Isoleucine production method, acetyl-hydroxy acid synthase mutant, and recombinant microorganism and application

Abstract

The present application relates to the technical field of microbial engineering, and particularly discloses a method for producing isoleucine, an acetoacetyl-hydroxy acid synthase mutant, a recombinant microorganism and application. The method for producing isoleucine comprises a step of fermenting and culturing the recombinant microorganism, wherein the recombinant microorganism expresses acetoacetyl-hydroxy acid synthase ilvIH or acetoacetyl-hydroxy acid synthase mutant ilvH F134L , ilvH F134V , or ilvH F134A compared with a starting strain, and the starting strain of the recombinant microorganism is coryneform bacteria. The method for producing isoleucine has high production efficiency, less by-products, and is suitable for industrial promotion.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial engineering, in particular to a method for producing isoleucine, an acetoacetyl-CoA synthase mutant, a recombinant microorganism and applications thereof. BACKGROUND

[0002] It is known that branched-chain amino acids (e.g., valine, leucine, isoleucine) are used in various industrial applications to produce various products, such as human nutritional enhancers, animal feed additives, medical product ingredients, and cosmetic ingredients. There are three methods for producing branched-chain amino acids: extraction, chemical synthesis, and microbial fermentation. The extraction and chemical synthesis methods are difficult to achieve industrial production due to limited raw material sources, high production costs, and environmental pollution. The microbial fermentation method has the advantages of low raw material cost, mild reaction conditions, and easy large-scale production, and is currently the most important method for producing branched-chain amino acids. However, the fermentation performance of the strain is still poor, and the byproduct content is high, resulting in a low conversion rate, which cannot meet the needs of large-scale industrial production.

[0003] The biosynthesis of branched-chain amino acids has a common precursor (pyruvate) and the same enzyme (acetoacetyl-CoA synthase), so it is difficult to biosynthesize a single type of branched-chain amino acid through microbial fermentation. Acetoacetyl-CoA synthase is the first enzyme in the biosynthesis of branched-chain amino acids and is also a key enzyme in the biosynthesis of branched-chain amino acids. It catalyzes the formation of acetoacetic acid from two molecules of pyruvic acid (acetoacetic acid is the precursor of valine and leucine), and also catalyzes the formation of a-acetylhydroxybutyric acid from a-ketobutyric acid and pyruvic acid (a-acetylhydroxybutyric acid is the precursor of isoleucine). Therefore, acetoacetyl-CoA synthase is a very important enzyme in the biosynthesis of branched-chain amino acids.

[0004] Acetoacetyl-CoA synthase is composed of two subunits, large and small subunits. The large subunit has catalytic activity, and the small subunit plays a regulatory role in feedback inhibition, which is inhibited by valine, leucine, and isoleucine. In Corynebacterium glutamicum, the large subunit is encoded by the ilvB gene, and the small subunit is encoded by the ilvN gene. In Escherichia coli, the large subunit is encoded by the ilvI gene, and the small subunit is encoded by the ilvH gene. Chinese patents CN 110506112 A and CN 110724679 A report acetoacetyl-CoA synthase mutants, microorganisms containing the same, and methods for producing L-branched-chain amino acids using the same, and disclose mutants of the large subunit of acetoacetyl-CoA synthase, IlvB protein. Chinese patent CN201610647430.1 reports a Corynebacterium glutamicum and applications, and discloses mutants of the small subunit of acetoacetyl-CoA synthase, IlvN protein. It is still necessary to further study the biosynthesis of branched-chain amino acids to provide a new method for increasing the yield of isoleucine and reducing the content of byproducts valine and leucine in branched-chain amino acids. SUMMARY

[0005] The present application aims to provide a recombinant microorganism and a method for efficiently producing branched-chain amino acids.

[0006] The technical solution of the present application is as follows:

[0007] An isoleucine production method, comprising a step of fermentation culture with a recombinant microorganism, wherein the recombinant microorganism expresses acetyl-hydroxy acid synthase ilvIH or acetyl-hydroxy acid synthase mutant ilvH F134L , ilvH F134V or ilvH F134A compared to a starting strain, and the starting strain of the recombinant microorganism is coryneform bacteria.

[0008] The amino acid sequence of acetyl-hydroxy acid synthase ilvIH is shown in SEQ ID NO. 21, the amino acid sequence of acetyl-hydroxy acid synthase mutant ilvH F134L is shown in SEQ ID NO. 22, the amino acid sequence of acetyl-hydroxy acid synthase mutant ilvH F134V is shown in SEQ ID NO. 23, and the amino acid sequence of acetyl-hydroxy acid synthase mutant ilvH F134A is shown in SEQ ID NO. 24.

[0009] The present application introduces acetyl-hydroxy acid synthase (encoded by ilvIH gene) from Escherichia coli str. K-12 substrate MG1655 into the cells of the starting strain, and introduces specific mutation sites in the ilvH gene, thereby successfully creating a new microorganism capable of efficiently producing isoleucine, and applying it to the production of isoleucine, so that isoleucine can be efficiently produced, and the content of by-products valine and leucine is reduced.

[0010] In the isoleucine production method of the present application, the nucleotide sequence of acetyl-hydroxy acid synthase ilvIH is shown in SEQ ID NO. 17, the nucleotide sequence of acetyl-hydroxy acid synthase mutant ilvH F134L is shown in SEQ ID NO. 18, the nucleotide sequence of acetyl-hydroxy acid synthase mutant ilvH F134V is shown in SEQ ID NO. 19, and the nucleotide sequence of acetyl-hydroxy acid synthase mutant ilvH F134A is shown in SEQ ID NO. 20.

[0011] The starting strain in the isoleucine production method of the present application is Corynebacterium glutamicum, Corynebacterium pekinense or Breviabacterium flavum.

[0012] The present application also provides an acetoacetyl-CoA synthase mutant, which has a substitution mutation of leucine, valine or alanine at position 134, with reference to the amino acid sequence of wild-type acetoacetyl-CoA synthase of Escherichia coli.

[0013] The present application mutates the amino acid at position 134 of acetoacetyl-CoA synthase from phenylalanine (F) to another amino acid other than phenylalanine, preferably leucine (L), valine (V) or alanine (A), so that the coryneform bacteria expressing the mutant can efficiently produce isoleucine.

[0014] When the amino acid at position 134 of acetoacetyl-CoA synthase gene ilvH is mutated from phenylalanine (F) to leucine (L), valine (V) or alanine (A), the yield of isoleucine is increased, and the yield of valine and leucine is decreased, especially when the amino acid is mutated from phenylalanine (F) to alanine (A).

[0015] The above mutation site can be applied to Corynebacterium glutamicum, but is not limited to Corynebacterium glutamicum, and can also be applied to, for example, Corynebacterium pekinense or Breviabacterium flavum, for the production of branched chain amino acids such as valine, isoleucine and leucine or their derivatives.

[0016] The acetoacetyl-CoA synthase mutant of the present application has an amino acid sequence as shown in any one of SEQ ID NO. 22-24.

[0017] The present application also provides a nucleic acid encoding the above-mentioned acetoacetyl-CoA synthase mutant.

[0018] Preferably, the nucleic acid has a nucleotide sequence as shown in any one of SEQ ID NO. 18-20.

[0019] The present application also provides a biological material containing the above-mentioned nucleic acid, which is an expression cassette, a vector or a host cell.

[0020] The present application also provides a recombinant microorganism expressing acetoacetyl-CoA synthase ilvIH or an acetoacetyl-CoA synthase mutant, wherein the acetoacetyl-CoA synthase ilvIH is as described above, and the acetoacetyl-CoA synthase mutant is as described above; and the starting strain of the recombinant microorganism is coryneform bacteria.

[0021] Preferably, the nucleic acid encoding the acetyl-hydroxy acid synthase mutant has a nucleotide sequence as shown in any one of SEQ ID NO. 18-20;

[0022] And / or, the starting strain of the recombinant microorganism is Corynebacterium glutamicum, Corynebacterium pekinense or Brevibacterium flavum.

[0023] The acetyl-hydroxy acid synthase encoded by the ilvIH gene is the first enzyme in the biosynthesis of branched-chain amino acids, and is also a key enzyme in the biosynthesis of branched-chain amino acids, catalyzing two molecules of pyruvic acid to generate acetyl lactate (acetyl lactate is a precursor of valine and leucine), and also catalyzing a-ketobutyric acid and pyruvic acid to generate a-acetyl hydroxy butyric acid (a-acetyl hydroxy butyric acid is a precursor of isoleucine).

[0024] The present application introduces the acetyl-hydroxy acid synthase (encoded by the ilvIH gene) from E. coli str. K-12 substr. MG1655 into the cell of the starting strain by genetic engineering means, to obtain an acetyl-hydroxy acid synthase enhanced strain, so that the ability of the microorganism to produce isoleucine is enhanced compared with the unmodified strain.

[0025] Further, the present application realizes the mutation of the acetyl-hydroxy acid synthase by modifying the ilvH gene, mutating the 134th amino acid of the acetyl-hydroxy acid synthase from phenylalanine (F) to leucine (L), valine (V) or alanine (A), to obtain an acetyl-hydroxy acid synthase mutant strain, so that the ability of the microorganism to produce by-products valine and leucine is reduced, and the ability of the microorganism to produce isoleucine is enhanced compared with the unmodified strain, and finally the yield of isoleucine is improved.

[0026] The present application also provides any one of the following applications of the above-mentioned acetyl-hydroxy acid synthase mutant, or nucleic acid, or biological material, or recombinant microorganism:

[0027] (1) application in the fermentation production of valine, leucine and / or isoleucine and derivatives thereof;

[0028] (2) application in the genetic breeding of microorganisms for producing valine, leucine and / or isoleucine and derivatives thereof;

[0029] (3) application in improving the yield of fermentation production of isoleucine and derivatives thereof.

[0030] The present application has at least the following beneficial effects:

[0031] The isoleucine yield of the acetyl-hydroxy acid synthase enhanced strain 2-ilvIH of the present application is significantly improved compared with the starting strain, and the isoleucine yield is 4.6 g / L, which is 1.9 g / L higher than the yield of the starting strain, and is increased by 70%.

[0032] The acetyl-hydroxy acid synthase mutant strain 2-ilvIH of the present application F134L , 2-ilvIH F134V , 2-ilvIH F134A The isoleucine yield and conversion rate of the acetyl-hydroxy acid synthase mutant strain 2-ilvIH are further improved compared with before the mutation, and the byproduct valine and leucine yield is greatly reduced, and the growth of the strain is normal, and the strain still maintains good growth performance. Among them, the acetyl-hydroxy acid synthase mutant strain 2-ilvIH F134A is the most outstanding, the isoleucine yield is 6.5 g / L, which is increased by 3.8 g / L compared with the starting strain, and is increased by 141%; the byproduct valine yield is 2.2 g / L, which is decreased by 63% compared with the valine yield of the starting strain; the byproduct leucine yield is 1.5 g / L, which is decreased by 46% compared with the leucine yield of the starting strain.

[0033] The present application provides a new method for efficiently producing isoleucine, which has less byproducts and high production efficiency, and is suitable for industrialization. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the following examples are given only for the purpose of illustration and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. The specific techniques or conditions not specified in the following examples are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not specified by the manufacturer are conventional products that can be purchased through regular channels.

[0035] The primer names and sequences involved in the examples of the present application are shown in Table 1.

[0036] Table 1 Primer sequences (SEQ ID No. 1-16)

[0037]

[0038]

[0039] Example 1 Construction of acetyl-hydroxy acid synthase enhanced strain 2-ilvIH

[0040] The acetyl-hydroxy acid synthase gene ilvIH from E. coli str. K-12 substr. MG1655 is introduced into the cells of MHZ-1012-2 as the starting strain to construct the acetyl-hydroxy acid synthase enhanced strain 2-ilvIH.

[0041] The wild-type ilvH nucleotide sequence is shown as SEQ ID NO. 17, and the amino acid sequence is shown as SEQ ID NO. 21.

[0042] The starting strain MHZ-1012-2 of the present application is Corynebacterium glutamicum, and the classification name is Corynebacterium glutamicum, which was preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 30, 2016, and the address of the preservation center is No. 1, Yihuang 3rd Road, Beijing City, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC No. 13406, and it is disclosed in Chinese patent CN201611250330.1.

[0043] The specific construction method is as follows:

[0044] 1. Construction of plasmid pK18mobsacB-ilvIH

[0045] The recombinant fragment UP-1 was prepared by using the genome of the starting strain MHZ-1012-2 as a template and using UP-1F / UP-1R as primers, the recombinant fragment DN-1 was prepared by using DN-4F / DN-4R as primers, the recombinant fragment ilvIH-2 was prepared by using the genome of Escherichia coli MG1655 (NCBI number: NC_000913.3) as a template and using ilvIH-2F / ilvIH-2R as primers, the recombinant fragment ilvIH-3 was prepared by using ilvIH-3F / ilvIH-3R as primers, and the recombinant fragment ilvIH was prepared by using the recombinant fragment ilvIH-2 and the recombinant fragment ilvIH-3 as a template and using ilvIH-2F / ilvIH-3R as primers, the fragment pk18-1 was obtained by using the plasmid pk18-mob-sacB as a template and using pk18-5F / pk18-5R as primers, and purified by an agarose gel recovery kit (Tiangen), and then the reaction was performed according to the Gibson assembly kit configuration system, and the reaction system is shown in Table 2.

[0046] Table 2 Gibson assembly reaction system

[0047] Components UP-1 DN-1 ilvIH pk18-1 CE Buffer CE Exnase Sterile water Volume / μL 1 1 1 2 4 2 9

[0048] The prepared reaction system was reacted at 37℃ for 30 min, 10 μL of the transformed Trans1T1 competent cells (TransGen Biotech) were taken, single colonies were picked, and the inserted fragment was identified to be correct by colony PCR, positive clones in which the fragment was inserted into pK18mobsacB were further identified by enzyme digestion, and finally the plasmid was sent to Jiweizhi Biotechnology Co., Ltd. for sequencing. The plasmid obtained by sequencing correctly was named as pK18mobsacB-ilvIH.

[0049] 2. Construction of acetylhydroxy acid synthase enhanced strain 2-ilvIH

[0050] The recombinant plasmid pK18mobsacB-ilvIH obtained by the method described in step 1 above was transformed into the starting strain MHZ-1012-2, and the exchange recombinants were selected on a selection medium containing 15 mg / L of kanamycin. The culture temperature was 30℃, and the culture was inverted. The transformants obtained by screening were cultured overnight in a general liquid brain heart infusion medium, and the culture temperature was 30℃, and the culture was shaken at 220 rpm. During this culture process, the transformants underwent a second recombination, and the vector sequence was removed from the genome by gene exchange. The culture was serially diluted (10 -2 to 10 -4 ), and the diluents were spread on a general solid brain heart infusion medium containing 10% sucrose, and incubated at 33℃ for 48 h. The transformants grown on this medium were identified. The target strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis, and was named as 2-ilvIH.

[0051] Example 2: Construction of acetylhydroxy acid synthase mutant strain 2-ilvIH F134L , 2-ilvIH F134V , 2-ilvIH F134A

[0052] Example 2: Construction of acetylhydroxy acid synthase mutant strain 2-ilvIH F134L , 2-ilvIH F134V , 2-ilvIH F134A .

[0053] ilvH F134L The mutant nucleotide sequence is shown as SEQ ID NO. 18, and the amino acid sequence is shown as SEQ ID NO. 22.

[0054] ilvH F134V The mutant nucleotide sequence is shown as SEQ ID NO. 19, and the amino acid sequence is shown as SEQ ID NO. 23.

[0055] ilvH F134A The mutant nucleotide sequence is shown as SEQ ID NO. 20, and the amino acid sequence is shown as SEQ ID NO. 24.

[0056] The specific construction method is as follows.

[0057] 1. Plasmid pK18mobsacB-ilvIH F134L , pK18mobsacB-ilvIH F134V , pK18mobsacB-ilvIH F134A Construction

[0058] According to the construction method of the plasmid pK18mobsacB-ilvIH described in Example 1, the primers ilvIH-2R and ilvIH-3F are replaced by ilvIH-2R and ilvIH-3F respectively, and the obtained plasmid is named as pK18mobsacB-ilvIH. F134L F134L F134L

[0059] According to the construction method of the plasmid pK18mobsacB-ilvIH described in Example 1, the primers ilvIH-2R and ilvIH-3F are replaced by ilvIH-2R and ilvIH-3F respectively, and the obtained plasmid is named as pK18mobsacB-ilvIH. F134V F134V F134V

[0060] According to the construction method of the plasmid pK18mobsacB-ilvIH described in Example 1, the primers ilvIH-2R and ilvIH-3F are replaced by ilvIH-2R and ilvIH-3F respectively, and the obtained plasmid is named as pK18mobsacB-ilvIH. F134A F134A F134A

[0061] 2. Acetohydroxy acid synthase mutant strain 2-ilvIH F134L , 2-ilvIH F134V , 2-ilvIH F134A Construction

[0062] According to the construction method of the acetohydroxy acid synthase enhanced strain 2-ilvIH described in the above Example 1, the plasmid pK18mobsacB-ilvIH is replaced by pK18mobsacB-ilvIH, pK18mobsacB-ilvIH, and pK18mobsacB-ilvIH respectively. F134L F134V ​​​​​​​​​​pK18mobsacB-ilvIH F134A , and the target mutant strain was obtained successively, and named as: 2-ilvIH F134L , 2-ilvIH F134V , 2-ilvIH F134A .

[0063] Example 3 Shake flask fermentation verification

[0064] The mutant strains obtained by the above construction were subjected to shake flask fermentation together with the starting strain MHZ-1012-2, and the production performance was compared.

[0065] 1. The culture medium used in shake flask fermentation was as follows:

[0066] (1) Solid activated plate: BHI medium 37 g / L, agar powder 20 g / L.

[0067] (2) Seed culture medium: glucose 20 g / L, proteose 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.

[0068] (3) Fermentation medium: glucose 100 g / L, soybean meal extract 9.75 g / L, corn syrup dry 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 0.01 g / L, (NH4)2SO4 50 g / L, prepared with distilled water, pH adjusted to 7.0.

[0069] 2. The method of shake flask fermentation was as follows:

[0070] (1) Seed culture: 1 ring of bacteria was scraped from the plate and inoculated into a 500 ml conical flask containing 50 ml of seed culture medium, and then cultured at 30°C with 110 rpm shaking for 15-17 h until the OD 562 value was 16-18;

[0071] (2) Shake flask fermentation: inoculated into a 500 ml conical flask containing 25 ml of fermentation medium at a 10% inoculation amount, and then cultured at 30°C with 135 rpm shaking for 48 hours.

[0072] (3) The concentration of bacteria was detected using a spectrophotometer at a wavelength of 562 nanometers, and was recorded as OD 562The content of amino acids in the fermentation liquor was detected by HPLC. The fermentation results are shown in Table 3.

[0073] Table 3 Fermentation results in shake flask

[0074]

[0075]

[0076] Note: * indicates a significant difference (P < 0.05) compared with the starting strain.

[0077] The above results show that the isoleucine yield of the starting strain MHZ-1012-2 is only 2.7 g / L, and the isoleucine yield and conversion rate of the acetyl-hydroxy acid synthase enhanced strain 2-ilvIH are significantly improved compared with the starting strain. The isoleucine yield is 4.6 g / L, which is 1.9 g / L higher than that of the starting strain, and is increased by 70%. The isoleucine yield and conversion rate of the acetyl-hydroxy acid synthase mutant strain 2-ilvIH F134L , 2-ilvIH F134V , 2-ilvIH F134A are further improved compared with before the mutation, and the byproduct valine and leucine yield is greatly reduced, and the strain still maintains good growth performance. Among them, the acetyl-hydroxy acid synthase mutant strain 2-ilvIH F134A performs the most outstanding, the isoleucine yield is 6.5 g / L, which is 3.8 g / L higher than that of the starting strain, and is increased by 141%; the byproduct valine yield is 2.2 g / L, which is decreased by 63% compared with the valine yield of the starting strain; the byproduct leucine yield is 1.5 g / L, which is decreased by 46% compared with the leucine yield of the starting strain.

[0078] It can be seen that the acetyl-hydroxy acid synthase mutant and the acetyl-hydroxy acid synthase mutant strain provided by the present application have a significant promoting effect on the yield of the target product isoleucine, and have a significant reducing effect on the byproduct valine and leucine yield. The acetyl-hydroxy acid synthase mutant and the recombinant microorganism provide a reference for the construction of production strains of valine, leucine, isoleucine and derivatives taking them as precursors.

[0079] Although the present application has been described in detail in the foregoing description with general principles and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection claimed by the present application. SEQUENCE LISTING <110> Langfang Meihua Biotechnology Development Co., Ltd. <120> A method for producing isoleucine, an acetyl-hydroxy acid synthase mutant, a recombinant microorganism and application <130> KHP211125949.4 <160> 24 <170> SIPOSequenceListing 1.0 <210> 1 <211> 46 <212> DNA <213> Artificial Sequence <400> 1 acaacgtcgt gactgggaaa accccttgga gcaaaggctg gcgcac 46 <210> 2 <211> 53 <212> DNA <213> Artificial Sequence <400> 2 catggcctgc ctcactgttt gacggaatta tgcctcggtc gattcaacgg cgg 53 <210> 3 <211> 53 <212> DNA <213> Artificial Sequence <400> 3 ccgccgttga atcgaccgag gcataattcc gtcaaacagt gaggcaggcc atg 53 <210> 4 <211> 43 <212> DNA <213> Artificial Sequence <400> 4 acatcgcgaa tcgatgctaa aaatgcatca agcttaccgc tgg 43 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <400> 5 ccagcggtaa gcttgatgca tttttagcat cgattcgcga tgt 43 <210> 6 <211> 47 <212> DNA <213> Artificial Sequence <400> 6 gaattagcca tcttgggtct ctcctcaacg cattatttta tcgccgc 47 <210> 7 <211> 47 <212> DNA <213> Artificial Sequence <400> 7 gcggcgataa aataatgcgt tgaggagaga cccaagatgg ctaattc 47 <210> 8 <211> 46 <212> DNA <213> Artificial Sequence <400> 8 cgtaatcatg tcatagctgt ttcccacatt tatgcagcag gtgcaa 46 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <400> 9 ttgcacctgc tgcataaatg tgggaaacag ctatgacatg attacg 46 <210> 10 <211> 46 <212> DNA <213> Artificial Sequence <400> 10 gtgcgccagc ctttgctcca aggggttttc ccagtcacga cgttgt 46 <210> 11 <211> 43 <212> DNA <213> Artificial Sequence <400> 11 acatcgcgaa tcgatgctaa aagtgcatca agcttaccgc tgg 43 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <400> 12 ccagcggtaa gcttgatgca cttttagcat cgattcgcga tgt 43 <210> 13 <211> 43 <212> DNA <213> Artificial Sequence <400> 13 acatcgcgaa tcgatgctaa aactgcatca agcttaccgc tgg 43 <210> 14 <211> 43 <212> DNA <213> Artificial Sequence <400> 14 ccagcggtaa gcttgatgca gttttagcat cgattcgcga tgt 43 <210> 15 <211> 43 <212> DNA <213> Artificial Sequence <400> 15 acatcgcgaa tcgatgctaa agctgcatca agcttaccgc tgg 43 <210> 16 <211> 43 <212> DNA <213> Artificial Sequence <400> 16 ccagcggtaa gcttgatgca gctttagcat cgattcgcga tgt 43 <210> 17 <211> 492 <212> DNA <213> Artificial Sequence <400> 17 atgcgccgga tattatcagt cttactcgaa aatgaatcag gcgcgttatc ccgcgtgatt 60 ggcctttttt cccagcgtgg ctacaacatt gaaagcctga ccgttgcgcc aaccgacgat 120 ccgacattat cgcgtatgac catccagacc gtgggcgatg aaaaagtact tgagcagatc 180 gaaaagcaat tacacaaact ggtcgatgtc ttgcgcgtga gtgagttggg gcagggcgcg 240 catgttgagc gggaaatcat gctggtgaaa attcaggcca gcggttacgg gcgtgacgaa 300 gtgaaacgta atacggaaat attccgtggg caaattatcg atgtcacacc ctcgctttat 360 accgttcaat tagcaggcac cagcggtaag cttgatgcat ttttagcatc gattcgcgat 420 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 TCTAGAGCGG TAAAGCGGTA ATAG 49 <210> 18 <211> 49 <212> DNA <213> Artificial Sequence <400> 18 ATGCCTCGTA TATATCAGTC TTACTCGAAA ATGAATCAGG CCGTTATCCC GCGTGATT 60 GGCCTTTTTT CCCAGCGTGG CTACAACATT GAAAGCCTGA CCCTTGCGCC AACCCTCGAT 120 CCGACATCAT CGCCTATGAC CATCCAGACC GTGGGCGATG AAAAAGTACT TGAGCAGATC 180 GAAAAGCAAT ACACAAACTG GTCGATGTCT TGCACGCGTA GTGAGTTGGG GCAGGGCGCG 240 CATGTTGAGC GGGAAATCAT GCTGCTGAAA ATTCAAGCCA GCGGTTACGG GCCTGACGAA 300 GTGAAACGTA TACGGAAATA TTCCGTGGGC AAATTATCGA TGTCACTCCT CGCTTTAT 360 ACCCTTCAAT TAGCAGGCAC CAGCAGTAAG CTTGATGCAC TTTTAGCATC GATTCCCGAT 420 GTGGCGAAAA TTGTGGAGGT TGCTCGCTCT GGTGTGGTGG ACTTTTGGCG CGCGGTAAGA 480 ATAATGCCTT GA 49 <210> 19 <211> 49 <212> DNA <213> Artificial Sequence <400> 19 atgcgccgga tattatcagt cttactcgaa aatgaatcag gcgcgttatc ccgcgtgatt 60 ggcctttttt cccagcgtgg ctacaacatt gaaagcctga ccgttgcgcc aaccgacgat 120 ccgacattat cgcgtatgac catccagacc gtgggcgatg aaaaagtact tgagcagatc 180 gaaaagcaat tacacaaact ggtcgatgtc ttgcgcgtga gtgagttggg gcagggcgcg 240 catgttgagc gggaaatcat gctggtgaaa attcaggcca gcggttacgg gcgtgacgaa 300 gtgaaacgta atacggaaat attccgtggg caaattatcg atgtcacacc ctcgctttat 360 accgttcaat tagcaggcac cagcggtaag cttgatgcag ttttagcatc gattcgcgat 420 gtggcgaaaa ttgtggaggt tgctcgctct ggtgtggtcg gactttcgcg cggcgataaa 480 ataatgcgtt ga 492 <210> 20 <211> 492 <212> DNA <213> Artificial Sequence <400> 20 atgcgccgga tattatcagt cttactcgaa aatgaatcag gcgcgttatc ccgcgtgatt 60 ggcctttttt cccagcgtgg ctacaacatt gaaagcctga ccgttgcgcc aaccgacgat 120 ccgacattat cgcgtatgac catccagacc gtgggcgatg aaaaagtact tgagcagatc 180 gaaaagcaat tacacaaact ggtcgatgtc ttgcgcgtga gtgagttggg gcagggcgcg 240 catgttgagc gggaaatcat gctggtgaaa attcaggcca gcggttacgg gcgtgacgaa 300 gtgaaacgta atacggaaat attccgtggg caaattatcg atgtcacacc ctcgctttat 360 accgttcaat tagcaggcac cagcggtaag cttgatgcag ctttagcatc gattcgcgat 420 gtggcgaaaa ttgtggaggt tgctcgctct ggtgtggtcg gactttcgcg cggcgataaa 480 ataatgcgtt ga 492 <210> 21 <211> 163 <212> PRT <213> Artificial Sequence <400> 21 Met Arg Arg Ile Leu Ser Val Leu Leu Glu Asn Glu Ser Gly Ala Leu 1 5 10 15 Ser Arg Val Ile Gly Leu Phe Ser Gln Arg Gly Tyr Asn Ile Glu Ser 20 25 30 Leu Thr Val Ala Pro Thr Asp Asp Pro Thr Leu Ser Arg Met Thr Ile 35 40 45 Gln Thr Val Gly Asp Glu Lys Val Leu Glu Gln Ile Glu Lys Gln Leu 50 55 60 His Lys Leu Val Asp Val Leu Arg Val Ser Glu Leu Gly Gln Gly Ala 65 70 75 80 His Val Glu Arg Glu Ile Met Leu Val Lys Ile Gln Ala Ser Gly Tyr 85 90 95 Gly Arg Asp Glu Val Lys Arg Asn Thr Glu Ile Phe Arg Gly Gln Ile 100 105 110 Ile Asp Val Thr Pro Ser Leu Tyr Thr Val Gln Leu Ala Gly Thr Ser 115 120 125 Gly Lys Leu Asp Ala Phe Leu Ala Ser Ile Arg Asp Val Ala Lys Ile 130 135 140 Val Glu Val Ala Arg Ser Gly Val Val Gly Leu Ser Arg Gly Asp Lys 145 150 155 160 Ile Met Arg <210> 22 <211> 163 <212> PRT <213> Artificial Sequence <400> 22 Met Arg Arg Ile Leu Ser Val Leu Leu Glu Asn Glu Ser Gly Ala Leu 1 5 10 15 Ser Arg Val lie Gly Leu Phe Ser Gin Arg Gly Tyr Asn lie Gin Ser 20 25 30 Leu Thr Val Ala Pro Thr Asp Asp Pro Thr Leu Ser Arg Met Thr lie 35 40 45 Gln Thr Val Gly Asp Glu Lys Val Leu Glu Gin lie Glu Lys Gin Leu 50 55 60 His Lys Leu Val Asp Val Leu Arg Val Ser Glu Leu Gly Gin Gly Ala 65 70 75 80 His Val Glu Arg Glu lie Met Leu Val Lys lie Gin Ala Ser Gly Tyr 85 90 95 Gly Arg Asp Glu Val Lys Arg Asn Thr Glu lie Phe Arg Gly Gin lie 100 105 110 lie Asp Val Thr Pro Ser Leu Tyr Thr Val Gin Leu Ala Gly Thr Ser 115 120 125 Gly Lys Leu Asp Ala Leu Leu Ala Ser lie Arg Asp Val Ala Lys lie 130 135 140 Val Glu Val Ala Arg Ser Gly Val Val Gly Leu Ser Arg Gly Asp Lys 145 150 155 160 lie Met Arg <210> 23 <211> 163 <212> PRT <213> Artificial Sequence <400> 23 Met Arg Arg Ile Leu Ser Val Leu Leu Glu Asn Glu Ser Gly Ala Leu 1 5 10 15 Ser Arg Val Ile Gly Leu Phe Ser Gln Arg Gly Tyr Asn Ile Glu Ser 20 25 30 Leu Thr Val Ala Pro Thr Asp Asp Pro Thr Leu Ser Arg Met Thr Ile 35 40 45 Gln Thr Val Gly Asp Glu Lys Val Leu Glu Gln Ile Glu Lys Gln Leu 50 55 60 His Lys Leu Val Asp Val Leu Arg Val Ser Glu Leu Gly Gln Gly Ala 65 70 75 80 His Val Glu Arg Glu Ile Met Leu Val Lys Ile Gln Ala Ser Gly Tyr 85 90 95 Gly Arg Asp Glu Val Lys Arg Asn Thr Glu Ile Phe Arg Gly Gln Ile 100 105 110 Ile Asp Val Thr Pro Ser Leu Tyr Thr Val Gln Leu Ala Gly Thr Ser 115 120 125 Gly Lys Leu Asp Ala Val Leu Ala Ser Ile Arg Asp Val Ala Lys Ile 130 135 140 Val Glu Val Ala Arg Ser Gly Val Val Gly Leu Ser Arg Gly Asp Lys 145 150 155 160 Ile Met Arg <210> 24 <211> 163 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 24 Met Arg Arg Ile Leu Ser Val Leu Leu Glu Asn Glu Ser Gly Ala Leu 1 5 10 15 Ser Arg Val Ile Gly Leu Phe Ser Gln Arg Gly Tyr Asn Ile Glu Ser 20 25 30 Leu Thr Val Ala Pro Thr Asp Asp Pro Thr Leu Ser Arg Met Thr Ile 35 40 45 Gln Thr Val Gly Asp Glu Lys Val Leu Glu Gln Ile Glu Lys Gln Leu 50 55 60 His Lys Leu Val Asp Val Leu Arg Val Ser Glu Leu Gly Gln Gly Ala 65 70 75 80 His Val Glu Arg Glu Ile Met Leu Val Lys Ile Gln Ala Ser Gly Tyr 85 90 95 Gly Arg Asp Glu Val Lys Arg Asn Thr Glu Ile Phe Arg Gly Gln Ile 100 105 110 Ile Asp Val Thr Pro Ser Leu Tyr Thr Val Gin Leu Ala Gly Thr Ser 115 120 125 Gly Lys Leu Asp Ala Ala Leu Ala Ser Ile Arg Asp Val Ala Lys Ile 130 135 140 Val Glu Val Ala Arg Ser Gly Val Val Gly Leu Ser Arg Gly Asp Lys 145 150 155 160 Ile Met Arg

Claims

1. A method for producing isoleucine, characterized in that, The step includes fermentation culture with recombinant microorganisms, wherein the recombinant microorganisms express the acetylhydroxyl synthase mutant ilvH compared to the starting strain. F134L ,ilvH F134V or ilvH F134A The starting strain of the recombinant microorganism is Corynebacterium. Acetylhydroxy acid synthase mutant ilvH F134L The amino acid sequence is as shown in SEQ ID NO.22, acetylhydroxyl synthase mutant ilvH F134V The amino acid sequence is shown in SEQ ID NO.23, acetylhydroxy acid synthase mutant ilvH F134A The amino acid sequence is shown in SEQ ID NO.

24.

2. The method for producing isoleucine according to claim 1, characterized in that, Acetylhydroxy acid synthase mutant ilvH F134L The nucleotide sequence is as shown in SEQ ID NO.18, acetylhydroxyl synthase mutant ilvH F134V The nucleotide sequence is shown in SEQ ID NO.19, acetylhydroxyl synthase mutant ilvH F134A The nucleotide sequence is shown in SEQ ID NO.

20.

3. The method for producing isoleucine according to claim 1 or 2, characterized in that, The starting strain is either Corynebacterium glutamicum or Corynebacterium pingeri.

4. An acetylhydroxy acid synthase mutant, characterized in that, Using the amino acid sequence of wild-type Escherichia coli acetylhydroxyl synthase as a reference sequence, the acetylhydroxyl synthase mutant contains a mutation that substitutes leucine, valine, or alanine at position 134; the amino acid sequence of the acetylhydroxyl synthase mutant is shown in any one of SEQ ID NO.22-24.

5. The nucleic acid encoding the acetylhydroxy acid synthase mutant of claim 4.

6. The nucleic acid according to claim 5, characterized in that, The nucleotide sequence is shown in any one of SEQ ID NO.18-20.

7. A biological material containing the nucleic acid of claim 5 or 6, wherein the biological material is an expression cassette, a vector, or a host cell.

8. A recombinant microorganism, characterized in that, The recombinant microorganism expresses an acetylhydroxy acid synthase mutant, which is as described in claim 4; the starting strain of the recombinant microorganism is Corynebacterium.

9. The recombinant microorganism according to claim 8, characterized in that, The nucleotide sequence encoding the acetylhydroxy acid synthase mutant is shown in any one of SEQ ID NO. 18-20; And / or, the starting strain of the recombinant microorganism is Corynebacterium glutamicum or Corynebacterium pingeiformis.

10. Any of the following applications of the acetylhydroxyl synthase mutant of claim 4, or the nucleic acid of claim 5 or 6, or the biological material of claim 7, or the recombinant microorganism of claim 8 or 9: (1) Application in the fermentation production of isoleucine; (2) Application in the genetic breeding of microorganisms for the production of isoleucine; (3) Application in increasing the yield of isoleucine produced by fermentation.

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