Threonine dehydratase mutants, recombinant microorganisms producing branched-chain amino acids, and use thereof

Abstract

The present application relates to the field of microbial technology, in particular to threonine dehydratase mutant, branched-chain amino acid-producing recombinant microorganism and application thereof. The present application provides threonine dehydratase mutant, taking wild-type threonine dehydratase of corynebacterium glutamicum as reference sequence, which contains any one or at least two combinations of mutations at positions 54, 131, 171, 218, 243, 381 and 383. The mutant can effectively eliminate the feedback inhibition of isoleucine on threonine dehydratase, significantly improve the catalytic activity of threonine dehydratase in the presence of high-concentration isoleucine, and improve the synthesis capacity, yield and conversion rate of branched-chain amino acids such as isoleucine of microorganism. The recombinant microorganism expressing the mutant provided by the present application can efficiently synthesize isoleucine, and the yield and conversion rate of isoleucine are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to threonine dehydratase mutants, recombinant microorganisms that produce branched-chain amino acids, and their applications. Background Technology

[0002] Branched-chain amino acids (BCAAs), including valine, leucine, and isoleucine, are essential amino acids for the human body, playing vital physiological functions. They are widely used in food, pharmaceuticals, health products, cosmetics, and animal feed, serving as nutritional enhancers, animal feed additives, ingredients in medical products, and cosmetics. Currently, BCAAs are primarily produced through microbial fermentation, with *Escherichia coli* and *Corynebacterium glutamicum* being the main production strains. However, *E. coli* often synthesizes endotoxins, potentially causing product contamination. Therefore, *Corynebacterium glutamicum* has become an ideal strain for producing BCAAs.

[0003] Taking the isoleucine synthesis pathway as an example, in *Corynebacterium glutamicum*, isoleucine is synthesized through ten steps starting from L-aspartic acid. Some of the synthases can also be used to synthesize valine and leucine. Therefore, strains that produce high levels of isoleucine are not easy to obtain.

[0004] To improve the L-isoleucine production capacity of bacterial strains, mutagenesis breeding is often employed. However, strains obtained through this method are prone to degeneration after passage. For example, patent application CN111197014A discloses an L-isoleucine-producing strain (accession number CCTCC NO: M 2019695) obtained through dual mutagenesis using ultraviolet irradiation and methylene biguanide. However, this strain requires the addition of neomycin during cultivation to prevent reversion mutations. Existing technologies also report the rational modification of Corynebacterium glutamicum for L-isoleucine production using genetic engineering (e.g., patent application CN111321100A discloses an L-isoleucine-producing strain with an acid production of 32.1 g / L, a conversion rate of 18.1%, and a fermentation cycle of 70-75 hours). However, the conversion rate remains low and the fermentation cycle is long, failing to meet the demands of industrial production. Summary of the Invention

[0005] One objective of this invention is to provide threonine dehydratase mutants and their applications. Another objective of this invention is to provide recombinant microorganisms expressing threonine dehydratase mutants and their applications.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] This invention provides a threonine dehydratase mutant, using the wild-type threonine dehydratase of Corynebacterium glutamicum as a reference sequence. The threonine dehydratase mutant contains any one or a combination of at least two of the following mutations: alanine at position 54 is replaced with an amino acid other than alanine; valine at position 131 is replaced with an amino acid other than valine; arginine at position 171 is replaced with an amino acid other than arginine; methionine at position 218 is replaced with an amino acid other than methionine; glycine at position 243 is replaced with an amino acid other than glycine; threonine at position 381 is replaced with an amino acid other than threonine; and phenylalanine at position 383 is replaced with an amino acid other than phenylalanine.

[0008] In this invention, threonine dehydratase catalyzes the dehydration and decomposition of threonine to produce ammonia and α-ketobutyrate. This enzyme is regulated by feedback inhibition from isoleucine. The threonine dehydratase in Corynebacterium glutamicum is encoded by the ilvA gene, and the amino acid sequence of the threonine dehydratase in wild-type Corynebacterium glutamicum is shown in SEQ ID NO. 1.

[0009] This invention discovers that mutations in amino acids at positions 54, 131, 171, 218, 243, 381, and 383 of threonine dehydratase can relieve the feedback inhibition of isoleucine on the enzyme, significantly improve the enzyme's activity in the presence of isoleucine, and thus enhance the synthesis capacity of branched-chain amino acids such as isoleucine.

[0010] Preferably, using the wild-type threonine dehydratase of Corynebacterium glutamicum as a reference sequence, the threonine dehydratase mutant contains any one or a combination of at least two of the following mutations: alanine to threonine at position 54, valine to alanine at position 131, arginine to histidine at position 171, methionine to isoleucine at position 218, glycine to arginine at position 243, threonine to alanine at position 381, and phenylalanine to alanine at position 383.

[0011] More preferably, the threonine dehydratase mutant has the amino acid sequence shown in SEQ ID NO.2. This mutant exhibits superior feedback inhibition of isoleucine, significantly enhancing the enzyme's activity in the presence of high concentrations of isoleucine, thereby promoting the efficient synthesis of branched-chain amino acids such as isoleucine.

[0012] Those skilled in the art should understand that adding a tagged protein to the N-terminus or C-terminus of the above-mentioned threonine dehydratase mutant sequence or fusing it with other proteins to form a fusion protein will not significantly change the activity of the above-mentioned threonine dehydratase mutant without altering its own structure. Therefore, the tagged protein or fusion protein mentioned above is also within the scope of protection of this invention.

[0013] The present invention also provides a gene encoding the threonine dehydratase mutant.

[0014] Based on the amino acid sequence of the threonine dehydratase mutant provided above, those skilled in the art can obtain the sequence of its encoding gene. Due to codon degeneracy, there is more than one gene sequence encoding the above amino acid sequence, and all genes capable of encoding the above mutant are within the protection scope of this invention.

[0015] As one embodiment of the present invention, the nucleotide sequence of the gene encoding the threonine dehydratase mutant is shown in SEQ ID NO.3.

[0016] The present invention also provides biological materials containing the said gene, wherein the biological material is recombinant DNA, a vector or a host cell.

[0017] The recombinant DNA may be recombinant DNA obtained by linking elements upstream or downstream of the gene to drive its transcription and translation. The vector may be an expression vector or a cloning vector, including but not limited to plasmid vectors, phage vectors, transposons, etc. The host cell may be a microbial cell.

[0018] Furthermore, the present invention provides a recombinant microorganism expressing the threonine dehydratase mutant.

[0019] The expression of the threonine dehydratase mutant described above can be achieved by integrating the coding gene of the threonine dehydratase mutant onto the chromosome, or by transferring an expression vector carrying the coding gene of the threonine dehydratase mutant.

[0020] This invention does not impose any special limitations on the location of chromosome integration or the type of expression vector, as long as the expression of the threonine dehydratase mutant can be achieved.

[0021] The threonine dehydratase encoding gene present in the starting strain can be inactivated, or it can be directly replaced with the gene encoding a threonine dehydratase mutant.

[0022] In a preferred embodiment of the present invention, the original threonine dehydratase encoding gene on the chromosome is replaced with a gene encoding the threonine dehydratase mutant.

[0023] To further enhance the expression level of the threonine dehydratase mutant, it is preferable to operatively link an optimized ilvA promoter upstream of the coding gene of the threonine dehydratase mutant, wherein the optimized ilvA promoter is obtained by mutating the -10 region (TACACT) of the original ilvA promoter to TATAAT.

[0024] Preferably, the nucleotide sequence of the optimized ilvA promoter is shown in SEQ ID NO.4; in the recombinant microorganism, an optimized ilvA promoter is inserted between the original ilvA promoter and the ilvA coding gene.

[0025] Compared with the original strain, the terminal synthesis pathway of L-isoleucine in the above recombinant microorganisms was significantly enhanced, and the yield and conversion rate of branched-chain amino acids such as isoleucine were significantly improved.

[0026] To further improve the synthesis capacity of branched-chain amino acids such as isoleucine, the above-mentioned recombinant microorganisms can be further modified.

[0027] Preferably, compared with the starting strain, the recombinant microorganism exhibits enhanced expression and / or enzyme activity of phosphoenolpyruvate carboxylase.

[0028] Phosphoenolpyruvate carboxylase (ppc) catalyzes the synthesis of oxaloacetate, a precursor of branched-chain amino acids, from phosphoenolpyruvate. This invention reveals that, in recombinant microorganisms modified with threonine dehydratase, compared to other modification targets, enhancing the expression and activity of phosphoenolpyruvate carboxylase significantly increases the metabolic flux of the isoleucine synthesis pathway, promoting the efficient synthesis of branched-chain amino acids such as isoleucine.

[0029] Furthermore, compared to the original strain, the recombinant microorganism exhibits enhanced expression and / or enzyme activity of glucose-6-phosphate dehydrogenase.

[0030] Glucose-6-phosphate dehydrogenase (GND) catalyzes the production of 6-phosphogluconate-δ-lactone from glucose-6-phosphate, generating reducing power in the process. This invention reveals that in recombinant microorganisms modified with threonine dehydratase, or in recombinant microorganisms jointly modified with threonine dehydratase and phosphoenolpyruvate carboxylase, enhancing the expression and activity of glucose-6-phosphate dehydrogenase and increasing the supply of reducing NADPH can significantly improve the synthesis of branched-chain amino acids such as isoleucine.

[0031] Furthermore, compared with the original strain, the recombinant microorganism exhibits enhanced expression and / or enzyme activity of acetylhydroxy acid synthase and dihydroxy acid reductase.

[0032] Acetylhydroxy acid synthase (ilvBN) and dihydroxy acid reductase (ilvC) are catalytic enzymes in the isoleucine synthesis pathway. In recombinant microorganisms modified with the above targets, further enhancing the expression and enzyme activity of the catalytic enzymes in the isoleucine terminal synthesis pathway is more conducive to the synthesis and accumulation of isoleucine.

[0033] In recombinant microorganisms expressing threonine dehydratase mutants, the above-mentioned modification targets can be combined in different ways. For example, in recombinant microorganisms expressing threonine dehydratase mutants, the expression and / or activity of phosphoenolpyruvate carboxylase can be enhanced; or, in recombinant microorganisms expressing threonine dehydratase mutants, the expression and / or activity of glucose-6-phosphate dehydrogenase can be enhanced; or, the expression and / or activity of phosphoenolpyruvate carboxylase and glucose-6-phosphate dehydrogenase can be enhanced simultaneously; or, in recombinant microorganisms expressing threonine dehydratase mutants, the expression and / or activity of acetylhydroxy acid synthase and dihydroxy acid reductase can be enhanced; or, the expression and / or activity of phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, acetylhydroxy acid synthase, and dihydroxy acid reductase can be enhanced simultaneously.

[0034] The enhancement of expression described above is achieved through any of the following methods:

[0035] (1) Increase the copy number of the target enzyme encoding gene in the starting strain;

[0036] (2) Replace the expression regulatory element of the target enzyme encoding gene in the starting strain with a more active regulatory element;

[0037] (3) Optimize the codons of the target enzyme encoding gene in the starting strain.

[0038] In (1) above, increasing the copy number can be achieved by integrating additional gene copies onto the chromosome or by introducing an expression plasmid carrying the target gene.

[0039] In (2) above, expression regulatory elements include, but are not limited to, promoters, ribosome binding sites (RBS), and enhancers. Among them, promoters with stronger activity include, but are not limited to, Ptac, Plac, Ptrp, and Ptrc.

[0040] Enzyme activity enhancement is achieved through any of the following mechanisms:

[0041] (1) Mutate one or more bases in the target enzyme encoding gene in the starting strain to enhance the enzyme activity of the target enzyme.

[0042] (2) Replace the target enzyme coding gene in the starting strain with the target enzyme coding gene from other biological sources to enhance the enzyme activity of the target enzyme.

[0043] As a preferred embodiment of the present invention, the present invention provides the following recombinant microorganisms:

[0044] The gene encoding threonine dehydratase in the starting strain is replaced with the gene encoding the threonine dehydratase mutant, and transcription of the gene encoding the threonine dehydratase mutant is initiated with the promoter shown in SEQ ID NO.4; and, compared with the starting strain, an additional copy of the ppc gene is integrated into the chromosome of the recombinant microorganism, with Ptac as the promoter of the ppc gene; and the promoter of the gnd gene of the recombinant microorganism is replaced by the original gnd gene promoter with the Ptac promoter, and the recombinant microorganism carries an expression plasmid expressing the ilvBNC gene.

[0045] Preferably, the recombinant microorganism is a bacterium of the genus Corynebacterium or the genus Brachybacterium.

[0046] The Corynebacterium species are preferably selected from Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, and Corynebacterium aminogenes. The Brevibacterium species are preferably selected from Brevibacterium flavum and Brevibacterium lactofermentum.

[0047] In this invention, the starting strain is preferably a bacterium capable of synthesizing and accumulating branched-chain amino acids such as isoleucine.

[0048] In one embodiment of the present invention, the starting strain is MHZ-1012-2. MHZ-1012-2 was deposited on November 30, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo.13406 and classified as *Corynebacterium glutamicum*. This strain is disclosed in patent application CN201611250330.1.

[0049] The recombinant microorganisms provided by the present invention can be constructed using conventional methods in the art. For example, mutations or substitutions of coding genes, replacements of promoters, and integration of additional gene copies into chromosomes can be performed using homologous recombination.

[0050] Based on the functions of the above-mentioned threonine dehydratase mutant and recombinant microorganism, the present invention provides any one of the following applications of the threonine dehydratase mutant, the gene, the biological material, or the recombinant microorganism:

[0051] (1) Application in the construction of engineered bacteria;

[0052] (2) Application in the fermentation production of branched-chain amino acids or their derivatives;

[0053] (3) Application in increasing the yield and / or conversion rate of branched-chain amino acids or their derivatives in strains.

[0054] Preferably, in the application described in (1) above, the engineered bacteria are engineered bacteria of branched-chain amino acids (especially isoleucine). Preferably, they are Corynebacterium glutamicum, Corynebacterium pekinense, or Breviabacterium flavum.

[0055] The branched-chain amino acid derivatives of this invention are substances synthesized using isoleucine, valine, and leucine as precursors. When the ability of recombinant microorganisms to synthesize branched-chain amino acids is significantly improved, the synthetic ability of downstream products using branched-chain amino acids as precursors is also enhanced. Among these, isoleucine derivatives include, but are not limited to, 4-hydroxyisoleucine, N-acetyl-DL-valine, 2-aminobutyric acid, and 2-hydroxyketobutyric acid; valine derivatives include, but are not limited to, pantothenic acid and 2-aminoisovaleric acid; and leucine derivatives include, but are not limited to, N-acetyl-L-leucine.

[0056] The present invention provides a method for fermenting to produce branched-chain amino acids or their derivatives, comprising: culturing the recombinant microorganisms to obtain a culture, and separating and extracting the culture to obtain branched-chain amino acids or their derivatives.

[0057] Specifically, the method for producing branched-chain amino acids or their derivatives by fermentation includes: inoculating the recombinant microorganisms into a seed culture medium for seed culture to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for culture to obtain a fermentation broth; and separating and extracting the fermentation broth to obtain the amino acids or their derivatives.

[0058] Preferably, the fermentation medium comprises the following components: glucose 80-120 g / L, (NH4)2SO4 5-20 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.3-0.8 g / L, corn steep liquor 20-40 g / L, biotin 0.05-0.2 mg / L, and vitamin C. B1 0.05-0.2 mg / L, pH 6.8-7.2.

[0059] Preferably, the seed culture medium comprises the following components: glucose 10-30 g / L, potassium chloride 0.3-0.8 g / L, magnesium sulfate 0.3-0.8 g / L, corn steep liquor 50-70 g / L, biotin 0.2-0.5 mg / L, vitamin C, and vitamin D. B1 1-3 mg / L, pH 6.8-7.2.

[0060] Preferably, the fermentation temperature is 30-33°C.

[0061] In this invention, the branched-chain amino acid is preferably isoleucine.

[0062] The beneficial effects of the present invention are as follows: the threonine dehydratase mutant provided by the present invention can effectively relieve the feedback inhibition of isoleucine on threonine dehydratase, significantly improve the catalytic activity of threonine dehydratase in the presence of high concentrations of isoleucine, and improve the synthesis capacity, yield and conversion rate of branched-chain amino acids such as isoleucine in microorganisms.

[0063] The recombinant microorganism expressing a threonine dehydratase mutant provided by this invention can efficiently synthesize branched-chain amino acids such as isoleucine, and the yield and conversion rate of isoleucine are significantly improved.

[0064] This invention utilizes a combination of metabolic engineering strategies to construct the isoleucine-producing engineered strain MHI-4 / pEKEX2-ilvBNMC. In fed-batch fermentation in a fermenter, this strain accumulates 36.2 g / L of L-isoleucine with a sugar-acid conversion rate of 0.276 g / g. Simultaneously, the content of byproducts L-leucine, L-valine, and L-threonine is significantly reduced. This strain is currently a highly competitive industrial production strain, laying the foundation for the industrial production of L-isoleucine. Detailed Implementation

[0065] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0066] All reagents and materials used in the following examples are commercially available products and can be purchased through commercial channels. The starting strain MHZ-1012-2 is *Corynebacterium glutamicum*, deposited on November 30, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo. 13406. The formulation of the ordinary liquid brain heart extract culture medium is 3.7% brain heart extract powder solution, and the formulation of the ordinary solid brain heart extract culture medium is 3.7% brain heart extract powder solution and 1.8% agar powder.

[0067] The primer sequences used in the following examples are shown in Table 1.

[0068] Table 1 Primer Sequences

[0069]

[0070]

[0071] Example 1: ilvA gene mutation and enhanced ilvA gene expression

[0072] Using MHZ-1012-2 as the starting strain, the ilvA gene in MHZ-1012-2 was mutated to encode the threonine dehydratase mutant shown in SEQ ID NO.2. An optimized promoter was then inserted between the original ilvA promoter and the ilvA encoding gene to enhance ilvA gene expression. The specific method is as follows:

[0073] A DNA fragment (SEQ ID NO.5) containing the optimized ilvA promoter (SEQ ID NO.4) and the mutated ilvA coding region (ORF) sequence (SEQ ID NO.3) was synthesized using a whole-genome synthesis method. The original ilvA gene coding region sequence in the starting strain MHZ-1012-2 was replaced with the synthesized DNA fragment. Specifically, the optimized promoter was inserted between the original ilvA promoter and the ilvA coding gene, and the ilvA coding region was replaced with the mutated coding region. The specific method is as follows:

[0074] Using a vector containing the sequence described in SEQ ID NO. 5 as a template, the optimized promoter region and the mutated ORF region sequence of ilvA were amplified using PI-ilvA-2f / PI-ilvA-2r primers, and named fragment 2 (1591 bp). The upper and lower homologous arms were amplified using the genome of strain MHZ-1012-2 as templates using primer pairs PI-ilvA-1f / PI-ilvA-1r and PI-ilvA-3f / PI-ilvA-3r, respectively, and named fragment 1 (855 bp) and fragment 3 (844 bp). The three fragments were fused by fusion PCR using primer pair PI-ilvA-1f / PI-ilvA-3r, and the fused fragment and vector pK18-mob-sacB were double-digested with restriction enzymes XbaI and BamHI and then recovered. The fragments were ligated using T4 ligase and transformed into T1 competent cells. The competent cells of the electroporated strain MHZ-1012-2, whose recombinant plasmids were correctly sequenced, were subjected to two rounds of screening to obtain modified strains in which the original ilvA gene promoter and its coding region sequence in the starting strain MHZ-1012-2 were replaced with the sequence shown in SEQ ID NO. 5. The appropriateness of the replacement was mainly determined by selecting amplification strains using primer combinations corresponding to the modified sequence (PI-ilvA-ID-F / PI-ilvA-ID-R). In addition, the appropriateness of the replacement was further confirmed by analyzing the modified sequence using primers PI-ilvA-F and PI-ilvA-R. The correctly sequenced strain was named MHI-1.

[0075] Example 2 Construction of strain containing threonine dehydratase mutant

[0076] Using MHZ-1012-2 as the starting strain, the ilvA gene in MHZ-1012-2 was mutated to encode the threonine dehydratase mutant shown in SEQ ID NO.2. The specific method is as follows:

[0077] Using the whole-gene synthesis fragment from Example 1 as a template, primer pair PI-ilvA was used. M -2f / PI-ilvA M -2r amplification yielded the ilvA gene containing the mutation sites Ala54Thr, Val131Ala, Arg171His, Met218Ile, Gly243Arg, Thr381Ala, and Phe383Ala. Using the genome of strain MHZ-1012-2 as a template, primer pair PI-ilvA was used for amplification. TM -1f / PI-ilvA TM -1r amplification yielded the upper homologous arm; using the genome of strain MHZ-1012-2 as a template, primer pair PI-ilvA was used. TM-3f / PI-ilvA TM -3r amplification yielded the lower homologous arm; using a mixture of the three fragments as a template, primer pair PI-ilvA was used. TM -1f / PI-ilvA TM Fusion PCR was performed at -3r to obtain the fusion fragment containing the ilvA mutant. The fusion fragment and the pK18-mob-sacB vector were double-digested with XbaI and BamHI. The two digestion products were ligated with T4 DNA Ligase for 1 hour, and the ligation product was transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-ilvA. M The correctly sequenced recombinant plasmid was electroporated into competent cells of strain MHZ-1012-2. After two rounds of selection, modified bacteria containing the ilvA mutant were obtained. The mutant was then further modified using primers PL-ilvA. TM -F and PL-ilvA TM -R analysis was performed on the modified sequence to confirm the appropriateness of the substitution, and it was named MHI-2.

[0078] Example 3: Increasing the copy number of the ppc gene

[0079] In the MHI-1 strain constructed in Example 1, the ppc gene with Ptac as the promoter was further integrated at site cg1507 to enhance the expression of the ppc gene. The specific method is as follows:

[0080] Using the genome of strain MHZ-1012-2 as a template, the upper homologous arm was amplified using primer pair PI-ppc-1f / PI-ppc-1r, and named fragment 1 (1024 bp); using the genome as a template, the ppc gene and its terminator sequence were amplified using primer pair PI-ppc-2f / PI-ppc-2r, and named fragment 2 (2907 bp); using pXMJ19 as a template, the promoter Ptac was amplified using primer pair PI-ppc-3f / PI-ppc-3r, and named fragment 3 (307 bp). Using the genome of strain MHZ-1012-2 as a template, homologous arms were amplified using primer pair PI-ppc-4f / I-ppc-4r and named fragment 4 (930 bp). Overlapping fragments were obtained using primer pair PI-ppc-1f / PI-ppc-4r with fragments 1-4 as templates. The vector pK18-mob-sacB was linearized using primer pair PI-pK18-F / PI-pK18-R, and then ligated to the linear vector using an assembly kit, followed by transformation into T1 competent cells. The correctly sequenced recombinant plasmid was electroporated into competent cells of strain MHI-1, and strains with integrated ppc genes were obtained after two rounds of screening. The suitability of integration was mainly determined by selecting amplified strains using primer combinations corresponding to the modified sequence (PI-ppc-ID-F / PI-ppc-4r). In addition, the appropriateness of the substitution was confirmed by analyzing the sequence of the selected strain using primers PI-ppc-F and PI-ppc-R. The correctly sequenced strain was named MHI-3.

[0081] Example 4: Enhancing the expression of the gnd gene

[0082] In the MHI-3 strain constructed in Example 3, the original promoter of the gnd gene was further replaced with the strong promoter Ptac to enhance the expression of the gnd gene. The specific method is as follows:

[0083] Using the genome of strain MHZ-1012-2 as a template, the upper homologous arm (986 bp) was amplified using primer pair PI-gnd-1f / PI-gnd-1r; Ptac (289 bp) was amplified using pXMJ19 as a template using primer pair PI-gnd-2f / PI-gnd-2r; and the lower homologous arm (884 bp) was amplified using the genome of MHZ-1012-2 as a template using primer pair PI-gnd-3f / PI-gnd-3r. The three fragments were fused using overlap PCR, and the fusion fragment was ligated to the pK18-mob-sacB vector using the BamHI / EcoRI restriction site, and transformed into T1 competent cells. The correctly sequenced recombinant plasmid was electroporated into competent cells of strain MHI-3. After two rounds of screening, the gnd gene-modified strain was obtained. The suitability of integration was mainly determined by selecting amplified strains using primer combinations corresponding to the modified sequence (PI-gnd-ID-F / PI-ppc-3r). In addition, the appropriateness of the substitution was confirmed by analyzing the sequence of the selected strain using primers PI-gnd-F and PI-gnd-R. The correctly sequenced strain was named MHI-4.

[0084] Example 5: Plasmid overexpression of ilvBNC

[0085] In the strain MHI-4 constructed in Example 4, a plasmid overexpressing ilvBNC was further introduced, as follows:

[0086] Using the MHZ-1012-2 genome as a template, the PilvB-ilvBNC fragment was amplified using primer pair PI-ilvBNC-F / PI-ilvBNC-R. The PilvB-ilvBNC fragment was ligated to plasmid pEKEX2 using the BamHI / EcoRI restriction sites, and then transformed into T1 competent cells to obtain the correctly sequenced expression plasmid pEKEX2-ilvBNC.

[0087] The plasmid pEKEX2-ilvBNC was electroporated into the competent cells of strain MHI-4 to obtain the final strain MHI-4 / pEKEX2-ilvBNC.

[0088] Example 6 Activity assay of threonine dehydratase mutant

[0089] Wild-type and mutant ilvA genes were amplified using primers PI-ilvA-f / PI-ilvA-r as templates from the whole-gene synthesis fragments of MHZ-1012-2 and Example 1, respectively. The ilvA gene fragments were then ligated into the vector pET28a using EcoRI and HindIII. The constructed vector pET28a-ilvA... TMpET28a-ilvA was transformed into E. coli BL21(DE3). Expression was induced using 0.5 mM IPTG, and the threonine dehydratase of MHZ-1012-2 and the threonine dehydratase mutant of this invention (amino acid sequence shown in SEQ ID NO.2) were purified and their activity was measured using the method reported in the literature (Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum).

[0090] The enzyme activity assay results are shown in Table 2. The relative enzyme activity is calculated as 100% of the activity of the wild-type threonine dehydratase without the addition of isoleucine. The results show that the threonine dehydratase mutant with the sequence shown in SEQ ID NO.2 essentially eliminates the feedback inhibition of isoleucine. Even in the presence of high concentrations of isoleucine, it retains high enzyme activity. Furthermore, in the absence of isoleucine, the enzyme activity of the threonine dehydratase mutant shown in SEQ ID NO.2 is 28.9% higher than that of the wild type.

[0091] Table 2 Enzyme activity detection results

[0092]

[0093] Example 7: Shake-flask fermentation verification

[0094] The strains MHI-1, MHI-2, MHI-3, MHI-4, MHI-4 / pEKEX2-ilvBNC obtained in Examples 1-5, as well as the starting strain MHZ-1012-2, were subjected to shake-flask fermentation, and the production performance of L-isoleucine was compared.

[0095] The culture medium used for shake flask fermentation is as follows:

[0096] Solid activation plates: BHI medium 37 g / L; agar powder 20 g / L;

[0097] Seed culture medium: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, urea 1.5 g / L, KH2PO4 4 g / L, K2HPO4 8 g / L, MgSO4·7H2O 0.5 g / L, biotin 100 μg / L, thiamine hydrochloride 1000 μg / L, calcium pantothenate 2000 μg / L, nicotinamide 2000 μg / L, pH adjusted to 7.0.

[0098] Fermentation medium: glucose 100 g / L, soybean meal extract 9.75 g / L, corn steep liquor powder 14.4 g / L, MgSO4·7H2O 2 g / L, KH2PO4·12H2O 2 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·H2O 0.01 g / L, V B1 Prepared with 0.01 g / L of (NH4)2SO4 and 50 g / L of distilled water, pH adjusted to 7.0.

[0099] Among them, the culture of strain MHI-4 / pEKEX2-ilvBNC requires the addition of 25 mg / L kanamycin.

[0100] The method for shake-flask fermentation is as follows:

[0101] Seed culture: Scrape one loopful of strains MHI-1, MHI-2, MHI-3, MHI-4, MHI-4 / pEKEX2-ilvBNC and the starting strain MHZ-1012-2 from the plate and inoculate them into Erlenmeyer flasks (500 ml) containing 50 ml of seed culture medium. Then, culture at 30-31℃ with shaking at 110 rpm for 15-17 h until the OD value is 16-18.

[0102] Shake-flask fermentation: A 10% inoculum was transferred to a 500ml Erlenmeyer flask containing 25ml of fermentation medium, and then cultured at 30-31℃ with shaking at 135rpm for 48 hours. After culture, the content of L-isoleucine and other amino acids in the fermentation broth was detected by HPLC. The fermentation results are shown in Table 3. The results showed that compared with the starting strain, strain MHI-1 produced 1.8 times more L-isoleucine, while the content of byproducts L-leucine and L-valine was significantly reduced, indicating that the modification of the ilvA gene was effective, shifting the metabolic flux from the valine and leucine pathways to the isoleucine synthesis pathway. The L-isoleucine production of strain MHI-2 increased by 54.2% compared with the starting strain, indicating that the ilvA mutant had a significant promoting effect on L-isoleucine production, and the enzyme activity assay results of the ilvA mutant corroborated this effect. The L-isoleucine production of strains MHI-3, MHI-4, and MHI-4 / pEKEX2-ilvBNC was further increased compared with strain MHI-1, by 56.7%, 92.5%, and 2 times, respectively, indicating that increasing the supply of precursors and reducing power has a significant promoting effect on increasing L-isoleucine production.

[0103] Table 3 Results of shake-flask fermentation

[0104]

[0105] Example 8: Fermentation Validation in a Fermenter

[0106] The starting strain MHZ-1012-2 and the engineered strain MHI-4 / pEKEX2-ilvBNC were fed-batch fermented in a 50-L BAILUN fermenter, and the specific method is as follows:

[0107] Simultaneously activate the starting strain MHZ-1012-2 and the engineered strain MHI-4 / pEKEX2-ilvBNC on two solid activation plates and incubate statically at 30°C for 36-48 hours. Transfer the bacterial culture to two 500-ml shake flasks containing 50ml of seed culture medium and incubate at 30°C and 200rpm for 6-8 hours. Transfer 100ml of seed culture to a 50-L seed tank containing 20L of seed culture medium at an inoculation rate of 0.5% (v / v) and incubate at 31-33°C with stirring at 200-800rpm. Then, transfer 2L of seed culture to a 50-L fermenter containing 20L of fermentation culture medium at an inoculation rate of 1%.

[0108] The seed culture medium formula is as follows: glucose 20 g / L, corn steep liquor 60 g / L, potassium chloride 0.5 g / L, magnesium sulfate 0.5 g / L, defoamer 0.3 mL / L, biotin 0.3 mg / L, vitamin C... B1 2 mg / L, the remainder is water.

[0109] The fermentation medium formula is as follows: glucose 80g / L, corn steep liquor 30g / L, ammonium sulfate 10g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, antifoaming agent 0.3mL / L, biotin 0.1mg / L, vitamin C... B1 0.1 mg / L, the remainder being water.

[0110] Fermentation conditions were as follows: tank pressure 0.05-0.1 MPa, aeration rate 10-25 L / min, stirring 200-800 rpm, temperature 31-33℃, pH 6.8-7.2. The residual sugar concentration in the fermentation broth was controlled below 0.5 g / L, and the sugar consumption rate was maintained at 6 g / L / h. When the content of miscellaneous acids (including valine, leucine, and threonine) reached 1 g / L, the sugar consumption rate was gradually reduced at a rate of 0.3 g / L / h until fermentation was completed after 48 hours. The content of L-isoleucine and other amino acids in the fermentation broth was detected by HPLC. The fermentation results are shown in Table 4.

[0111] Table 4 Fermentation results in the fermenter

[0112]

[0113] The above results indicate that the genetically engineered strain MHI-4 / pEKEX2-ilvBNC constructed in this invention can achieve efficient synthesis and accumulation of L-isoleucine during fermentation. At the same time, the content of byproducts L-leucine, L-valine and L-threonine is significantly reduced, and this strain has good prospects for industrial application.

[0114] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Hu Dan <120> Threonine dehydratase mutants, recombinant microorganisms producing branched-chain amino acids and their applications <130> KHP211120023.2 <160> 44 <170> SIPOSequenceListing 1.0 <210> 1 <211> 436 <212> PRT <213> Artificial Sequence <400> 1 Met Ser Glu Thr Tyr Val Ser Glu Lys Ser Pro Gly Val Met Ala Ser 1 5 10 15 Gly Ala Glu Leu Ile Arg Ala Ala Asp Ile Gln Thr Ala Gln Ala Arg 20 25 30 Ile Ser Ser Val Ile Ala Pro Thr Pro Leu Gln Tyr Cys Pro Arg Leu 35 40 45 Ser Glu Glu Thr Gly Ala Glu Ile Tyr Leu Lys Arg Glu Asp Leu Gln 50 55 60 Asp Val Arg Ser Tyr Lys Ile Arg Gly Ala Leu Asn Ser Gly Ala Gln 65 70 75 80 Leu Thr Gln Glu Gln Arg Asp Ala Gly Ile Val Ala Ala Ser Ala Gly 85 90 95 Asn His Ala Gln Gly Val Ala Tyr Val Cys Lys Ser Leu Gly Val Gln 100 105 110 Gly Arg Ile Tyr Val Pro Val Gln Thr Pro Lys Gln Lys Arg Asp Arg 115 120 125 Ile Met Val His Gly Gly Glu Phe Val Ser Leu Val Val Thr Gly Asn 130 135 140 Asn Phe Asp Glu Ala Ser Ala Ala Ala His Glu Asp Ala Glu Arg Thr 145 150 155 160 Gly Ala Thr Leu Ile Glu Pro Phe Asp Ala Arg Asn Thr Val Ile Gly 165 170 175 Gln Gly Thr Val Ala Ala Glu Ile Leu Ser Gln Leu Thr Ser Met Gly 180 185 190 Lys Ser Ala Asp His Val Met Val Pro Val Gly Gly Gly Gly Leu Leu 195 200 205 Ala Gly Val Val Ser Tyr Met Ala Asp Met Ala Pro Arg Thr Ala Ile 210 215 220 Val Gly Ile Glu Pro Ala Gly Ala Ala Ser Met Gln Ala Ala Leu His 225 230 235 240 Asn Gly Gly Pro Ile Thr Leu Glu Thr Val Asp Pro Phe Val Asp Gly 245 250 255 Ala Ala Val Lys Arg Val Gly Asp Leu Asn Tyr Thr Ile Val Glu Lys 260 265 270 Asn Gln Gly Arg Val His Met Met Ser Ala Thr Glu Gly Ala Val Cys 275 280 285 Thr Glu Met Leu Asp Leu Tyr Gln Asn Glu Gly Ile Ile Ala Glu Pro 290 295 300 Ala Gly Ala Leu Ser Ile Ala Gly Leu Lys Glu Met Ser Phe Ala Pro 305 310 315 320 Gly Ser Val Val Val Cys Ile Ile Ser Gly Gly Asn Asn Asp Val Leu 325 330 335 Arg Tyr Ala Glu Ile Ala Glu Arg Ser Leu Val His Arg Gly Leu Lys 340 345 350 His Tyr Phe Leu Val Asn Phe Pro Gln Lys Pro Gly Gln Leu Arg His 355 360 365 Phe Leu Glu Asp Ile Leu Gly Pro Asp Asp Asp Ile Thr Leu Phe Glu 370 375 380 Tyr Leu Lys Arg Asn Asn Arg Glu Thr Gly Thr Ala Leu Val Gly Ile 385 390 395 400 His Leu Ser Glu Ala Ser Gly Leu Asp Ser Leu Leu Glu Arg Met Glu 405 410 415 Glu Ser Ala Ile Asp Ser Arg Arg Leu Glu Pro Gly Thr Pro Glu Tyr 420 425 430 Glu Tyr Leu Thr 435 <210> 2 <211> 436 <212> PRT <213> Artificial Sequence <400> 2 Met Ser Glu Thr Tyr Val Ser Glu Lys Ser Pro Gly Val Met Ala Ser 1 5 10 15 Gly Ala Glu Leu Ile Arg Ala Ala Asp Ile Gln Thr Ala Gln Ala Arg 20 25 30 Ile Ser Ser Val Ile Ala Pro Thr Pro Leu Gln Tyr Cys Pro Arg Leu 35 40 45 Ser Glu Glu Thr Gly Thr Glu Ile Tyr Leu Lys Arg Glu Asp Leu Gln 50 55 60 Asp Val Arg Ser Tyr Lys Ile Arg Gly Ala Leu Asn Ser Gly Ala Gln 65 70 75 80 Leu Thr Gln Glu Gln Arg Asp Ala Gly Ile Val Ala Ala Ser Ala Gly 85 90 95 Asn His Ala Gln Gly Val Ala Tyr Val Cys Lys Ser Leu Gly Val Gln 100 105 110 Gly Arg Ile Tyr Val Pro Val Gln Thr Pro Lys Gln Lys Arg Asp Arg 115 120 125 Ile Met Ala His Gly Gly Glu Phe Val Ser Leu Val Val Thr Gly Asn 130 135 140 Asn Phe Asp Glu Ala Ser Ala Ala Ala His Glu Asp Ala Glu Arg Thr 145 150 155 160 Gly Ala Thr Leu Ile Glu Pro Phe Asp Ala His Asn Thr Val Ile Gly 165 170 175 Gln Gly Thr Val Ala Ala Glu Ile Leu Ser Gln Leu Thr Ser Met Gly 180 185 190 Lys Ser Ala Asp His Val Met Val Pro Val Gly Gly Gly Gly Leu Leu 195 200 205 Ala Gly Val Val Ser Tyr Met Ala Asp Ile Ala Pro Arg Thr Ala Ile 210 215 220 Val Gly Ile Glu Pro Ala Gly Ala Ala Ser Met Gln Ala Ala Leu His 225 230 235 240 Asn Gly Arg Pro Ile Thr Leu Glu Thr Val Asp Pro Phe Val Asp Gly 245 250 255 Ala Ala Val Lys Arg Val Gly Asp Leu Asn Tyr Thr Ile Val Glu Lys 260 265 270 Asn Gln Gly Arg Val His Met Met Ser Ala Thr Glu Gly Ala Val Cys 275 280 285 Thr Glu Met Leu Asp Leu Tyr Gln Asn Glu Gly Ile Ile Ala Glu Pro 290 295 300 Ala Gly Ala Leu Ser Ile Ala Gly Leu Lys Glu Met Ser Phe Ala Pro 305 310 315 320 Gly Ser Val Val Val Cys Ile Ile Ser Gly Gly Asn Asn Asp Val Leu 325 330 335 Arg Tyr Ala Glu Ile Ala Glu Arg Ser Leu Val His Arg Gly Leu Lys 340 345 350 His Tyr Phe Leu Val Asn Phe Pro Gln Lys Pro Gly Gln Leu Arg His 355 360 365 Phe Leu Glu Asp Ile Leu Gly Pro Asp Asp Asp Ile Ala Leu Ala Glu 370 375 380 Tyr Leu Lys Arg Asn Asn Arg Glu Thr Gly Thr Ala Leu Val Gly Ile 385 390 395 400 His Leu Ser Glu Ala Ser Gly Leu Asp Ser Leu Leu Glu Arg Met Glu 405 410 415 Glu Ser Ala Ile Asp Ser Arg Arg Leu Glu Pro Gly Thr Pro Glu Tyr 420 425 430 Glu Tyr Leu Thr 435 <210> 3 <211> 1311 <212> DNA <213> Artificial Sequence <400> 3 atgagtgaaa catacgtgtc tgagaaaagt ccaggagtga tggctagcgg agcggagctg 60 attcgtgccg ccgacattca aacggcgcag gcacgaattt cctccgtcat tgcaccaact 120 ccattgcagt attgccctcg tctttctgag gaaaccggaa cggaaatcta ccttaagcgt 180 gaggatctgc aggatgttcg ttcctacaag atccgcggtg cgctgaactc tggagcgcag 240 ctcactcagg agcagcgcga tgcaggtatc gttgccgcat ctgcaggtaa ccatgcccag 300 ggcgtggcct atgtgtgcaa gtccttgggc gttcagggac gcatctatgt tcctgtgcag 360 actccaaagc aaaagcgtga ccgcatcatg gctcacggcg gagagtttgt ctccttggtg 420 gtcactggca ataacttcga cgaagcatcg gctgcagcgc atgaagatgc agagcgcacc 480 ggcgcaacgc tgatcgagcc tttcgatgct cacaacaccg tcatcggtca gggtacagtg 540 gctgctgaga tcttgtcgca gctgacttcc atgggcaaga gtgcagatca cgtgatggtt 600 ccagtcggcg gtggcggact tcttgcaggt gtggtcagct acatggctga tatagcacct 660 cgcactgcga tcgttggtat cgaaccagcg ggagcagcat ccatgcaggc tgcattgcac 720 aatggtagac caatcacttt ggagactgtt gatccctttg tggacggcgc agcagtcaaa 780 cgtgtcggag atctcaacta caccatcgtg gagaagaacc agggtcgcgt gcacatgatg 840 agcgcgaccg agggcgctgt gtgtactgag atgctcgatc tttaccaaaa cgaaggcatc 900 atcgcggagc ctgctggcgc gctgtctatc gctgggttga aggaaatgtc ctttgcacct 960 ggttctgtcg tggtgtgcat catctctggt ggcaacaacg atgtgctgcg ttatgcggaa 1020 atcgctgagc gctccttggt gcaccgcggt ttgaagcact acttcttggt gaacttcccg 1080 caaaagcctg gtcagttgcg tcacttcctg gaagatatcc tgggaccgga tgatgacatc 1140 gcgctggcag agtacctcaa gcgcaacaac cgtgagaccg gtactgcgtt ggtgggtatt 1200 cacttgagtg aagcatcagg attggattct ttgctggaac gtatggagga atcggcaatt 1260 gattcccgtc gcctcgagcc gggcacccct gagtacgaat acttgaccta a 1311 <210> 4 <211> 280 <212> DNA <213> Artificial Sequence <400> 4 gtttgatggt agttggaggg cggtgcatcg gttgcgtgtg caggcgttgg atgcccatta 60 ccggcagttc ccgcagcggt atgtgggaca gccggtggtt caggaagttg ctggtgtggt 120 gcgtcttaat ggtgcgcgtg atgatgggtc tgtacaggag agggttggtg gtgtagcgtc 180 gctgttaagt gcttgagtta gcacgtgttc ttatcgcccc cctggttcac aaacccctgg 240 cagcgagcgc aattctagga gaggattata atagtcaacc 280 <210> 5 <211> 1591 <212> DNA <213> Artificial Sequence <400> 5 ttaggtcaag tattcgtact caggggtgcc cggctcgagg cgacgggaat caattgccga 60 ttcctccata cgttccagca aagaatccaa tcctgatgct tcactcaagt gaatacccac 120 caacgcagta ccggtctcac ggttgttgcg cttgaggtac tctgccagcg cgatgtcatc 180 atccggtccc aggatatctt ccaggaagtg acgcaactga ccaggctttt gcgggaagtt 240 caccaagaag tagtgcttca aaccgcggtg caccaaggag cgctcagcga tttccgcata 300 acgcagcaca tcgttgttgc caccagagat gatgcacacc acgacagaac caggtgcaaa 360 ggacatttcc ttcaacccag cgatagacag cgcgccagca ggctccgcga tgatgccttc 420 gttttggtaa agatcgagca tctcagtaca cacagcgccc tcggtcgcgc tcatcatgtg 480 cacgcgaccc tggttcttct ccacgatggt gtagttgaga tctccgacac gtttgactgc 540 tgcgccgtcc acaaagggat caacagtctc caaagtgatt ggtctaccat tgtgcaatgc 600 agcctgcatg gatgctgctc ccgctggttc gataccaacg atcgcagtgc gaggtgctat 660 atcagccatg tagctgacca cacctgcaag aagtccgcca ccgccgactg gaaccatcac 720 gtgatctgca ctcttgccca tggaagtcag ctgcgacaag atctcagcag ccactgtacc 780 ctgaccgatg acggtgttgt gagcatcgaa aggctcgatc agcgttgcgc cggtgcgctc 840 tgcatcttca tgcgctgcag ccgatgcttc gtcgaagtta ttgccagtga ccaccaagga 900 gacaaactct ccgccgtgag ccatgatgcg gtcacgcttt tgctttggag tctgcacagg 960 aacatagatg cgtccctgaa cgcccaagga cttgcacaca taggccacgc cctgggcatg 1020 gttacctgca gatgcggcaa cgatacctgc atcgcgctgc tcctgagtga gctgcgctcc 1080 agagttcagc gcaccgcgga tcttgtagga acgaacatcc tgcagatcct cacgcttaag 1140 gtagatttcc gttccggttt cctcagaaag acgagggcaa tactgcaatg gagttggtgc 1200 aatgacggag gaaattcgtg cctgcgccgt ttgaatgtcg gcggcacgaa tcagctccgc 1260 tccgctagcc atcactcctg gacttttctc agacacgtat gtttcactca tggttgacta 1320 ttataatcct ctcctagaat tgcgctcgct gccaggggtt tgtgaaccag gggggcgata 1380 agaacacgtg ctaactcaag cacttaacag cgacgctaca ccaccaaccc tctcctgtac 1440 agacccatca tcacgcgcac cattaagacg caccacacca gcaacttcct gaaccaccgg 1500 ctgtcccaca taccgctgcg ggaactgccg gtaatgggca tccaacgcct gcacacgcaa 1560 ccgatgcacc gccctccaac taccatcaaa c 1591 <210> 6 <211> 253 <212> DNA <213> Artificial Sequence <400> 6 tctagagtcg acctgcaggc atgcaagctt aattaattct gtttcctgtg tgaaattgtt 60 atccgctcac aattccacac attatacgag ccgatgatta attgtcaaca gctcatttca 120 gaatatttgc cagaaccgtt atgatgtcgg cgcaaaaaac attatccaga acgggagtgc 180 gccttgagcg acacgaatta tgcagtgatt tacgacctgc acagccatac cacagcttcc 240 gatggctgcc tga 253 <210> 7 <211> 2760 <212> DNA <213> Artificial Sequence <400> 7 ctagccagag ttgcgcagtg cagtggaaag accgttcatg gtcagctgga tgttgcggga 60 tacttgctcg ctttggtcgc cttttcggta gcgtcgcatc atctctacct ggatcacgtt 120 gagtggaagc aggtaggggt atcggcgctg gacggatcgt gcgagaagcg ggttgtcatc 180 aagcagatca tcagaaccgg tgattacgca gaacatcttc ttggtcagga agtattcctc 240 gcggatgacg gcataaacgc gctcagctac ttccctatct gggatcaggt ctgcgtagag 300 ctttgccaaa cgcagctctg ccttggacat cacctgagcc atgttatcca acactgaggt 360 gaaaaatggc caggactcgt tgagtgtttg tagctcggca atgcgctggg tggcctgctc 420 cccttcgcca atccattgct caagtgcggt gccgacacca aaccagcccg gcagcatgac 480 acgagactgg gaccaactga gcacccacgg gattgctcgc aaatcttcca ccgaggaggt 540 ctgcttgcgt gaggaaggcc tggatccgat gttgagggat ccaatctcct gcagcggcgt 600 ggactgggtg aagtaatcga tgaagccttg atcctcgtgc accaaggagg cgtacttctt 660 caagctgagc tcagagatct cactcatgat gtcgtacgcg cgttggtgat cggtgagttc 720 ggagacgtcg agaagcgatg cctcaagcgt tgctgagacc agagcttcga ggtttcggcg 780 cgcggtttcg gggttgccgt acttagcgga gatgatctcg ccctgctcgg tgatgcgcac 840 ggaaccttgg acagcccccc tgggctgggc aagaatcgcg tcgtaggaag gtccgccacc 900 gcggccgacg gtgccaccac ggccgtggaa caggcgaagc ttgaccccgg ctgatcggca 960 tagttcgacg agctgcagtt ccgcgtcgta aagcgcccag tttgcggaga aatatccgcc 1020 atccttgttg gaatcggagt aaccgagcat gacttcctgg acgttgtcgc gctgcaggag 1080 gtagttgcgg taaagatcaa ttttccacag ttcgtcgagg attccggcgc cggcctggag 1140 atcttcgatg gtttcgaaca gtgggatgac atcgacggtg ccgcgtgggt tgtcgccgtt 1200 ggctgcaatg aggccgaatt ccttgagcaa taccatcggc tcgagcacat cggtgaccga 1260 tgatgccatg gagatgatgc agtgaggcac catccgtggc ccgaatttct taacagcctc 1320 cgacgcggtg cggaagatgc cgagctcgcg gtcggtgacc tcgctgtatt catctgaacc 1380 gtgcgggatc agcggacgag ggctgcgcag ttccttcagc agcacctcaa gcttctctgc 1440 ttcagacagc tcgcggtagt ttgcggtgac ttgggcgcgt tcgaaaagct cggtgaggac 1500 gtcctcgtag ctttcggagt tttggcgcag atccagtgcg taaaggttga atccaaagct 1560 ctcgatggca gaaatcagca cagacaaacg atcatcggca atgagaacgt cattggattc 1620 acgcagaga tgatcaatgg tcaacgcatc gtttaagaat tcttccggag atgcgtatgg 1680 agtaaagacc ttgaaccaca cgccctcaac ggcgtcctcg ccgatcagct cggccgtcgt 1740 cgcgaggata cgtccgcgaa cgccatggac ggcgcgtcga taaggctcat ccacgcggct 1800 tggcacgtcg ttgtgcccgg catctgccag cgcaagcagc tgcggggtga ccttatcat 1860 gcggtccgac aggctgagct catgctcgag ggaatgcagc tggcgtgcat agtacttgag 1920 cacggtttcc gcagcgcggt gagtggataa ctcaactgtt tccgcggtga cataagggtt 1980 accgtcgtgg tctccaccaa tccaggaacc tggcttgacc acgggcttca aaggaacatc 2040 ctcgccgaaa cgctcacgaa gctcaacagc cacatcacgg ttgatacgtg gaatctcttc 2100 caaaaggctc agcttgtagt agcgcagccc tacttcgatc tcgtcctcga tacgtgggcg 2160 ggccacacga atcaacgcgg tctgccacaa aatggtgatg cgaccgcgga tgttcttctc 2220 gatctcatcc aacttgcttt gcgtacgagc ggtaggctcc gcagactgca aagcgtggcg 2280 ttcacgcatg tgggtggtga tccacttttg cgcatcaaaa acagtgcggc ggcgagtctc 2340 agttgggtgc gcagtcagaa ccggcgccac ctcagcattg cgcagcacat cggccacagc 2400 ttctgcgcca acattgccct cattgagttt cagccaggtg gcatcaagag tgctgtccgg 2460 aggggtgtcg cctgcatcga gagcctgttc acgaagctct tcatcgtaga ggtcttccgc 2520 caggttagcc agcagagcga agtgggaaaa tgcgcgagca atcggtgttg ccttggctgg 2580 agtaatgccg tcgaaaacct gaaccaggct atccatttcg gcgttgccct tggcgatatc 2640 aaaagaagtc aggcgcgctt gttcgaccag ttcataaacc tcctggcctt cttgttccgc 2700 aattacctca ccgaggattt gaccgaggaa cctgatgtca tcgcgtaaaa aatcagtcat 2760 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <400> 8 cgggatcctg gcctggatgc acagtggg 28 <210> 9 <211> 47 <212> DNA <213> Artificial Sequence <400> 9 ctgagtacga atacttgacc taaacatagc tgaaggccac ctcaatc 47 <210> 10 <211> 47 <212> DNA <213> Artificial Sequence <400> 10 gattgaggtg gccttcagct atgtttaggt caagtattcg tactcag 47 <210> 11 <211> 41 <212> DNA <213> Artificial Sequence <400> 11 agaggattac actagtcaac cgtttgatgg tagttggagg g 41 <210> 12 <211> 41 <212> DNA <213> Artificial Sequence <400> 12 ccctccaact accatcaaac ggttgactag tgtaatcctc t 41 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <400> 13 gctctagatg ccaagcagca caagttgt 28 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 tagccatcac tcctggactt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 cacacgccag tggttcgaag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 gtcgtcggtg cgtcaagcgt 20 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <400> 17 tctagagtcg acctgcaggc caaaccaccg ctaatgatgt 40 <210> 18 <211> 42 <212> DNA <213> Artificial Sequence <400> 18 acggcaaagg aacattttcc acgggtactg catatgattg gg 42 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <400> 19 cccaatcata tgcagtaccc gtggaaaatg ttcctttgcc gt 42 <210> 20 <211> 57 <212> DNA <213> Artificial Sequence <400> 20 acaattagaa ggagatcaga gtaatgactg attttttacg cgatgacatc aggttcc 57 <210> twenty one <211> 57 <212> DNA <213> Artificial Sequence <400> twenty one ggaacctgat gtcatcgcgt aaaaaatcag tcattactct gatctccttc taattgt 57 <210> twenty two <211> 40 <212> DNA <213> Artificial Sequence <400> twenty two ttgttgcgaa aacaccgaaa ggacgtttga gctgttgaca 40 <210> twenty three <211> 40 <212> DNA <213> Artificial Sequence <400> twenty three tgtcaacagc tcaaacgtcc tttcggtgtt ttcgcaacaa 40 <210> twenty four <211> 40 <212> DNA <213> Artificial Sequence <400> twenty four ggccagtgcc aagcttgcat ttctgtcata tgcgaagctt 40 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 cggtccttga tcaacacctt 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <400> 26 ccgtcccttt ttgtcactct 20 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <400> 27 cggaattctc ttcgacgaac tgtgcctt 28 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <400> 28 gtcgactcta gaatgactaa tggagataat ctc 33 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <400> 29 gattatctcc attagtcatt ctagagtcga cctgcaggca 40 <210> 30 <211> 40 <212> DNA <213> Artificial Sequence <400> 30 cactaccccc aaatggttca ggcagccatc ggaagctgtg 40 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <400> 31 gatggctgcc tgaaccattt gggggtagtg gccat 35 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <400> 32 cgggatccga gatctgatca acagagag 28 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 aacttagcgc gaatgatgca 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 gtctccaaag acccatccac 20 <210> 35 <211> 28 <212> DNA <213> Artificial Sequence <400> 35 cgggatcctt aagcggtttc tgcgcgag 28 <210> 36 <211> 32 <212> DNA <213> Artificial Sequence <400> 36 cggaattcgg gtgggaattt tcgtgcgttg tg 32 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <400> 37 cgggatccgc acagtgggtt gacgatat 28 <210> 38 <211> 40 <212> DNA <213> Artificial Sequence <400> 38 gtacgaatac ttgacctaaa catagctgaa ggccacctca 40 <210> 39 <211> 40 <212> DNA <213> Artificial Sequence <400> 39 tgaggtggcc ttcagctatg tttaggtcaa gtattcgtac 40 <210> 40 <211> 40 <212> DNA <213> Artificial Sequence <400> 40 gaggattaca ctagtcaacc atgagtgaaa catacgtgtc 40 <210> 41 <211> 40 <212> DNA <213> Artificial Sequence <400> 41 gacacgtatg tttcactcat ggttgactag tgtaatcctc 40 <210> 42 <211> 28 <212> DNA <213> Artificial Sequence <400> 42 gctctagagg tggcgtgttg ccaagcag 28 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <400> 43 cacacgccag tggttcgaag 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <400> 44 agccagcagt gtcgtcggtg 20

Claims

1. A threonine dehydratase mutant, characterized in that, The amino acid sequence of the threonine dehydratase mutant is shown in SEQ ID NO.

2. Using the wild-type threonine dehydratase of Corynebacterium glutamicum as a reference sequence, the mutations of the threonine dehydratase mutant are as follows: alanine at position 54 is mutated to threonine, valine at position 131 is mutated to alanine, arginine at position 171 is mutated to histidine, methionine at position 218 is mutated to isoleucine, glycine at position 243 is mutated to arginine, threonine at position 381 is mutated to alanine, and phenylalanine at position 383 is mutated to alanine.

2. The gene encoding the threonine dehydratase mutant of claim 1.

3. A biomaterial containing the gene of claim 2, characterized in that, The biological material is recombinant DNA, a vector, or a host cell.

4. A recombinant microorganism, characterized in that, It expresses the threonine dehydratase mutant of claim 1; the microorganism is Corynebacterium glutamicum.

5. The recombinant microorganism according to claim 4, characterized in that, An optimized ilvA promoter can be operatively linked upstream of the coding gene of the threonine dehydratase mutant; The optimized ilvA promoter nucleotide sequence is shown in SEQ ID NO.

4.

6. The recombinant microorganism according to claim 5, characterized in that, In the recombinant microorganism, an optimized ilvA promoter is inserted between the original ilvA promoter and the ilvA coding gene.

7. The recombinant microorganism according to any one of claims 4 to 6, characterized in that, Compared with the original strain, the recombinant microorganism showed enhanced expression and / or enzyme activity of phosphoenolpyruvate carboxylase.

8. The recombinant microorganism according to claim 7, characterized in that, The enhanced expression of phosphoenolpyruvate carboxylase is achieved by increasing the copy number of the phosphoenolpyruvate carboxylase encoding gene in the starting strain.

9. The recombinant microorganism according to claim 7, characterized in that, Compared with the original strain, the recombinant microorganism showed enhanced expression and / or enzyme activity of glucose-6-phosphate dehydrogenase.

10. The recombinant microorganism according to claim 9, characterized in that, The enhanced expression of glucose-6-phosphate dehydrogenase is achieved by replacing the promoter of the glucose-6-phosphate dehydrogenase encoding gene in the starting strain with a more active promoter.

11. The recombinant microorganism according to claim 9 or 10, characterized in that, Compared with the original strain, the recombinant microorganism showed enhanced expression and / or enzyme activity of acetylhydroxy acid synthase and dihydroxy acid reductase.

12. The recombinant microorganism according to claim 11, characterized in that, The enhanced expression of acetylhydroxy acid synthase and dihydroxy acid reductase isomerase is achieved by increasing the copy number of the genes encoding acetylhydroxy acid synthase and dihydroxy acid reductase in the starting strain.

13. Any of the following applications of the threonine dehydratase mutant of claim 1, the gene of claim 2, the biomaterial of claim 3, or the recombinant microorganism of any one of claims 4 to 12: (1) Application in the fermentation production of isoleucine; (2) Application in improving the yield and / or conversion rate of isoleucine in strains.

14. A method for producing isoleucine by fermentation, characterized in that, include: The recombinant microorganisms according to any one of claims 4 to 12 are cultured to obtain a culture, and isoleucine is obtained by separation and extraction of the culture.

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