctataca 9300 aatatcaagc tatgtaaata atatgcaaaa aaattataaa atatcaatct ttccttttcc 9360 tgtttttgcc cctgcaaata aataataata aaaagtatac tctgtgaact gaaaccaccc 9420 ttttttcatc ctctggtttg atcacaacgg cacgtcgtat ccaagaatca tgcttccgat 9480 caagtacaag ttgcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 9540 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 9600 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 9660 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 9720 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 9780 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 9840 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 9900 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 9960 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 10020 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 10080 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 10140 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 10200 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 10260 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 10320 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 10380 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attack gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaatga agttttaaat caatctaaag fathers taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctattcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacggggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 10920 tattaattgt tgccgggaag ctaggtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 11040 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 11160 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 11220 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 11280 ttgcccggcg tcaatacggg samaaccgc gccacatagc agaactttaa aagtgctcat 11340 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 11400 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 11460 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 11520 gaaatgttga atactcatac tcttccttt tcaatattat tgaagcattt atcagggtta 11580 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 11640 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 11700 aacctataaa aataggcgta tcacgaggcc ctttcgtc 11738 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> GOGAT_KO-F primer <400> 4 ttcctgagaa taagtcttgg gcttccgcat tgccagtcgc tagctaactg cagctaatca 60 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> GOGAT_KO-R primer <400> 5 acaggcgtct ttttctaact ctgggttgta taaaccttta tgattagctg cagttagcta 60 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GDH-F Primer <400> 6 atggtcttgc cttacgaacc 20 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> GDH-R Primers <400> 7 ttagaagacg tcaccttggt c 21 <210> 8 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Upstream homologous sequence <400> 8 tacttggtac aaactggaaa agcatataca aaatgaagaa aaagagaaca attgcacaaa 60 gcagaacaat caacgactgc tacccctttt ataccgcgtt tcctgtttac tatcatttta 120 ctcgcttaag ttcgacgcaa atcagacgaa aatcccaact tcccccaact cccccttcaa 180 ttcacgtcaa accaccacaa acaggggaac catctcctga tggcccatgt cggagaaacc 240 aaaaaaaatc aatccgacag cggcaggatt cgaacctgcg cgggcgaaac ccaaaagata 300 tctagagagc ggataacaat ttcacacagg ctcgag 336 <210> 9 <211> 1038 <212> DNA <213> Artificial Sequence <220> <223> Promoter sequence <400> 9 gagagcggat aacaatttca cacaggctcg agttactctg agaaaaactt gaacaatatc 60 gattatcgtc cgataaattg tctgagcttg tccgtacgag cctctgatat tcttgcaagt 120 gccgacgctt gcaatccatg tgagagtatc aacaggagag atggagtttg aacccaccca 180 gctagtcctc ttgggcattc cgcggattgt catcagcgaa ctccggtggc tggtgggttg 240 gaggtcaatt agcgggtggt attgtcttgc atcagtcacg tggacaaatc gagcgagaga 300 gaaaaaaaaa ttgtcctcgg taaagaatct tattgtccat gtgaaggcag tacctcggca 360 acagcgcgta gaaggagatc gatgtgtgtg tgcccacccc cacctttttc tctcctggag 420 gtaagccgac agaaaggaaa aataaggcga cggagttagt gtccgttatg tgacacattt 480 ttcattggcg gagattatac cgagctccaa ttgtaatatt tcgggagaaa tatcgttggg 540 gtaaaacaac agagagagag agggagagat ggttctggta gaattataat ctggttgttg 600 caaatgctac tgatcgactc tggcaatgtc tgtagctcgc tagttgtatg caacttaggt 660 gttatgcata cacacggtta ttcggttgaa ttgtggagta aaaattgtct gagttgtgtc 720 ttagctactg gctggccccc cgcgaaagat aatcaaaatt acacttgtga atttttgcac 780 acacaccgat taacatttcc cttttttgtc caccgataca cgcttgcctc ttcttttttt 840 tctctgtgct tccccctcct gtgacttttt ccaccattga tataaaatca actccatttc 900 cctaaaatct ccccagattc taaaaacaac ttcttctctt ctgcttttcc tttttttttg 960 ttatatttat ttaccatccc ttttttttga atagttattc cccactaaca ttgttcaaat 1020 cttcacgaca tactgcag 1038 <210> 10 <211> 1441 <212> DNA <213> Artificial Sequence <220> <223> GDH coding sequence <400> 10 actaacattg ttcaaatctt cacgacatac tgcagatggt cttgccttac gaaccagaat 60 tccaacaagc ttacgacgaa ttggtctccg ccatcgaaga ctccaccttg ttccaagaag 120 aaccaaagta caagaaggtc atcccagtcg tctccgtccc tgagagatcc gtccaattca 180 gagtcgcttg ggaaaacgac aagggcgaaa tcgaaatcaa ccacggttac agagtccaat 240 acaactccgc cttgggtcca tacaagggtg gtttgcgttt ccacccaacc gtcaacttgt 300 ccatcttgaa gttcttgggt ttcgagcaga tcttcaagaa tgccttgacc ggcttgtcca 360 tgggtggtgg taaaggtggt tccgacttca acccaaaggg cagaaccgac agcgaaatca 420 gaagattctg tgtcgccttc atgagacaat tggccagata cattggccca gacactgatg 480 tcccagccgg tgacattggt gttggtggta gagaagtcgg cttcatgttt ggtgcataca 540 agcaaatgca aaacaactgg gccggtatct tgaccggtaa aggtttgtcc tggggtggtt 600 ctttgatcag accagaagcc accggttacg gttgtgtcta ctacgtcgaa aagatgattg 660 aaaaggccac cgacggtaag gaatccttca agggcaagag agttgccatc tccggttccg 720 gtaacgttgc tcaatacgcc gctttgaagg tcatcgaatt gggcggtatc gttgtctcct 780 tgtccgactc caagggctcc atcatctcca agaacggtat caccgtcgac caagtccacg 840 ccattgccgc tgccaagttg aagttcaagt ccttggaaga aatcaccaaa gactccgccc 900 acttgttctc cggcgacaac tctgtcgaat acttggctgg tgtccgtcca tggaccaagg 960 ttggtgccgt cgacattgcc ttgccatctg ctacccaaaa cgaagtcagc ggtgacgaag 1020 ccaaggcctt ggttgaagcc ggctgtaaat tcattgctga aggttccaac atgggttcca 1080 ccaaggaagc cattgacgtt ttcgaagcca acagagaatc caacggtgtt tggtacgccc 1140 caggtaaggc cgccaactgt ggtggtgttg ctgtctccgg tttggaaatg gcccaaaact 1200 cccagagagt ccaatggacc gaagccgagg tcgacgccaa gttgaaggac atcatgtaca cttgtttcga caactgttac tccactgctc agaaatactc tgttgaaaag aacagcaacg gcttgccatc cttgttgaag ggtgccaaca ttgctggttt catcaaggtt gccgatgcca tgtttgacca aggtgacgtc ttctaaggat ccgaatagaa gagagtgact cttttgataa g 1441 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Enclosure TER-F <400> 11 needs gagtgactct tttgatas gtc <210> 12 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Indicator TER-R <400> 12 accataccac gttggtaccg cggcgctagc aaggtggttt aaatactgtt tatgcatcg <210> 13 <211> 302 <212> DNA <213> Artificial Sequence <220> <223> Properties of snowflakes1 <400> 13 gathering gagtgactct tttgathering gtcgcaaatt tgatttcata agtatattt cattatgtaa agtagtaaat ggaaaattca ttaaaaaaaa agcaaatttc cgttgtatgc atactccga cacaaaacta gccccggaa aacccttagt tgatagttgc gaatttaggt 240. cgacctctca tgatttcaac cttaccccca cgtctcataa ctcaaggccg gtcatgacta 300. ttgctagcgc cgcggtacca acgtggtatg gt 302 <210> 14 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Section TER-2F <400> 14 ttcaaatctt cacgacatac tgcaggaata gaagagagtg actcttttga taagagtc <210> 15 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> Equipment Scale2 <400> 15 60. ttcaaatctt cacgacatac tgcaggaata gaagagagtg actcttttga taagagtcgc aaatttgatt tcataagtat atattcatta tgtaaagtag taaatggaaa attcattaaa aaaaaagcaa atttccgttg tatgcatact ccgaacacaa aactagcccc ggaaaaaccc 180 ttagttgata gttgcgaatt taggtcgacc tctcatgatt tcaaccttac ccccacgtct 240 cataactcaa ggccggtcat gactacaccg atgcataaac agtatttaaa ccaccttgct 300 agcgccgcgg taccaacgtg gtatggt 327 <210> 16 <211> 325 <212> DNA <213> Artificial Sequence <220> <223> Downstream homologous sequence <400> 16 ggtaccaacg tggtatggtt gtaagctaat ctttctcttt aaccactcag acacgctgcc 60 tcattcgtgg aaatttatcc ttactgaagt gaccaccgaa gtctggtaga atctaccaac 120 cagcatcaca cgcgtcaaca tcacgtcagt aacaactcac atttcctctc tgtgtttata 180 ttatcttatt gcttactttc tagcgtgtgt tgttaaggtt ttcggtagta tacaggggtc 240 ttctacattc agtttgtcac gtgatcagcc tctcgaaatc ctcgtaatca acctgtaaat 300 cctctaaaca tgcacagaag gcaac 325 <210> 17 <211> 3327 <212> DNA <213> Artificial Sequence <220> <223> Fusion sequence 1 <400> 17 tacttggtac aaactggaaa agcatataca aaatgaagaa aaagagaaca attgcacaaa 60 gcagaacaat caacgactgc tacccctttt ataccgcgtt tcctgtttac tatcatttta 120 ctcgcttaag ttcgacgcaa atcagacgaa aatcccaact tcccccaact cccccttcaa 180 ttcacgtcaa accaccacaa acaggggaac catctcctga tggcccatgt cggagaaacc 240 aaaaaaaatc aatccgacag cggcaggatt cgaacctgcg cgggcgaaac ccaaaagata 300 tctagagagc ggataacaat ttcacacagg ctcgagttac tctgagaaaa acttgaacaa 360 tatcgattat cgtccgataa attgtctgag cttgtccgta cgagcctctg atattcttgc 420 aagtgccgac gcttgcaatc catgtgagag tatcaacagg agagatggag tttgaaccca 480 cccagctagt cctcttgggc attccgcgga ttgtcatcag cgaactccgg tggctggtgg 540 gttggaggtc aattagcggg tggtattgtc ttgcatcagt cacgtggaca aatcgagcga 600 gagagaaaaa aaaattgtcc tcggtaaaga atcttattgt ccatgtgaag gcagtacctc 660 ggcaacagcg cgtagaagga gatcgatgtg tgtgtgccca cccccacctt tttctctcct 720 ggaggtaagc cgacagaaag gaaaaataag gcgacggagt tagtgtccgt tatgtgacac 780 atttttcatt ggcggagatt ataccgagct ccaattgtaa tatttcggga gaaatatcgt 840 tggggtaaaa caacagagag agagagggag agatggttct ggtagaatta taatctggtt 900 gttgcaaatg ctactgatcg actctggcaa tgtctgtagc tcgctagttg tatgcaactt 960 aggtgttatg catacacacg gttattcggt tgaattgtgg agtaaaaatt gtctgagttg 1020 tgtcttagct actggctggc cccccgcgaa agataatcaa aattacactt gtgaattttt 1080 gcacacacac cgattaacat ttcccttttt tgtccaccga tacacgcttg cctcttcttt 1140 tttttctctg tgcttccccc tcctgtgact ttttccacca ttgatataaa atcaactcca 1200 tttccctaaa atctccccag attctaaaaa caacttcttc tcttctgctt ttcctttttt 1260 tttgttatat ttatttacca tccctttttt ttgaatagtt attccccact aacattgttc 1320 aaatcttcac gacatactgc agatggtctt gccttacgaa ccagaattcc aacaagctta 1380 cgacgaattg gtctccgcca tcgaagactc caccttgttc caagaagaac caaagtacaa 1440 gaaggtcatc ccagtcgtct ccgtccctga gagatccgtc caattcagag tcgcttggga 1500 aaacgacaag ggcgaaatcg aaatcaacca cggttacaga gtccaataca actccgcctt 1560 gggtccatac aagggtggtt tgcgtttcca cccaaccgtc aacttgtcca tcttgaagtt 1620 cttgggtttc gagcagatct tcaagaatgc cttgaccggc ttgtccatgg gtggtggtaa 1680 aggtggttcc gacttcaacc caaagggcag aaccgacagc gaaatcagaa gattctgtgt 1740 cgccttcatg agacaattgg ccagatacat tggcccagac actgatgtcc cagccggtga 1800 cattggtgtt ggtggtagag aagtcggctt catgtttggt gcatacaagc aaatgcaaaa 1860 caactgggcc ggtatcttga ccggtaaagg tttgtcctgg ggtggttctt tgatcagacc 1920 agaagccacc ggttacggtt gtgtctacta cgtcgaaaag atgattgaaa aggccaccga 1980 cggtaaggaa tccttcaagg gcaagagagt tgccatctcc ggttccggta acgttgctca 2040 atacgccgct ttgaaggtca tcgaattggg cggtatcgtt gtctccttgt ccgactccaa 2100 gggctccatc atctccaaga acggtatcac cgtcgaccaa gtccacgcca ttgccgctgc 2160 caagttgaag ttcaagtcct tggaagaat caccaaagac tcccccact tgttctccgg 2220 cgacaactct gtcgaatact tggctggtgt ccgtccatgg accaaggttg gtgccgtcga 2280 cattgccttg ccatctgcta cccaaaacga agtcagcggt gacgaagcca aggccttggt 2340 tgaagccggc tgtaattca tgctgagg ttccacatg ggttccacca aggaagccat 2400 tgacgttttc gaagccaaca gagaatccaa cggtgttttgg tacgccccag gtaggccgc 2460 siactgtggt gtgttgctg tctccggtttt ggaatggcc caaactccc agagagtcca 2520 atggaccgaa gccgaggtcg acgccaagtt gaaggacacatc atgtacactt gtttcgacaa 2580 ctgttactcc actgctcaga atactctt tgaaagac agcaacggct tgccatcctt 2640 gttgaagggt gccacattg ctggttcat caggttgcc gatgccatgt ttgaccagg 2700 tgacgtcttc taaggatccg atagaagag agtgactctt ttgataagag tcgcaaattt 2760 gatttcataa gtatattattc attagtaaa gtagtaaatg gaaaattcaat taaaaaaaaa 2820 gcaaatttcc gttgtatgca tactccgaac acaaaactag ccccggaaaa acccttagtt 2880 gatagttgcg aatttaggtc gacctctcat gatttcaacc ttacccccac gtctcataac 2940 tcaaggccgg tcatgactac accgatgcat aaacagtatt taaaccacct tgctagcgcc 3000 gcggtaccaa cgtggtatgg ttgtaagcta atctttctct ttaaccactc agacacgctg 3060 cctcattcgt ggaaatttat ccttactgaa gtgaccaccg aagtctggta gaatctacca 3120 accagcatca cacgcgtcaa catcacgtca gtaacaactc acatttcctc tctgtgttta 3180 tattatctta ttgcttactt tctagcgtgt gttgttaagg ttttcggtag tatacagggg 3240 tcttctacat tcagtttgtc acgtgatcag cctctcgaaa tcctcgtaat caacctgtaa 3300 atcctctaaa catgcacaga aggcaac 3327 <210> 18 <211> 1950 <212> DNA <213> Artificial Sequence <220> <223> Fusion Sequence 2 <400> 18 tacttggtac aaactggaaa agcatataca aaatgaagaa aaagagaaca attgcacaaa 60 gcagaacaat caacgactgc taccctttt ataccgcgtt tcctgtttac tatcatttta 120 ctcgcttaag ttcgacgcaa atcagacgaa aatcccaact tcccccaact cccccttcaa 180 ttcacgtcaa accaccacaa acagggaac catctcctga tggcccatgt cggagaaacc 240 aaaaaaaatc aatccgacag cggcaggatt cgaacctgcg cgggcgaaac ccaaaagata 300 tctagagagc ggataacaat ttcacacagg ctcgagttac tctgagaaaa acttgaacaa 360 tatcgattat cgtccgataa attgtctgag cttgtccgta cgagcctctg atattcttgc 420 aagtgccgac gcttgcaatc catgtgagag tatcaacagg agagatggag tttgaaccca 480 cccagctagt cctcttgggc attccgcgga ttgtcatcag cgaactccgg tggctggtgg 540 gttggaggtc aattagcggg tggtattgtc ttgcatcagt cacgtggaca aatcgagcga 600 gagagaaaaa aaaattgtcc tcggtaaaga atcttattgt ccatgtgaag gcagtacctc 660 ggcaacagcg cgtagaagga gatcgatgtg tgtgtgccca cccccacctt tttctctcct 720 ggaggtaagc cgacagaaag gaaaaataag gcgacggagt tagtgtccgt tatgtgacac 780 atttttcatt ggcggagatt ataccgagct caattgtaa tatttcgggga gaaatatcgt 840 tggggtaaaa caacagagag agagaggggag agatggttct ggtagaatta taatctggtt 900 gttgcaaatg ctactgatcg actctggcaa tgtctgtagc tcgctagttg tatgcaactt 960 aggtgttatg catacacacg gttattcggt tgaattgtgg agtaaaaatt gtctgagttg 1020 tgtcttagct actggctggc cccccgcgaa agataatcaa aattacactt gtgaattttt 1080 gcacacacac cgattaacat ttcccttttt tgtccaccga tacacgcttg cctcttcttt 1140 ttttctctg tgcttccccc tcctgtgact tttccacca ttgatataaa atcaactcca 1200 tttccctaaa atctccccag attctaaaaa caacttctttc tcttctgcttt ttcctttttt 1260 tttgttatat ttatttacca tcccttttt ttgaatagtt attccccact aacattgttc 1320 aaatcttcac gacatactgc aggaatagaa gagagtgact cttttgataa gagtcgcaaa 1380 tttgatttca tagtatata ttcattatgt aaagtagtaa atggaaaatt cattaaaaaa 1440 aaagcaaatt tccgttgtat gcatactccg aacacaaaac tagccccgga aaaaccctta 1500 gttgatagtt gcgaatttag gtcgacctct catgatttca accttacccc cacgtctcat 1560 aactcaaggc cggtcatgac tacaccgatg cataaacagt atttaaacca ccttgctagc 1620 gccgcggtac caacgtggta tggttgtaag ctaatctttc tctttaacca ctcagacacg 1680 ctgcctcatt cgtggaaatt tatccttact gaagtgacca ccgaagtctg gtagaatcta 1740 ccaaccagca tcacacgcgt caacatcacg tcagtaacaa ctcacatttc ctctctgtgt 1800 ttatattatc ttattgctta ctttctagcg tgtgttgtta aggttttcgg tagtatacag 1860 gggtcttcta cattcagttt gtcacgtgat cagcctctcg aaatcctcgt aatcaacctg 1920 taaatcctct aaacatgcac agaaggcaac 1950 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TGR_INTA-F primer <400> 19 taccacccaa aagatagtta caaa 24 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TGR_INTA-R primer <400> 20 aaactttgta actatctttt gggt <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GOGAT_SEQ-F function <400> 21 ccatccgcat tgccagtcgc go <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GOGAT_SEQ-R specification <400> 22 aacttagcga ctggcaatgc gga <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> INTA-F switch <400> 23 aacaaggagc tgtaacgggg <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> INTA-R Building <400> 24 agttgatgag aagcggtggg <210> 25 <211> 6378 <212> DNA <213> Artificial Sequence <220> <223> GOGAT nucleotide sequence <400> 25 atgagtccaa cctcatccta cttgccagaa gaagagttta gtacaactag tgtttacaac 60 tatgacgaag ttcctgagaa taagtcttgg gcttccgcat tgccagtcgc taaaggttta 120 tacaacccag agttagaaaa agacgcctgt ggtgtcggtt tcacctgcca cttgaaaggt 180 gtgccttctc acaaaattgt gtcggacgcc aaaaacttgt tgtgtaatat gacccacaga 240 ggtggtgagt tgaaccccaa cgacggtgac ggtgctggtt tgttgtcttc gatcccacac 300 agattcttga aaagagagtt ccaatactac tgcaacgttg acttgccagc caagggccag 360 tatggtgttg gtaacgtttt cttcaagaag gacgatgctg tcttcgacaa gtccaagaaa 420 acttttgaaa acattgctga ttctttgggc ttaaaagttt tgggctggag agaagttcct 480 cacgactcct ccatcttggg cgctgctgca ttgtccagag aaccatacat cttgcaacca 540 gccgttgtct acaaggaaat atggggccaa gacatctctc ctgaagagtt tgactccaag 600 cacagaaagg atttcgaaaa aagattattc atcttgagaa agcaagcttc tcacaccatt 660 ggtttgcaca actggttcta catttgttcg ctttccaaca agaccattgt ttacaagggt caattggctc caaaacaagt gtactcgtac taccatgatt tggtcaatgc cgagtacgaa 840. tcccatttcg cattggtgca ctcccgtttc tccaccaaca ccttcccttc atgggacaga 840 gctcagcctt tgaggttggc tgcccacaat ggtgaaatca acaccttgag aggcaacaag aactggatga gagccaaaga aggtgtcatg aaatccgaat tgtttggcga tgaaatggac aaattgttcc caatcatcga agaggtggt tccgactccg ctgctttcga taacgttttg gaattgttgg tcataaacgg tgttgtctcc ttgccagaag ccgtcatgat gatgattcca gagcatggc aaaatgacga gtatatcgac ccaaagaaga aggctttcta cgagtgggct gtttgtttga tggaaccatg ggatggtcca gctttgttta cttttgctga tgacagatac tgtggtgcca acttggatcg taatggtttg agaccatgtc gttactatgt tctcgacgac gaccgtatca tctgtgcttc cgaagttggt gttattgaag tgcagccaga aaaggttttg caaaaaggta gattgcaacc aggtaagatg ttgttggttg acaccaaaga aggtcgtatt gttgacgaca aagaattgaa gagcagagtc tcttccaagt acgatttcaa gtcctgggtc 1440 ttggccaaca tgatctcctt acaagacttg aatgaaaaat tggcctccag aagcattgat 1500 gtttctgcca agccagtcga caccaccatc aatgtgcaat ccgacccaag attggttgcc 1560 tctggttact cccacgaaca gcttttgttt gtcttggccc caatggctga aggtaacgaa 1620 gcattgggtt ctatgggtaa cgacaatgcc ttggcttgta tttccgaaca accaaaattg 1680 ttgtacgaat acttccgtca attgttcgcc caagtcacca accctccaat cgatccaatc 1740 agaagaaaaga ttgtcatgtc tttggaatgt tatgtcggtc cacagggtaa cttgttggaa 1800 atgaaaccaa accaattgaa cagattgttg ttgaaatctc caattttgac caactcagaa 1860 ttggttgcca tcaagaacat tgaaactgtc tacccatcct ggtctgtcgc taacatcgac 1920 atcacttttg acaagtctga aggcattcaa ggttacctca acaccattga cagaatctgt 1980 caggctgctt ctagagccat tgccgacgac aaccaaatca tcatcttgag tgatgttaac 2040 actggtccag agagattgcc aatttctgct ttgattgccg ttggtgctgt gcaccaccac 2100 ttggtcagac aaaagcagcg ttcgaaggtt gctcttatca ttgagactca agaagccaag 2160 gaagtccatc acgcttgttg tttggttggt tatggtgccg atggtatcaa cccatatttg 2220 gccatggaaa ctttggttag aatgaagcaa caaggcttgt tgaagaacga gtccttgact 2280 gaagagaaaa tcatctccaa ctataaatct tctattgatg ctggtatctt gaaggttatg 2340 tccaagatgg gtatctcaac cttggcttct tacaaaggtg cccaaatttt tgaagctttg 2400 ggtatcgaca actcagtaat tgacaggtgc tttgctggta ccgcctctag aatcaagggt 2460 gtcactttg agtacattgc ccaagatgct ttcactttgc acgaaagagg ttacccaacc 2520 agagaaacta tcaaaccaat tggattgcca gaaactggtg agtaccactg gagagatggt 2580 ggtgatgccc acatcaacga tccagctgcc attgctttct tgcaagatgc cgttcgtaac 2640 aagaatgaac gagcttacga cgcttatgcc aagaaggaaa acgaagctgt tcgtaactgt 2700 actttgcgtg ggttgttaga tttcgatttc gagtcttcca ctgaagtccc agttgaccaa 2760 gtggaaccat ggactgagat tgtccgtcgt ttcttcacgg gtgccatgtc ctatggttct 2820 atttcgatgg aatcacactc caccttggcc gttgccatga acagattggg tggtaagtcc 2880 aacactggtg aaggtggtga agatgccgcc agatcccaag ttagagacaa tggtgacacc 2940 atgagatccg ctatcaagca agttgcctct ggtagattcg gtgttacttc atactacttg 3000 gccgatgccg acgagttgca aatcaagatg gcccaaggtg ccaagccagg tgaaggtggt 3060 gaattgcccg gtcacaaagt gtctgttgaa attggtaaga ccagacactc tactccaggt 3120 gtcggtttga tttctccacc accacatcac gatatctatt ccatcgagga tttgaagcaa 3180 ttgttgtacg acttgaagtg tgccaaccca cgagcaagaa cttctgtcaa gttggtttct 3240 gaagttggtg tcggtatcgt cgccgctggt gtcgccaagg cggggtccga gaacattttg 3300 gtttctggtg gtgacggtgg tactggtgcc gccaaattga cttccatcaa gtacgctggt 3360 ttgccatggg aattgggttt ggctgaatcc catcaaactt tggttttgaa cgatttgaga 3420 ggcagagtta ttttgcaaac cgatggtcaa atcagaactg gtagagatat cgcattgcc 3480 tgtttgttgg gtgctgaaga atggggttc gctacttctc cattgatcgc catgggttgc 3540 atttacatga gaaagtgtca tttgggtact tgtccagttg gtattgctac ccaagaccca 3600 gagttgagaa agaagtttga aggtactcca gagcacgtca tcaacttttt ctactatttg 3660 gctaacgact tgagaaagtt catggccaaa ttgggtttca gaacaatcaa cgagatggtt 3720 ggtagaactg aaaaattgaa ggtcagagac gattttagaa ccaccaagaa tgccaacatt 3780 gacttgtctc caattttgac tcctgcccac accattagac caggcgttgt cactcattgt 3840 gttagaaagc aagaccacaa attgcatgtc agagtcgaca acaagttgat cgacgaatct 3900 gattgactt tggctaaagg attgccagtc accatcgatt gtgacgttgt caacactgac 3960 cgttccttgg gtaccacttt gtcctaccgt gtctccaaga cttttggtga aagtggcttg 4020 ccacacgaca ccatccacgt caatgtcacc ggttctgctg gtcaatcttt tggtgccttc 4080 ttggcctcgg gtgtcacttt agagttagag ggtgatgcca acgattacat cggtaaaggt 4140 ttgtctggtg gtagaatcat tgtctaccca ccaaaggaggt ccaagttcaa ggctgaggac 4200 cagatcattg ctggtaacac tgccttcttt ggtgctactt ccggttctgc tttcatcaga 4260 ggtattgtcg ctgaaagatt tgctgttaga aactctggtg ccttcattgt cgccgaaggt 4320 actggtgacc acggttgtga gtacatgtct ggtggtagag ttgtcatctt gggtgccact 4380 ggtagaaact ttgcttctgg tatgtgtggt ggtattgctt atgtcttgga catggctcaa 4440 gatttcagcg acaaggtcaa caaggctcaa gttgaattgt ctcaagttac tgaaactgaa 4500 gaaattgcct tcttgagaaa cttgattgaa gaccaccgtc actacactgc ttctgaagtt 4560 gccgacagca tcttgaacga cttcaacaga tacttgccaa gattcgtcaa ggtgttgcca 4620 aacgactata agaaggtttt ggagaaggaa aagcagatgg ctgccgaagc caagaagaag 4680 gaattgagca gcttcttgaa gtccatcaag gaagacccag aagctgatgt aaccaacggt 4740 gaagcttaca agatcaagaa gggtcatgtt cacaaaccag tcaacaagca agaaccaaag 4800 gttgccgatg tcgaagacac tgttttcaac accgaatttg agaagaaggc accagtcaag 4860 ttggacaagc ttagaggttt catgaagtac aagcgtagaa acgaaaagta cagagaagcc 4920 aagtcaagaa ctaaggactg gaacgaaatg acgtctagat tgaccaagga agagttgaag 4980 tatgaaactg ctagatgtat ggactgtggt gttccattct gtacctccga taccggttgt 5040 ccaatctcca acgttattcc aaagtggaac gaattggtgt tcaacgacag atggtacgat 5100 gctttgcaaa gattgcaaat gaccaacaac ttccctgaat tcactggtag aatttgtcca 5160 gcaccatgta acggtgcctg tgttttgggt atcaatgaag acccagtcaa catcaagtcc 5220 gttgaatgtg ccattatcga ccatggtttc gaacagggct ggatcaaacc aaacccacca 5280 actcacagaa ctggcaagac cgttgccgtt attggttctg gtccatccgg tttggctgct 5340 gctgaccaat tgaacaaggc tggtcactct gtcactgtct atgaaagaag cgacagacca 5400 ggtggtttgt tgatgtacgg tattccaaac atgaaattgg acaagggtat cgtcaagaga 5460 agaaccgact tgttggctgc tgaaggcatt gagtttgttt gcaacaccac cgtcggtgaa 5520 gatattactg tttctgaatt gaaatctcaa ttcgacgctg ttgtcttcgc tgttggttct 5580 accatcccaa gagacttgag aatcccaggt agagagttga agaacattgg ctttgctatg 5640 caattattgc actccaacac caaggcattg ttggaagacc aattggaaga cattagaaag 5700 accttggagg gcaagaatgt tgttgtcatt ggtggtggtg ataccggtaa cgattgtttg 5760 ggtacttcta ccagacacgg tgcgaagtcc gtcaccaact ttgagttgtt gccacaccca 5820 ccaaacgcta gaccaaagga caatccatgg ccacaatggc caagaatttt cagagtcgat 5880 tacggtcaca ccgaagtcac tgctcactat ggtaaggacc caagggagta ctcgatcttg 5940 tctaaagagt ttgttggtga tgaagaaggt aatgtcaagg gtatcaagac tgttagagtt 6000 gaatggaagc gttccgactc tggcgcttgg caaatggctg aggtcccagg aagcgaagaa 6060 ttcttcccag ccgacgttgt cttgttgtcc atgggtttcg ttggtcctga atctgacaac 6120 ttggaagttg aaaagaccaa gagaggtacc atttccacca ttgatccaaa tgtttacaag 6180 gttgacagag acgacaatgt tttcgccgct ggtgattgta gaagaggtca atctttggtt 6240 gtctggggta tccaagaagg tcgtcaatgt gctagagaag ttgacgagta tttgatgggt 6300 tctactagat tgccaggtaa cggttctgtt gaacaaagaa actacaagtt gttggaagaa 6360 ttggctgaaa aggtctaa 6378 <210> 26 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> GDH full gene sequence <400> 26 atggtcttgc cttacgaacc agaattccaa caagcttacg acgaattggt ctccgccatc 60 gaagactcca ccttgttcca agaagaacca aagtacaaga aggtcatccc agtcgtctcc 120 gtccctgaga gatccgtcca attcagagtc gcttgggaaa acgacaaggg cgaaatcgaa 180 atcaaccacg gttacagagt ccaatacaac tccgccttgg gtccatacaa gggtggtttg 240 cgtttccacc caaccgtcaa cttgtccatc ttgaagttct tgggtttcga gcagatcttc 300 aagaatgcct tgaccggctt gtccatgggt ggtggtaaag gtggttccga cttcaaccca 360 aagggcagaa ccgacagcga aatcagaaga ttctgtgtcg ccttcatgag acaattggcc 420 agatacattg gcccagacac tgatgtccca gccggtgaca ttggtgttgg tggtagagaa 480 gtcggcttca tgtttggtgc atacaagcaa atgcaaaaca actgggccgg tatcttgacc 540 ggtaaaggtt tgtcctgggg tggttctttg atcagaccag aagccaccgg ttacggttgt 600 gtctactacg tcgaaaagat gattgaaaag gccaccgacg gtaaggaatc cttcaagggc 660 aagagagttg ccatctccgg ttccggtaac gttgctcaat acgccgcttt gaaggtcatc 720 gaattgggcg gtatcgttgt ctccttgtcc gactccaagg gctccatcat ctccaagaac 780 ggtatcaccg tcgaccaagt ccacgcatt gccgctgcca agttgaagtt caagtccttg 840 gaagaaatca ccaaagactc cgcccacttg ttctccggcg acaactctgt cgaatacttg 900 gctggtgtcc gtccatggac caaggttggt gccgtcgaca ttgccttgcc atctgctacc 960 caaaacgaag tcagcggtga cgaagccaag gccttggttg aagccggctg taaattcatt 1020 gctgaaggtt ccaacatggg ttccaccaag gaagccattg acgtttttcga agccaacaga 1080 gaatccaacg gtgtttggta cgccccaggt aaggccgcca actgtggtgg tgttgctgtc 1140 tccggtttgg aaatggccca aaactcccag agagtccaat ggaccgaagc cgaggtcgac 1200 gccaagttga aggacatcat gtacacttgt ttcgacaact gttactccac tgctcagaaa 1260 tactctgttg aaaagaacag caacggcttg ccatccttgt tgaagggtgc caacattgct 1320 ggtttcatca aggttgccga tgccatgttt gaccaaggtg acgtcttcta a 1371 <210> 27 <211> 2125 <212> PRT <213> Artificial Sequence <220> <223> GOGAT amino acid sequence <400> 27 Met Ser Pro Thr Ser Ser Tyr Leu Pro Glu Glu Glu Phe Ser Thr Thr 1 5 10 15 Ser Val Tyr Asn Tyr Asp Glu Val Pro Glu Asn Lys Ser Trp Ala Ser 20 25 30 Ala Leu Pro Val Ala Lys Gly Leu Tyr Asn Pro Glu Leu Glu Lys Asp 35 40 45 Ala Cys Gly Val Gly Phe Thr Cys His Leu Lys Gly Val Pro Ser His 50 55 60 Lys Ile Val Ser Asp Ala Lys Asn Leu Leu Cys Asn Met Thr His Arg 65 70 75 80 Gly Gly Glu Leu Asn Pro Asn Asp Gly Asp Gly Ala Gly Leu Leu Ser 85 90 95 Ser Ile Pro His Arg Phe Leu Lys Arg Glu Phe Gln Tyr Tyr Cys Asn 100 105 110 Val Asp Leu Pro Ala Lys Gly Gln Tyr Gly Val Gly Asn Val Phe Phe 115 120 125 Lys Lys Asp Asp Ala Val Phe Asp Lys Ser Lys Lys Thr Phe Glu Asn 130 135 140 Ile Ala Asp Ser Leu Gly Leu Lys Val Leu Gly Trp Arg Glu Val Pro 145 150 155 160 His Asp Ser Ser Ile Leu Gly Ala Ala Ala Leu Ser Arg Glu Pro Tyr 165 170 175 Ile Leu Gln Pro Ala Val Val Tyr Lys Glu Ile Trp Gly Gln Asp Ile 180 185 190 Ser Pro Glu Glu Phe Asp Ser Lys His Arg Lys Asp Phe Glu Lys Arg 195 200 205 Leu Phe Ile Leu Arg Lys Gln Ala Ser His Thr Ile Gly Leu His Asn 210 215 220 Trp Phe Tyr Ile Cys Ser Leu Ser Asn Lys Thr Ile Val Tyr Lys Gly 225 230 235 240 Gln Leu Ala Pro Lys Gln Val Tyr Ser Tyr Tyr His Asp Leu Val Asn 245 250 255 Ala Glu Tyr Glu Ser His Phe Ala Leu Val His Ser Arg Phe Ser Thr 260 265 270 Asn Thr Phe Pro Ser Trp Asp Arg Ala Gln Pro Leu Arg Leu Ala Ala 275 280 285 His Asn Gly Glu Ile Asn Thr Leu Arg Gly Asn Lys Asn Trp Met Arg 290 295 300 Ala Lys Glu Gly Val Met Lys Ser Glu Leu Phe Gly Asp Glu Met Asp 305 310 315 320 Lys Leu Phe Pro Ile Ile Glu Glu Gly Gly Ser Asp Ser Ala Ala Phe 325 330 335 Asp Asn Val Leu Glu Leu Leu Val Ile Asn Gly Val Val Ser Leu Pro 340 345 350 Glu Ala Val Met Met Met Ile Pro Glu Ala Trp Gln Asn Asp Glu Tyr 355 360 365 Ile Asp Pro Lys Lys Lys Ala Phe Tyr Glu Trp Ala Val Cys Leu Met 370 375 380 Glu Pro Trp Asp Gly Pro Ala Leu Phe Thr Phe Ala Asp Asp Arg Tyr 385 390 395 400 Cys Gly Ala Asn Leu Asp Arg Asn Gly Leu Arg Pro Cys Arg Tyr Tyr 405 410 415 Val Leu Asp Asp Asp Arg Ile Ile Cys Ala Ser Glu Val Gly Val Ile 420 425 430 Glu Val Gln Pro Glu Lys Val Leu Gln Lys Gly Arg Leu Gln Pro Gly 435 440 445 Lys Met Leu Leu Val Asp Thr Lys Glu Gly Arg Ile Val Asp Asp Lys 450 455 460 Glu Leu Lys Ser Arg Val Ser Ser Lys Tyr Asp Phe Lys Ser Trp Val 465 470 475 480 Leu Ala Asn Met Ile Ser Leu Gln Asp Leu Asn Glu Lys Leu Ala Ser 485 490 495 Arg Ser Ile Asp Val Ser Ala Lys Pro Val Asp Thr Thr Ile Asn Val 500 505 510 Gln Ser Asp Pro Arg Leu Val Ala Ser Gly Tyr Ser His Glu Gln Leu 515 520 525 Leu Phe Val Leu Ala Pro Met Ala Glu Gly Asn Glu Ala Leu Gly Ser 530 535 540 Met Gly Asn Asp Asn Ala Leu Ala Cys Ile Ser Glu Gln Pro Lys Leu 545 550 555 560 Leu Tyr Glu Tyr Phe Arg Gln Leu Phe Ala Gln Val Thr Asn Pro Pro 565 570 575 Ile Asp Pro Ile Arg Glu Lys Ile Val Met Ser Leu Glu Cys Tyr Val 580 585 590 Gly Pro Gln Gly Asn Leu Leu Glu Met Lys Pro Asn Gln Leu Asn Arg 595 600 605 Leu Leu Leu Lys Ser Pro Ile Leu Thr Asn Ser Glu Leu Val Ala Ile 610 615 620 Lys Asn Ile Glu Thr Val Tyr Pro Ser Trp Ser Val Ala Asn Ile Asp 625 630 635 640 Ile Thr Phe Asp Lys Ser Glu Gly Ile Gln Gly Tyr Leu Asn Thr Ile 645 650 655 Asp Arg Ile Cys Gln Ala Ala Ser Arg Ala Ile Ala Asp Asp Asn Gln 660 665 670 Ile Ile Ile Leu Ser Asp Val Asn Thr Gly Pro Glu Arg Leu Pro Ile 675 680 685 Ser Ala Leu Ile Ala Val Gly Ala Val His His His Leu Val Arg Gln 690 695 700 Lys Gln Arg Ser Lys Val Ala Leu Ile Ile Glu Thr Gln Glu Ala Lys 705 710 715 720 Glu Val His His Ala Cys Cys Leu Val Gly Tyr Gly Ala Asp Gly Ile 725 730 735 Asn Pro Tyr Leu Ala Met Glu Thr Leu Val Arg Met Lys Gln Gln Gly 740 745 750 Leu Leu Lys Asn Glu Ser Leu Thr Glu Glu Lys Ile Ile Ser Asn Tyr 755 760 765 Lys Ser Ser Ile Asp Ala Gly Ile Leu Lys Val Met Ser Lys Met Gly 770 775 780 Ile Ser Thr Leu Ala Ser Tyr Lys Gly Ala Gln Ile Phe Glu Ala Leu 785 790 795 800 Gly Ile Asp Asn Ser Val Ile Asp Arg Cys Phe Ala Gly Thr Ala Ser 805 810 815 Arg Ile Lys Gly Val Thr Phe Glu Tyr Ile Ala Gln Asp Ala Phe Thr 820 825 830 Leu His Glu Arg Gly Tyr Pro Thr Arg Glu Thr Ile Lys Pro Ile Gly 835 840 845 Leu Pro Glu Thr Gly Glu Tyr His Trp Arg Asp Gly Gly Asp Ala His 850 855 860 Ile Asn Asp Pro Ala Ala Ile Ala Ser Leu Gln Asp Ala Val Arg Asn 865 870 875 880 Lys Asn Glu Arg Ala Tyr Asp Ala Tyr Ala Lys Lys Glu Asn Glu Ala 885 890 895 Val Arg Asn Cys Thr Leu Arg Gly Leu Leu Asp Phe Asp Phe Glu Ser 900 905 910 Ser Thr Glu Val Pro Val Asp Gln Val Glu Pro Trp Thr Glu Ile Val 915 920 925 Arg Arg Phe Phe Thr Gly Ala Met Ser Tyr Gly Ser Ile Ser Met Glu 930 935 940 Ser His Ser Thr Leu Ala Val Ala Met Asn Arg Leu Gly Gly Lys Ser 945 950 955 960 Asn Thr Gly Glu Gly Gly Glu Asp Ala Ala Arg Ser Gln Val Arg Asp 965 970 975 Asn Gly Asp Thr Met Arg Ser Ala Ile Lys Gln Val Ala Ser Gly Arg 980 985 990 Phe Gly Val Thr Ser Tyr Tyr Leu Ala Asp Ala Asp Glu Leu Gln Ile 995 1000 1005 Lys Met Ala Gln Gly Ala Lys Pro Gly Glu Gly Gly Glu Leu Pro 1010 1015 1020 Gly His Lys Val Ser Val Glu Ile Gly Lys Thr Arg His Ser Thr 1025 1030 1035 Pro Gly Val Gly Leu Ile Ser Pro Pro Pro His His Asp Ile Tyr 1040 1045 1050 Ser Ile Glu Asp Leu Lys Gln Leu Leu Tyr Asp Leu Lys Cys Ala 1055 1060 1065 Asn Pro Arg Ala Arg Thr Ser Val Lys Leu Val Ser Glu Val Gly 1070 1075 1080 Val Gly Ile Val Ala Ala Gly Val Ala Lys Ala Gly Ser Glu Asn 1085 1090 1095 Ile Leu Val Ser Gly Gly Asp Gly Gly Thr Gly Ala Ala Lys Leu 1100 1105 1110 Thr Ser Ile Lys Tyr Ala Gly Leu Pro Trp Glu Leu Gly Leu Ala 1115 1120 1125 Glu Ser His Gln Thr Leu Val Leu Asn Asp Leu Arg Gly Arg Val 1130 1135 1140 Ile Leu Gln Thr Asp Gly Gln Ile Arg Thr Gly Arg Asp Ile Ala 1145 1150 1155 Ile Ala Cys Leu Leu Gly Ala Glu Glu Trp Gly Phe Ala Thr Ser 1160 1165 1170 Pro Leu Ile Ala Met Gly Cys Ile Tyr Met Arg Lys Cys His Leu 1175 1180 1185 Gly Thr Cys Pro Val Gly Ile Ala Thr Gln Asp Pro Glu Leu Arg 1190 1195 1200 Lys Lys Phe Glu Gly Thr Pro Glu His Val Ile Asn Phe Phe Tyr 1205 1210 1215 Tyr Leu Ala Asn Asp Leu Arg Lys Phe Met Ala Lys Leu Gly Phe 1220 1225 1230 Arg Thr Ile Asn Glu Met Val Gly Arg Thr Glu Lys Leu Lys Val 1235 1240 1245 Arg Asp Asp Phe Arg Thr Thr Lys Asn Ala Asn Ile Asp Leu Ser 1250 1255 1260 Pro Ile Leu Thr Pro Ala His Thr Ile Arg Pro Gly Val Val Thr 1265 1270 1275 His Cys Val Arg Lys Gln Asp His Lys Leu His Val Arg Val Asp 1280 1285 1290 Asn Lys Leu Ile Asp Glu Ser Glu Leu Thr Leu Ala Lys Gly Leu 1295 1300 1305 Pro Val Thr Ile Asp Cys Asp Val Val Asn Thr Asp Arg Ser Leu 1310 1315 1320 Gly Thr Thr Leu Ser Tyr Arg Val Ser Lys Thr Phe Gly Glu Ser 1325 1330 1335 Gly Leu Pro His Asp Thr Ile His Val Asn Val Thr Gly Ser Ala 1340 1345 1350 Gly Gln Ser Phe Gly Ala Phe Leu Ala Ser Gly Val Thr Leu Glu 1355 1360 1365 Leu Glu Gly Asp Ala Asn Asp Tyr Ile Gly Lys Gly Leu Ser Gly 1370 1375 1380 Gly Arg Ile Ile Val Tyr Pro Pro Lys Glu Ser Lys Phe Lys Ala 1385 1390 1395 Glu Asp Gln Ile Ile Ala Gly Asn Thr Ala Phe Phe Gly Ala Thr 1400 1405 1410 Ser Gly Ser Ala Phe Ile Arg Gly Ile Val Ala Glu Arg Phe Ala 1415 1420 1425 Val Arg Asn Ser Gly Ala Phe Ile Val Ala Glu Gly Thr Gly Asp 1430 1435 1440 His Gly Cys Glu Tyr Met Ser Gly Gly Arg Val Val Ile Leu Gly 1445 1450 1455 Ala Thr Gly Arg Asn Phe Ala Ser Gly Met Cys Gly Gly Ile Ala 1460 1465 1470 Tyr Val Leu Asp Met Ala Gln Asp Phe Ser Asp Lys Val Asn Lys 1475 1480 1485 Ala Gln Val Glu Leu Ser Gln Val Thr Glu Thr Glu Glu Ile Ala 1490 1495 1500 Phe Leu Arg Asn Leu Ile Glu Asp His Arg His Tyr Thr Ala Ser 1505 1510 1515 Glu Val Ala Asp Ser Ile Leu Asn Asp Phe Asn Arg Tyr Leu Pro 1520 1525 1530 Arg Phe Val Lys Val Leu Pro Asn Asp Tyr Lys Val Leu Glu 1535 1540 1545 Lys Glu Lys Gln Met Ala Ala Glu Ala Lys Lys Glu Leu Ser 1550 1555 1560 Ser Phe Leu Lys Ser Ile Lys Glu Asp Pro Glu Ala Asp Val Thr 1565 1570 1575 Asn Gly Glu Ala Tyr Lys Ile Lys Gly His Val His Lys Pro 1580 1585 1590 Val Asn Lys Gln Glu Pro Lys Val Ala Asp Val Glu Asp Thr Val 1595 1600 1605 Phe Asn Thr Glu Phe Glu Lys Ala Pro Val Lys Leu Asp Lys 1610 1615 1620 Leu Arg Gly Phe Met Lys Tyr Lys Arg Asn Glu Lys Tyr Arg 1625 1630 1635 Glu Ala Lys Ser Arg Thr Lys Asp Trp Asn Glu Met Thr Ser Arg 1640 1645 1650 Leu Thr Lys Glu Glu Leu Lys Tyr Glu Thr Ala Arg Cys Met Asp 1655 1660 1665 Cys Gly Val Pro Phe Cys Thr Ser Asp Thr Gly Cys Pro Ile Ser 1670 1675 1680 Asn Val Ile Pro Lys Trp Asn Glu Leu Val Phe Asn Asp Arg Trp 1685 1690 1695 Tyr Asp Ala Leu Gln Arg Leu Gln Met Thr Asn Asn Phe Pro Glu 1700 1705 1710 Phe Thr Gly Arg Ile Cys Pro Ala Pro Cys Asn Gly Ala Cys Val 1715 1720 1725 Leu Gly Ile Asn Glu Asp Pro Val Asn Ile Lys Ser Val Glu Cys 1730 1735 1740 Ala Ile Ile Asp His Gly Phe Glu Gln Gly Trp Ile Lys Pro Asn 1745 1750 1755 Pro Pro Thr His Arg Thr Gly Lys Thr Val Ala Val Ile Gly Ser 1760 1765 1770 Gly Pro Ser Gly Leu Ala Ala Ala Asp Gln Leu Asn Lys Ala Gly 1775 1780 1785 His Ser Val Thr Val Tyr Glu Arg Ser Asp Arg Pro Gly Gly Leu 1790 1795 1800 Leu Met Tyr Gly Ile Pro Asn Met Lys Leu Asp Lys Gly Ile Val 1805 1810 1815 Lys Arg Arg Thr Asp Leu Leu Ala Ala Glu Gly Ile Glu Phe Val 1820 1825 1830 Cys Asn Thr Thr Val Gly Glu Asp Ile Thr Val Ser Glu Leu Lys 1835 1840 1845 Ser Gln Phe Asp Ala Val Val Phe Ala Val Gly Ser Thr Ile Pro 1850 1855 1860 Arg Asp Leu Arg Ile Pro Gly Arg Glu Leu Lys Asn Ile Gly Phe 1865 1870 1875 Ala Met Gln Leu Leu His Ser Asn Thr Lys Ala Leu Leu Glu Asp 1880 1885 1890 Gln Leu Glu Asp Ile Arg Lys Thr Leu Glu Gly Lys Asn Val Val 1895 1900 1905 Val Ile Gly Gly Gly Asp Thr Gly Asn Asp Cys Leu Gly Thr Ser 1910 1915 1920 Thr Arg His Gly Ala Lys Ser Val Thr Asn Phe Glu Leu Leu Pro 1925 1930 1935 His Pro Pro Asn Ala Arg Pro Lys Asp Asn Pro Trp Pro Gln Trp 1940 1945 1950 Pro Arg Ile Phe Arg Val Asp Tyr Gly His Thr Glu Val Thr Ala 1955 1960 1965 His Tyr Gly Lys Asp Pro Arg Glu Tyr Ser Ile Leu Ser Lys Glu 1970 1975 1980 Phe Val Gly Asp Glu Glu Gly Asn Val Lys Gly Ile Lys Thr Val 1985 1990 1995 Arg Val Glu Trp Lys Arg Ser Asp Ser Gly Ala Trp Gln Met Ala 2000 2005 2010 Glu Val Pro Gly Ser Glu Glu Phe Phe Pro Ala Asp Val Val Leu 2015 2020 2025 Leu Ser Met Gly Phe Val Gly Pro Glu Ser Asp Asn Leu Glu Val 2030 2035 2040 Glu Lys Thr Lys Arg Gly Thr Ile Ser Thr Ile Asp Pro Asn Val 2045 2050 2055 Tyr Lys Val Asp Arg Asp Asp Asn Val Phe Ala Ala Gly Asp Cys 2060 2065 2070 Arg Arg Gly Gln Ser Leu Val Val Trp Gly Ile Gln Glu Gly Arg 2075 2080 2085 Gln Cys Ala Arg Glu Val Asp Glu Tyr Leu Met Gly Ser Thr Arg 2090 2095 2100 Leu Pro Gly Asn Gly Ser Val Glu Gln Arg Asn Tyr Lys Leu Leu 2105 2110 2115 Glu Glu Leu Ala Glu Lys Val 2120 2125 <210> 28 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> GDH full gene amino acid sequence <400> 28 Met Val Leu Pro Tyr Glu Pro Glu Phe Gln Gln Ala Tyr Asp Glu Leu 1 5 10 15 Val Ser Ala Ile Glu Asp Ser Thr Leu Phe Gln Glu Glu Pro Lys Tyr 20 25 30 Lys Lys Val Ile Pro Val Val Ser Val Pro Glu Arg Ser Val Gln Phe 35 40 45 Arg Val Ala Trp Glu Asn Asp Lys Gly Glu Ile Glu Ile Asn His Gly 50 55 60 Tyr Arg Val Gln Tyr Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly Leu 65 70 75 80 Arg Phe His Pro Thr Val Asn Leu Ser Ile Leu Lys Phe Leu Gly Phe 85 90 95 Glu Gln Ile Phe Lys Asn Ala Leu Thr Gly Leu Ser Met Gly Gly Gly 100 105 110 Lys Gly Gly Ser Asp Phe Asn Pro Lys Gly Arg Thr Asp Ser Glu Ile 115 120 125 Arg Arg Phe Cys Val Ala Phe Met Arg Gln Leu Ala Arg Tyr Ile Gly 130 135 140 Pro Asp Thr Asp Val Pro Ala Gly Asp Ile Gly Val Gly Gly Arg Glu 145 150 155 160 Val Gly Phe Met Phe Gly Ala Tyr Lys Gln Met Gln Asn Asn Trp Ala 165 170 175 Gly Ile Leu Thr Gly Lys Gly Leu Ser Trp Gly Gly Ser Leu Ile Arg 180 185 190 Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Tyr Val Glu Lys Met Ile 195 200 205 Glu Lys Ala Thr Asp Gly Lys Glu Ser Phe Lys Gly Lys Arg Val Ala 210 215 220 Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu Lys Val Ile 225 230 235 240 Glu Leu Gly Gly Ile Val Val Ser Leu Ser Asp Ser Lys Gly Ser Ile 245 250 255 Ile Ser Lys Asn Gly Ile Thr Val Asp Gln Val His Ala Ile Ala Ala 260 265 270 Ala Lys Leu Lys Phe Lys Ser Leu Glu Glu Ile Thr Lys Asp Ser Ala 275 280 285 His Leu Phe Ser Gly Asp Asn Ser Val Glu Tyr Leu Ala Gly Val Arg 290 295 300 Pro Trp Thr Lys Val Gly Ala Val Asp Ile Ala Leu Pro Ser Ala Thr 305 310 315 320 Gln Asn Glu Val Ser Gly Asp Glu Ala Lys Ala Leu Val Glu Ala Gly 325 330 335 Cys Lys Phe Ile Ala Glu Gly Ser Asn Met Gly Ser Thr Lys Glu Ala 340 345 350 Ile Asp Val Phe Glu Ala Asn Arg Glu Ser Asn Gly Val Trp Tyr Ala 355 360 365 Pro Gly Lys Ala Ala Asn Cys Gly Gly Val Ala Val Ser Gly Leu Glu 370 375 380 Met Ala Gln Asn Ser Gln Arg Val Gln Trp Thr Glu Ala Glu Val Asp 385 390 395 400 Ala Lys Leu Lys Asp Ile Met Tyr Thr Cys Phe Asp Asn Cys Tyr Ser 405 410 415 Thr Ala Gln Lys Tyr Ser Val Glu Lys Asn Ser Asn Gly Leu Pro Ser 420 425 430 Leu Leu Lys Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala Asp Ala 435 440 445 Met Phe Asp Gln Gly Asp Val Phe 450 455

Claims

1. A genetically engineered bacterium for producing a dibasic acid, characterized in that: The method is obtained by down-regulating the expression of GOGAT gene and over-expressing GDH gene in a strain producing dibasic acid; the strain belongs to the genus Candida.

2. The genetically engineered bacterium according to claim 1, characterized in that The method of down-regulating the expression of GOGAT gene includes: i) changing the nucleotide sequence encoding the GOGAT gene; ii) altering the regulatory sequence of the GOGAT gene; iii) i) and ii) are used simultaneously; iv) RNA interference technology reduces the expression of GOGAT gene; And / or, the method of overexpressing the GDH gene includes: i) increasing the copy number of the gene encoding GDH; ii) changing the regulatory sequence of the GDH gene; iii) i) and ii) are used simultaneously.

3. The genetically engineered bacterium according to claim 2, characterized in that: The homologous or heterologous GDH gene is integrated into the genome of the strain or an expression vector carrying the homologous or heterologous GDH gene is transformed into the strain to increase the copy number of the GDH gene.

4. The genetically engineered bacterium according to claim 2, characterized in that: The number of promoters and / or enhancers is increased to change the regulatory sequence of the GDH gene.

5. The genetically engineered bacterium according to claim 1, characterized in that The method of down-regulating the expression of the GOGAT gene is to delete the nucleotide sequence encoding the GOGAT gene; and the method of over-expressing the GDH gene is to over-express the nucleotide sequence encoding the GDH gene.

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

7. The genetically engineered bacterium according to claim 6, characterized in that The Candida visweiss is the Candida visweiss with a preservation number of CCTCCNO: 2020048.

8. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that The dibasic acid is selected from one or more of C9-C22 long-chain dibasic acids.

9. The genetically engineered bacterium according to claim 8, characterized in that The dibasic acid is one or more of C9-C18 long-chain dibasic acids.

10. The genetically engineered bacterium according to claim 8, characterized in that: The dibasic acid is one or more of C10-C16 long-chain dibasic acids.

11. The genetically engineered bacterium according to claim 8, characterized in that: The dibasic acid is sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedicarboxylic acid or hexadecanedioic acid.

12. A method for preparing a dibasic acid, characterized in that: The dibasic acid is obtained by fermenting and culturing the genetically engineered bacteria according to any one of claims 1 to 11.

13. The preparation method according to claim 12, characterized in that: The temperature of the fermentation culture is 25-35°C, and the pH is 7-9.

14. The preparation method according to claim 13, characterized in that: The fermentation temperature is 30°C.

15. The preparation method according to claim 13, characterized in that: The pH of the fermentation culture is 8.

16. The preparation method according to any one of claims 12 to 15, characterized in that: The culture medium used in the fermentation culture contains 200 to 400 mL / L of alkane.

17. The preparation method according to claim 16, characterized in that: The alkane is one or more of C9-C22 long-chain alkanes.

18. The preparation method according to claim 16, characterized in that: The alkane is one or more of C10-C16 long-chain alkanes.

19. The preparation method according to claim 18, characterized in that: The alkane is decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane or n-hexadecane.

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