CN115678820A - Recombinant microorganism producing valine and construction method and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to a valine-producing recombinant microorganism and a construction method and application thereof. Compared with the original strain, the promoter of the gene brnFE in the recombinant microorganism is mutated into a DNA molecule shown in SEQ ID NO.1, and/or the gene brnQ coded intein in the recombinant microorganism is mutated to form a mutant, wherein the mutant contains mutation that the amino acid sequence of the intein coded by the wild type gene brnQ of the original strain is taken as a reference sequence, and the 112 th alanine is replaced by threonine, serine or tyrosine. The recombinant microorganism is an L-valine high-producing strain, can effectively accumulate L-valine, improves the yield of the L-valine, lays a foundation for industrial production of the L-valine, and has wide industrial application prospects.

Description

Recombinant microorganism producing valine and construction method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a valine-producing recombinant microorganism and a construction method and application thereof.

Background

L-valine (L-valine) with chemical name of L-alpha-amino isovaleric acid and molecular formula of C ₅ H ₁₁ NO ₂ The relative molecular mass was 117.15. L-valine is white crystal or crystalline powder, has no odor and bitter taste, and has solubility in water: 88.5g/L at 25 deg.C, 96.2g/L at 50 deg.C, and insoluble in cold ethanol, diethyl ether, and acetone. L-valine has an isoelectric point of 5.96 and a melting point of 315 ℃.

L-valine is one of eight essential amino acids in human body, is one of three branched chain amino acids (including valine, leucine and isoleucine), and has a particularly important position in human life metabolism due to the special structure and function. L-valine can be widely applied to the pharmaceutical industry, the food industry, the feed industry and the like. In the pharmaceutical industry, L-valine can be used as main ingredient of amino acid infusion and synthetic amino acid preparation, and can be used for treating hepatic failure and central nervous system dysfunction. In the food industry, L-valine is used as a food additive, a nutritional supplement solution, a flavor, and the like. L-valine can also be used as amino acid functional beverage and athlete beverage, and has effects of forming muscle, strengthening liver function, and relieving muscle fatigue. In the feed industry, it has an important promoting effect on the milk secretion of mammary tissues of animals.

There are three current methods for producing L-valine: extraction method, chemical synthesis method, and microorganism fermentation method. The extraction method and the chemical synthesis method are difficult to realize industrial production due to the limited raw material sources, high production cost and environmental pollution. The method for producing L-valine by using a microbial fermentation method has the advantages of low raw material cost, mild reaction conditions, easiness in realizing large-scale production and the like, and is the most important method for producing L-valine at present. However, the fermentation performance of the current L-valine strain is still poor, the conversion rate of the L-valine is still low, and the requirement of large-scale industrial production cannot be met.

Disclosure of Invention

The invention aims to provide a mutant and a corynebacterium capable of realizing high yield of valine and a construction method and application thereof aiming at the defects in the prior art.

Specifically, the invention provides the following technical scheme:

compared with an original strain, the recombinant microorganism has the advantages that a promoter of a gene brnFE in the recombinant microorganism is mutated into a DNA molecule shown as SEQ ID NO.1, and/or an endogenous protein coded by the gene brnQ in the recombinant microorganism is mutated to form a mutant, the mutant contains a mutation that the amino acid sequence of the endogenous protein coded by the wild-type gene brnQ of the original strain is taken as a reference sequence, and 112 th alanine is replaced by threonine, serine or tyrosine.

The invention makes point mutation to the brnQ gene 1, so that the 112 th amino acid is mutated from alanine to threonine or serine or tyrosine Tyr, and the specific codon is mutated from GCG to GCA or GCT or TAC. Thereby weakening the pathway of a valine internal transportation system, being beneficial to increasing the yield of the valine and avoiding the valine from being reused by the strain; and/or 2, introducing specific point mutation into a promoter region of a gene brnFE of the branched chain amino acid export protein, thereby improving the efflux efficiency of valine and finally obtaining the genetic engineering bacteria with high L-valine yield.

In the invention, the mutant has an amino acid sequence shown in any one of SEQ ID NO. 2-4.

The starting strain is corynebacterium or brevibacterium;

preferably, the Corynebacterium genus bacterium is Corynebacterium glutamicum (Corynebacterium glutamicum) or Corynebacterium beijing (Corynebacterium pekinense); the brevibacterium is brevibacterium flavum (brevibacterium flavum);

more preferably, the starting strain is a bacterium of the genus Corynebacterium which is capable of accumulating valine.

The invention also provides a protein mutant which is an internal transport protein mutant coded by the gene brnQ, and the specific amino acid sequence is as described above.

It will be understood by those skilled in the art that the addition of a tag protein to the N-terminus or C-terminus of the mutant sequence of the above protein or the fusion of the tag protein with other proteins to form a fusion protein is also within the scope of the present invention without altering the activity of the above mutant protein itself.

The invention also provides a nucleic acid for coding the protein mutant, preferably, the nucleic acid has a nucleotide sequence shown in any one of SEQ ID NO. 5-7.

Based on the amino acid sequences of the protein mutants provided above, the skilled person is able to obtain the sequences of the nucleic acids encoding them. Based on the degeneracy of the codon, more than one nucleic acid sequence encoding the above amino acid sequences is included in the scope of the present invention, and all nucleic acids encoding the above protein mutants are included in the scope of the present invention.

The invention also provides a DNA molecule which is a promoter mutant of the gene brnFE and has a nucleotide sequence shown as SEQ ID NO. 1.

The invention also provides a biological material containing the nucleic acid and/or DNA molecule, wherein the biological material is an expression cassette, a vector or a host cell.

The expression cassette is a recombinant nucleic acid molecule which is connected with elements for driving the transcription and expression of the nucleic acid and/or DNA molecule at the upstream or downstream.

The vector may be an expression vector or a cloning vector, including but not limited to a plasmid vector, a phage vector, a transposon, and the like.

Such host cells include, but are not limited to, microbial cells.

The invention also provides the application of the recombinant microorganism or the protein mutant or the nucleic acid or the DNA molecule or the biological material in improving the yield of the microbial branched-chain amino acid, wherein the branched-chain amino acid is valine, leucine or isoleucine, and preferably valine.

The invention also provides a construction method of the recombinant microorganism, which comprises the following steps: the promoter of brnFE gene in the original strain is mutated into DNA molecule shown in SEQ ID NO.1, and/or the brnQ gene in the original strain is mutated into the gene for coding the protein mutant.

The invention also provides a method for producing valine, which comprises the steps of inoculating the recombinant microorganism into a seed culture medium for seed culture, and then transferring the seed culture into a fermentation culture medium for fermentation culture;

preferably, the seed culture medium comprises the following components: 10-20g/L of soybean meal extract, 15-25g/L of glucose, 6-8g/L of ammonium sulfate, 0.4-0.6g/L of magnesium sulfate, 0.9-1.1g/L of monopotassium phosphate, 0.9-1.1g/L of dipotassium phosphate, 1.8-2.2g/L of urea and the balance of water, wherein the pH value is 7.2-7.5;

the fermentation medium comprises the following components: 10-20g/L of soybean meal extract, 65-75g/L of glucose, 15-25g/L of ammonium sulfate, 0.4-0.6g/L of magnesium sulfate, 0.9-1.1g/L of potassium dihydrogen phosphate, 0.9-1.1g/L of dipotassium hydrogen phosphate, 1.8-2.2g/L of urea, 35-45g/L of calcium carbonate, 14-16mg/L of VB3, and V _H 45-55 mug/L, VB1 & HCl 90-110 mug/L, and the balance of water, wherein the pH value is 7.2-7.5.

More preferably, the seed culture medium comprises the following components: 15g/L of soybean meal extract, 20g/L of glucose, 7g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea and the balance of water, wherein the pH value is 7.2.

The fermentation medium comprises the following components: 15g/L of soybean meal extract, 60g/L of glucose, 20g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea, 40g/L of calcium carbonate, 315mg/L of VB, V _H 50μg/L，VB 1. HCl 100. Mu.g/L, balance water, pH7.2.

In the invention, the seed culture is performed for 10-12h at 30 ℃ and 220rpm/min with shaking. The fermentation culture is carried out for 48 hours at 30 ℃ and 220r/min in a shaking way.

The invention has the beneficial effects that:

the invention provides a construction method and application of corynebacterium capable of producing valine with high yield, which comprises the steps of preparing a brnQ A112T, A112S and A112Y point mutation gene segment and/or a brnFE mutant promoter, connecting the gene brnQ A112T, A112S and A112Y point mutation gene segment and/or brnFE mutant promoter with a vector to obtain a site mutation recombinant vector, and transforming the corynebacterium to obtain the corynebacterium with target site mutation. Experiments show that the corynebacterium sp is an L-valine high-yield strain, can effectively accumulate L-valine, improves the yield of the L-valine, lays a foundation for industrial production of the L-valine, and has wide industrial application prospects. When the two mutations are used in combination, the effect is better.

Furthermore, these mutations can be used in other host bacteria such as Corynebacterium glutamicum and Escherichia coli, and can also be used in the production of branched-chain amino acids such as leucine and isoleucine.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Meanwhile, since the plasmid pK18mobsacB-brnQ (M1)/(M2)/(M3) is different only at the mutation point, a schematic diagram of pK18mobsacB-brnQ (M) is given to illustrate introduction of the series of plasmids.

FIG. 1 is a schematic representation of the recombinant plasmid pK18mobsacB-PbrnFE (M) -brnFE;

FIG. 2 is a schematic diagram of recombinant plasmid pK18mobsacB-brnQ (M).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The invention discloses corynebacterium capable of producing valine with high yield, a construction method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations and modifications, as appropriate, may be made to the methods described herein to achieve and use the techniques of the invention without departing from the spirit and scope of the invention.

For further understanding of the present invention, the present invention is described in detail below with reference to specific examples, and unless otherwise specified, all reagents involved in the examples of the present invention are commercially available and commercially available. The starting strain MHZ-1012-3 in the embodiment of the invention is corynebacterium glutamicum, the formula of the common liquid brain and heart infusion medium is 3.7% brain and heart infusion powder solution, and the formula of the common solid brain and heart infusion medium is 3.7% brain and heart infusion powder solution and 1.8% agar powder. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

The names and sequences of the primers of SEQ ID NO.8-31 referred to in the following examples are shown in Table 1.

TABLE 1 primer sequences

The strains referred to in the following examples are as follows:

the starting strain MHZ-1012-3 is a valine producing strain, and the strain is obtained by mutating alanine (A) at the 1 st position of isopropyl malate synthase gene leuA of the starting strain MHZ-1012-2 into glycine (G). The specific construction method is disclosed in Chinese patent CN110982772A. MHZ-1012-2 is preserved in China general microbiological culture Collection center (CGMCC) at 2016 (11 months and 30 days), wherein the preservation center address is No.3 of West Lu No.1 of Beijing, facing the Yangtze district, and the preservation number is CGMCC No.13406. And (3) classification and naming: corynebacterium glutamicum, corynebacterium glutamicum.

The Corynebacterium glutamicum MHZ-1012-2 (CGMCC No. 13406) mentioned in the invention is disclosed in Chinese patent publication No. CN 106520655A.

Example 1 construction of plasmid pK18mobsacB-PbrnFE (M) -brnFE and construction of recombinant Strain MHZ-1012-4

The sequence of the brnFE original promoter is shown in SEQ ID NO. 32. The sequence of the brnFE mutant promoter is shown as SEQ ID NO. 1.

The specific construction process is as follows:

phusion ultra-fidelity polymerase (New England BioLabs), genome of Corynebacterium glutamicum MHZ-1012-3 is used as a template, PV370-UP-1F/PV371-DOWN-1R is used as a primer to prepare a recombinant fragment UP-1, PV372-UP-2F/PV373-DOWN-2R is used as a primer to prepare a recombinant fragment DOWN-1, plasmid pk18-mob-sacB is used as a template, PV374-UP-3F/PV375-DOWN-3R is used as a primer to obtain fragment pk18-1, the fragment pk18-1 is purified by an agarose gel recovery kit (Tiangen), and then the reaction is carried out according to a Gipson assembly kit configuration system, wherein the reaction system is as shown in the following table 2:

TABLE 2 Gepton Assembly reaction System-1

Components	UP-1	DOWN-1	pk18-1	CE Buffer	CE Exnase	Sterile water
							Volume/. Mu.L	1	1	2	4	2	9

After the system is prepared, the reaction is carried out for 30min at 37 ℃, 10 mu L of transformed Trans1T1 competent cells (TransGen Biotech) are sucked, kanamycin-resistant clones are picked, the correctness of the inserted fragment is identified by sequencing with P82/P85 primer (Invitrogen company), a positive clone with the fragment inserted into pK18mobsacB is obtained by further EcoRI/SalI enzyme digestion identification, finally the plasmid is sent to a Jinzhi sequencing company for sequencing, and the obtained plasmid with the correct sequencing is named as pK18mobsacB-PbrnFE (M) -brnFE. The plasmid pK18mobsacB-PbrnFE (M) -brnFE is schematically shown in FIG. 1.

pK18mobsacB-PbrnFE (M) -brnFE was transferred into Corynebacterium glutamicum MHZ-1012-3, and the recombinant was selected for replacement on selection medium containing 15mg/L kanamycin. The temperature of the culture was 30 ℃ and the culture was inverted. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the culture temperature of 30 ℃ and performing shaking culture on a rotary shaking table at 220 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. Cultures were serially diluted in gradient (10) ^-2 Continuously diluting to 10 ^-4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10 percent of sucrose, and is subjected to static culture at 30 ℃ for 48 hours. Further performing phenotype verification on the screened strains, selecting a point mutation recombinants for the recombinants of KanS by utilizing PV376-ID-F/PV-TEST-brnFE-R, obtaining recombinants containing point mutation by groping annealing temperature, amplifying and sequencing the obtained positive recombinants by using PV-TEST-brnFE-F/PV-TEST-brnFE-R, and verifying to obtain the recombinantsIs the objective mutant strain and is named MHZ-1012-4.

Example 2 fermentation production of L-valine by L-valine genetically engineered bacterium MHZ-1012-4

1. Culture medium

Seed culture medium: 15g/L of soybean meal extract, 20g/L of glucose, 7g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea and the balance of water, wherein the pH value is 7.2.

Fermentation medium: 15g/L of soybean meal extract, 60g/L of glucose, 20g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea, 40g/L of calcium carbonate, 15mg/L of VB3 and V _H 50. Mu.g/L, VB 1. Mu.g/L HCl 100. Mu.g/L, balance water, pH7.2.

2. MHZ-1012-4 shake flask fermentation production of L-valine

(1) Seed culture: selecting MHZ-1012-3 and MHZ-1012-4 slant seeds 1, circularly inoculating to a 500mL triangular flask filled with 50mL seed culture medium, and performing shaking culture at 30 ℃ and 220r/min for 10-12h;

(2) Fermentation culture: 5mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 50mL of the fermentation medium, and cultured at 30 ℃ under shaking at 220r/min for 48 hours.

(3) 1mL of fermentation liquor is centrifuged (12000rpm, 2min), supernatant is collected, and L-valine in the fermentation liquor of the engineering bacteria MHZ-1012-4 and the starting bacteria MHZ-1012-3 (control bacteria) is detected by HPLC, and the concentration of the L-valine is shown in the following table 3.

TABLE 3L-valine concentration in fermentation broths

Strain of bacillus	L-val(g/L)	The acid yield is improved%	Conversion in g/g
				MHZ-1012-3	7.6	--	0.124
MHZ-1012-4	8.9	17.1％	0.139

The result shows that the accumulation amount of the L-valine of the starting strain MHZ-1012-3 is only 7.6g/L, while the yield of the L-valine of the engineering strain MHZ-1012-4 of the invention is 8.9g/L, which is 1.3g/L and 17.1% higher than that of the starting strain, and the conversion rate of the valine reaches 0.139g/g (0.139 g valine per gram glucose); therefore, the mutation of the promoter of brnFE can enhance the export capacity of valine, so that the cells can more efficiently export the produced valine to the outside of the cells, and the detectable product L-valine is increased.

Example 3 construction of plasmid pK18mobsacB-brnQ (M) and construction of recombinant Strain MHZ-1012-5-1/2/3

The mutated CDS region nucleic acid sequence of brnQ is shown in SEQ ID NO.5-7, and the mutated CDS region amino acid sequence of brnQ is shown in SEQ ID NO. 2-4.

The mutation A112T is introduced into the brnQ gene, and the specific construction process is as follows:

phusion ultra fidelity polymerase (New England BioLabs), genome of Corynebacterium glutamicum MHZ-1012-3 is used as a template, PV377-UP-1F/PV378-DOWN-1R is used as a primer to prepare a recombinant fragment UP-2, PV379-UP-2F/PV380-DOWN-2R is used as a primer to prepare a recombinant fragment DOWN-2, plasmid pk18-mob-sacB is used as a template, PV381-UP-3F/PV382-DOWN-3R is used as a primer to obtain a fragment pk18-2, the fragment pk18-2 is purified by an agarose gel recovery kit (Tiangen), and then the reaction is carried out according to a Gepton assembly kit configuration system, wherein the reaction system is as shown in the following table 4:

TABLE 4 Gepton Assembly reaction System-2

Components	UP-2	DOWN-2	pk18-2	CE Buffer	CE Exnase	Sterile water
							Volume/. Mu.L	1	1	2	4	2	9

After the system was prepared, the reaction was carried out at 37 ℃ for 30min, 10. Mu.L of the transformed Trans1T1 competent cells (TransGen Biotech) were aspirated, the kanamycin-resistant clones were picked up, the inserted fragment was confirmed to be correct by sequencing with P82/P85 primers (Invitrogen), the inserted fragment was further confirmed by XbaI/NheI digestion to obtain positive clones with the fragment inserted into pK18mobsacB, and finally the plasmid was sent to Kinzyme sequencer for sequencing, and the obtained correctly sequenced plasmid was named pK18mobsacB-brnQ (M1). Transfer of pK18mobsacB-brnQ (M1) into Corynebacterium glutamicum MHZ-1012-3, selecting the replacement recombinants on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30 ℃ and the culture was inverted. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the culture temperature of 30 ℃ and performing shaking culture on a rotary shaking table at 220 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10) ^-2 Continuously diluting to 10 ^-4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10 percent of sucrose, and is subjected to static culture at 30 ℃ for 48 hours. Further performing phenotype verification on the screened strains, selecting a point mutation recon of KanS by utilizing PV383-ID-F/PV-TEST-brnQ-R, obtaining a recon containing point mutation by groping the annealing temperature, performing amplification sequencing on the obtained positive recon by using PV-TEST-brnQ-F/PV-TEST-brnQ-R, verifying the obtained mutant strain to be a target mutant strain, and naming the mutant strain as MHZ-1012-5-1.

Example 3.1: the introduction of the mutations A112S and A112Y into the brnQ gene was constructed in the same manner as described in example 3, except that primers for the mutant brnQ (A112S) were used to replace PV378-DOWN-1R and PV379-UP-2F in example 3 with PV378-DOWN-1R-1 and PV379-UP-2F-1, respectively, to construct the vector pK18mobsacB-brnQ (M2); mutant brnQ (A112S) primers used were PV378-DOWN-1R and PV379-UP-2F in example 3 were replaced with PV378-DOWN-1R-2 and PV379-UP-2F-2, respectively, to construct vector pK18mobsacB-brnQ (M3) and the finally constructed engineered strains were named MHZ-1012-5-2 and MHZ-1012-5-3, respectively.

The plasmids pK18mobsacB-brnQ (M) (pK 18mobsacB-brnQ (M1), pK18mobsacB-brnQ (M2), pK18mobsacB-brnQ (M3)) are schematically shown in FIG. 2.

Example 4 fermentation production of L-valine by the genetically engineered bacteria MHZ-1012-5-1, MHZ-1012-5-2 and MHZ-1012-5-3

1. Culture medium

Seed culture medium: 15g/L of soybean meal extract, 20g/L of glucose, 7g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea and the balance of water, wherein the pH value is 7.2.

Fermentation cultureAnd (3) nutrient medium: 15g/L of soybean meal extract, 60g/L of glucose, 20g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea, 40g/L of calcium carbonate, 15mg/L of VB3 and V _H 50. Mu.g/L, VB 1. Mu.g/L HCl 100. Mu.g/L, balance water, pH7.2.

2. MHZ-1012-5-1/2/3 shake flask fermentation production of L-valine

(1) Seed culture: selecting MHZ-1012-3 and MHZ-1012-5-1/2/3 slant seeds 1, circularly inoculating to a 500mL triangular flask filled with 50mL seed culture medium, and performing shake culture at 30 ℃ and 220r/min for 10-12h;

(2) Fermentation culture: 5mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 50mL of the fermentation medium, and cultured at 30 ℃ under shaking at 220r/min for 48 hours.

(3) 1mL of fermentation broth is centrifuged (12000rpm, 2min), the supernatant is collected, and L-valine in the fermentation broth of the engineering bacteria MHZ-1012-5-1/2/3 and the development bacteria MHZ-1012-3 (control bacteria) is detected by HPLC, and the concentration of the L-valine is shown in the following table 5.

TABLE 5L-valine concentration in fermentation broths

Strain of bacillus	L-val(g/L)	The acid yield is improved%	Conversion in g/g
				MHZ-1012-3	7.6	--	0.124
MHZ-1012-5-1	8.5	11.8％	0.135
				MHZ-1012-5-2	8.2	7.9％	0.129
MHZ-1012-5-3	8.4	10.5％	0.134

The result shows that the valine yield of the target engineering bacterium MHZ-1012-5-1 is 8.5g/L, which is improved by 0.9g/L and 11.8% compared with the yield of the starting strain MHZ-1012-4, the maximum improvement range is realized, and the conversion rate is also improved from 0.124g/g to 0.135g/g; the yields of the strains MHZ-1012-5-2 and MHZ-1012-5-3 are respectively improved to 8.2g/L and 8.4g/L after Ser and Tyr are replaced, and the conversion rates are also improved in different ranges, which is shown in Table 5. Therefore, the mutant of brnQ reduces the internal transportation and consumption of valine, so that the cells can accumulate the valine more efficiently, and the detectable product L-valine is increased.

EXAMPLE 5 construction of recombinant Strain MHZ-1012-6

The specific construction process is as follows:

pK18mobsacB-brnQ (M1) was transferred into Corynebacterium glutamicum MHZ-1012-4 and the replacement recombinants were selected on selection medium containing 15mg/L kanamycin. The temperature of the culture was 30 ℃ and the culture was inverted. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the culture temperature of 30 ℃ and performing shaking culture on a rotary shaking table at 220 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10) ^-2 Continuously diluting to 10 ^-4 ) The diluted solution is coated on a common solution containing 10% sucroseAnd (3) standing and culturing for 48 hours at 30 ℃ on a solid brain heart infusion culture medium. Further performing phenotype verification on the screened strains, selecting a point mutation recon of KanS by utilizing PV383-ID-F/PV-TEST-brnQ-R, obtaining a recon containing point mutation by groping the annealing temperature, performing amplification sequencing on the obtained positive recon by using PV-TEST-brnQ-F/PV-TEST-brnQ-R, verifying the obtained mutant strain to be the target mutant strain, and naming the mutant strain as MHZ-1012-6.

EXAMPLE 6L-valine Gene-engineering bacterium MHZ-1012-6 fermentation production of L-valine

1. Culture medium

Seed culture medium: 15g/L of soybean meal extract, 20g/L of glucose, 7g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea and the balance of water, wherein the pH value is 7.2.

Fermentation medium: 15g/L of soybean meal extract, 60g/L of glucose, 20g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea, 40g/L of calcium carbonate, 15mg/L of VB3 and V _H 50. Mu.g/L, VB 1. Mu.HCl 100. Mu.g/L, balance water, pH7.2.

2. MHZ-1012-6 shake flask fermentation production of L-valine

(1) Seed culture: selecting MHZ-1012-3, MHZ-1012-4, MHZ-1012-5-1 and MHZ-1012-6 slant seeds 1, circularly inoculating the seeds into a 500mL triangular flask filled with 50mL seed culture medium, and carrying out shaking culture at 30 ℃ and 220r/min for 10-12h;

(2) Fermentation culture: 5mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 50mL of the fermentation medium, and the mixture was subjected to shaking culture at 30 ℃ and 220r/min for 48 hours.

(3) 1mL of fermentation broth was centrifuged (12000rpm, 2min), the supernatant was collected, and L-valine in the fermentation broth of the engineered bacteria was detected by HPLC, the concentrations of which are shown in Table 6 below.

TABLE 6L-valine concentration in fermentation broth

Bacterial strains	L-val(g/L)	The acid yield is improved%	Conversion in g/g
				MHZ-1012-3	7.6	--	0.124
MHZ-1012-4	8.9	17.1％	0.139
				MHZ-1012-5-1	8.5	11.8％	0.135
MHZ-1012-6	11.5	51.3％	0.178

The result shows that the valine yield of the target engineering bacterium MHZ-1012-6 is 11.5g/L, which is improved by 3.9g/L and 51.3% compared with the yield of the starting strain MHZ-1012-3, and the conversion rate is improved to 0.178g/g and 43.5% from 0.124g/g before modification; therefore, after the mutation site A112T of brnQ and the promoter region of brnFE of the export protein are introduced simultaneously, the internal transportation of valine is successfully reduced and the export efficiency is improved respectively, so that the cells can more efficiently accumulate valine and the detectable product L-valine is increased.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ser Lys Lys Ser Val Leu Ile Thr Ser Leu Met Leu Phe Ser Met

1 5 10 15

Phe Phe Gly Ala Gly Asn Leu Ile Phe Pro Pro Met Leu Gly Leu Ser

20 25 30

Ala Gly Thr Asn Tyr Leu Pro Ala Ile Leu Gly Phe Leu Ala Thr Ser

35 40 45

Val Leu Leu Pro Val Leu Ala Ile Ile Ala Val Val Leu Ser Gly Glu

50 55 60

Asn Val Lys Asp Met Ala Ser Arg Gly Gly Lys Ile Phe Gly Leu Val

65 70 75 80

Phe Pro Ile Ala Ala Tyr Leu Ser Ile Gly Ala Phe Tyr Ala Leu Pro

85 90 95

Arg Thr Gly Ala Val Ser Tyr Ser Thr Ala Val Gly Val Asp Asn Thr

100 105 110

Leu Tyr Ser Gly Leu Phe Asn Phe Val Phe Phe Ala Val Ala Leu Ala

115 120 125

Leu Ser Trp Asn Pro Asn Gly Ile Ala Asp Lys Leu Gly Lys Trp Leu

130 135 140

Thr Pro Ala Leu Leu Thr Leu Ile Val Val Leu Val Val Leu Ser Val

145 150 155 160

Ala Lys Leu Asp Gly Thr Pro Gly Glu Pro Ser Ser Ala Tyr Ala Gln

165 170 175

Gln Pro Ala Gly Ala Gly Leu Leu Glu Gly Tyr Met Thr Met Asp Ala

180 185 190

Ile Ala Ala Leu Ala Phe Gly Ile Val Val Ile Ser Ala Phe Lys Tyr

195 200 205

Gln Lys Val Asn Lys Val Arg Thr Ala Thr Val Val Ser Ala Phe Ile

210 215 220

Ala Gly Ile Leu Leu Ala Leu Val Tyr Leu Gly Leu Gly Ser Ile Gly

225 230 235 240

Gln Val Val Asn Gly Glu Phe Ala Asp Gly Thr Ala Ile Leu Asn Tyr

245 250 255

Ala Ala Leu Ser Thr Met Gly Gln Ala Gly Arg Ile Met Phe Val Ala

260 265 270

Ile Leu Ile Leu Ala Cys Met Thr Thr Ala Val Gly Leu Ile Ser Ala

275 280 285

Thr Ser Glu Phe Phe Asn Ser Leu Leu Pro Gly Val Lys Tyr His Val

290 295 300

Trp Ala Thr Val Phe Ala Leu Ile Ser Phe Gly Val Ala Thr Met Gly

305 310 315 320

Leu Asp Thr Val Leu Ala Val Ala Ala Pro Val Ile Ser Phe Ile Tyr

325 330 335

Pro Ser Ala Ile Thr Leu Val Phe Leu Ser Leu Ile Glu Pro Leu Leu

340 345 350

Phe Arg Leu Lys Trp Thr Tyr Leu Phe Gly Ile Trp Thr Ala Val Val

355 360 365

Trp Ala Leu Phe Met Ser Ile Pro Ala Leu Asn Pro Phe Ile Glu Trp

370 375 380

Ala Pro Leu His Ser Met Ser Leu Gly Trp Val Val Pro Val Leu Val

385 390 395 400

Ala Ser Ala Ile Gly Leu Ala Ile Asp Trp Asn Lys Lys Gly Ala Gln

405 410 415

Ser Val Ala Glu Lys Glu Ser Ile Ser Val

420 425

<210> 3

<211> 426

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ser Lys Lys Ser Val Leu Ile Thr Ser Leu Met Leu Phe Ser Met

1 5 10 15

Phe Phe Gly Ala Gly Asn Leu Ile Phe Pro Pro Met Leu Gly Leu Ser

20 25 30

Ala Gly Thr Asn Tyr Leu Pro Ala Ile Leu Gly Phe Leu Ala Thr Ser

35 40 45

Val Leu Leu Pro Val Leu Ala Ile Ile Ala Val Val Leu Ser Gly Glu

50 55 60

Asn Val Lys Asp Met Ala Ser Arg Gly Gly Lys Ile Phe Gly Leu Val

65 70 75 80

Phe Pro Ile Ala Ala Tyr Leu Ser Ile Gly Ala Phe Tyr Ala Leu Pro

85 90 95

Arg Thr Gly Ala Val Ser Tyr Ser Thr Ala Val Gly Val Asp Asn Ser

100 105 110

Leu Tyr Ser Gly Leu Phe Asn Phe Val Phe Phe Ala Val Ala Leu Ala

115 120 125

Leu Ser Trp Asn Pro Asn Gly Ile Ala Asp Lys Leu Gly Lys Trp Leu

130 135 140

Thr Pro Ala Leu Leu Thr Leu Ile Val Val Leu Val Val Leu Ser Val

145 150 155 160

Ala Lys Leu Asp Gly Thr Pro Gly Glu Pro Ser Ser Ala Tyr Ala Gln

165 170 175

Gln Pro Ala Gly Ala Gly Leu Leu Glu Gly Tyr Met Thr Met Asp Ala

180 185 190

Ile Ala Ala Leu Ala Phe Gly Ile Val Val Ile Ser Ala Phe Lys Tyr

195 200 205

Gln Lys Val Asn Lys Val Arg Thr Ala Thr Val Val Ser Ala Phe Ile

210 215 220

Ala Gly Ile Leu Leu Ala Leu Val Tyr Leu Gly Leu Gly Ser Ile Gly

225 230 235 240

Gln Val Val Asn Gly Glu Phe Ala Asp Gly Thr Ala Ile Leu Asn Tyr

245 250 255

Ala Ala Leu Ser Thr Met Gly Gln Ala Gly Arg Ile Met Phe Val Ala

260 265 270

Ile Leu Ile Leu Ala Cys Met Thr Thr Ala Val Gly Leu Ile Ser Ala

275 280 285

Thr Ser Glu Phe Phe Asn Ser Leu Leu Pro Gly Val Lys Tyr His Val

290 295 300

Trp Ala Thr Val Phe Ala Leu Ile Ser Phe Gly Val Ala Thr Met Gly

305 310 315 320

Leu Asp Thr Val Leu Ala Val Ala Ala Pro Val Ile Ser Phe Ile Tyr

325 330 335

Pro Ser Ala Ile Thr Leu Val Phe Leu Ser Leu Ile Glu Pro Leu Leu

340 345 350

Phe Arg Leu Lys Trp Thr Tyr Leu Phe Gly Ile Trp Thr Ala Val Val

355 360 365

Trp Ala Leu Phe Met Ser Ile Pro Ala Leu Asn Pro Phe Ile Glu Trp

370 375 380

Ala Pro Leu His Ser Met Ser Leu Gly Trp Val Val Pro Val Leu Val

385 390 395 400

Ala Ser Ala Ile Gly Leu Ala Ile Asp Trp Asn Lys Lys Gly Ala Gln

405 410 415

Ser Val Ala Glu Lys Glu Ser Ile Ser Val

420 425

<210> 4

<211> 426

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ser Lys Lys Ser Val Leu Ile Thr Ser Leu Met Leu Phe Ser Met

1 5 10 15

Phe Phe Gly Ala Gly Asn Leu Ile Phe Pro Pro Met Leu Gly Leu Ser

20 25 30

Ala Gly Thr Asn Tyr Leu Pro Ala Ile Leu Gly Phe Leu Ala Thr Ser

35 40 45

Val Leu Leu Pro Val Leu Ala Ile Ile Ala Val Val Leu Ser Gly Glu

50 55 60

Asn Val Lys Asp Met Ala Ser Arg Gly Gly Lys Ile Phe Gly Leu Val

65 70 75 80

Phe Pro Ile Ala Ala Tyr Leu Ser Ile Gly Ala Phe Tyr Ala Leu Pro

85 90 95

Arg Thr Gly Ala Val Ser Tyr Ser Thr Ala Val Gly Val Asp Asn Tyr

100 105 110

Leu Tyr Ser Gly Leu Phe Asn Phe Val Phe Phe Ala Val Ala Leu Ala

115 120 125

Leu Ser Trp Asn Pro Asn Gly Ile Ala Asp Lys Leu Gly Lys Trp Leu

130 135 140

Thr Pro Ala Leu Leu Thr Leu Ile Val Val Leu Val Val Leu Ser Val

145 150 155 160

Ala Lys Leu Asp Gly Thr Pro Gly Glu Pro Ser Ser Ala Tyr Ala Gln

165 170 175

Gln Pro Ala Gly Ala Gly Leu Leu Glu Gly Tyr Met Thr Met Asp Ala

180 185 190

Ile Ala Ala Leu Ala Phe Gly Ile Val Val Ile Ser Ala Phe Lys Tyr

195 200 205

Gln Lys Val Asn Lys Val Arg Thr Ala Thr Val Val Ser Ala Phe Ile

210 215 220

Ala Gly Ile Leu Leu Ala Leu Val Tyr Leu Gly Leu Gly Ser Ile Gly

225 230 235 240

Gln Val Val Asn Gly Glu Phe Ala Asp Gly Thr Ala Ile Leu Asn Tyr

245 250 255

Ala Ala Leu Ser Thr Met Gly Gln Ala Gly Arg Ile Met Phe Val Ala

260 265 270

Ile Leu Ile Leu Ala Cys Met Thr Thr Ala Val Gly Leu Ile Ser Ala

275 280 285

Thr Ser Glu Phe Phe Asn Ser Leu Leu Pro Gly Val Lys Tyr His Val

290 295 300

Trp Ala Thr Val Phe Ala Leu Ile Ser Phe Gly Val Ala Thr Met Gly

305 310 315 320

Leu Asp Thr Val Leu Ala Val Ala Ala Pro Val Ile Ser Phe Ile Tyr

325 330 335

Pro Ser Ala Ile Thr Leu Val Phe Leu Ser Leu Ile Glu Pro Leu Leu

340 345 350

Phe Arg Leu Lys Trp Thr Tyr Leu Phe Gly Ile Trp Thr Ala Val Val

355 360 365

Trp Ala Leu Phe Met Ser Ile Pro Ala Leu Asn Pro Phe Ile Glu Trp

370 375 380

Ala Pro Leu His Ser Met Ser Leu Gly Trp Val Val Pro Val Leu Val

385 390 395 400

Ala Ser Ala Ile Gly Leu Ala Ile Asp Trp Asn Lys Lys Gly Ala Gln

405 410 415

Ser Val Ala Glu Lys Glu Ser Ile Ser Val

420 425

<210> 5

<211> 1281

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgagtaaaa agtctgtcct gattacttct ttgatgctgt tttccatgtt cttcggagct 60

ggaaacctca tcttcccgcc gatgcttgga ttgtcggcag gaaccaacta tctaccagct 120

atcttaggat ttctagcaac gagtgttctg ctcccggtgc tggcgattat cgcggtggtg 180

ttgtcgggag aaaatgtcaa ggacatggct tctcgtggcg gtaagatctt tggcctggtg 240

tttcctattg ctgcctattt gtccatcggt gcgttttacg cgctgccgag gactggggcg 300

gtgagctatt cgacggcggt tggcgtcgat aatacgcttt attcgggctt gtttaacttt 360

gtgttttttg cggtggcact ggcgttgtcg tggaatccga atggcattgc agacaagttg 420

ggtaagtggc tcacgccagc gttgctcacg ttgattgtgg tgctggtggt gttgtcggta 480

gccaagttgg atggcacgcc aggtgagcca agtagtgcgt atgcgcagca gcctgcgggg 540

gcgggtttgc ttgagggcta catgacgatg gatgcgattg ctgcgttggc gtttggcatc 600

gtggtgattt ctgcgttcaa gtaccaaaag gttaacaagg tccgcacggc aactgtcgtg 660

tcggcgttca ttgccggaat tttgttggcg ctggtttatc ttggtttggg ctcaatcggt 720

caagtagtaa acggtgagtt cgctgatggc accgcaattt tgaactacgc tgcactgtcc 780

acgatgggtc aggctggtcg catcatgttc gtggccattt tgatccttgc atgtatgacc 840

accgcagttg gtctgatcag tgcgacgtct gagtttttca attcgctgct gccaggtgtc 900

aagtaccacg tctgggccac tgttttcgcg ctgatttcct ttggcgttgc cacgatggga 960

ttggatacgg tgttggccgt tgcggctcca gtgattagtt tcatttaccc atcggccatc 1020

accttggtgt tcttgtcgct catcgagccc ctgctgttcc gtctcaagtg gacctaccta 1080

ttcggcattt ggactgcagt tgtgtgggcg ctgttcatgt ctatccctgc gctgaatcca 1140

ttcatcgaat gggcgccgct gcacagcatg tctttgggtt gggttgtccc agttctcgtg 1200

gcctctgcca tcggtttggc tattgattgg aacaagaaag gtgcccagtc tgttgcagag 1260

aaggaatcca tttccgtcta a 1281

<210> 6

<211> 1281

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgagtaaaa agtctgtcct gattacttct ttgatgctgt tttccatgtt cttcggagct 60

ggaaacctca tcttcccgcc gatgcttgga ttgtcggcag gaaccaacta tctaccagct 120

atcttaggat ttctagcaac gagtgttctg ctcccggtgc tggcgattat cgcggtggtg 180

ttgtcgggag aaaatgtcaa ggacatggct tctcgtggcg gtaagatctt tggcctggtg 240

tttcctattg ctgcctattt gtccatcggt gcgttttacg cgctgccgag gactggggcg 300

gtgagctatt cgacggcggt tggcgtcgat aattcgcttt attcgggctt gtttaacttt 360

gtgttttttg cggtggcact ggcgttgtcg tggaatccga atggcattgc agacaagttg 420

ggtaagtggc tcacgccagc gttgctcacg ttgattgtgg tgctggtggt gttgtcggta 480

gccaagttgg atggcacgcc aggtgagcca agtagtgcgt atgcgcagca gcctgcgggg 540

gcgggtttgc ttgagggcta catgacgatg gatgcgattg ctgcgttggc gtttggcatc 600

gtggtgattt ctgcgttcaa gtaccaaaag gttaacaagg tccgcacggc aactgtcgtg 660

tcggcgttca ttgccggaat tttgttggcg ctggtttatc ttggtttggg ctcaatcggt 720

caagtagtaa acggtgagtt cgctgatggc accgcaattt tgaactacgc tgcactgtcc 780

acgatgggtc aggctggtcg catcatgttc gtggccattt tgatccttgc atgtatgacc 840

accgcagttg gtctgatcag tgcgacgtct gagtttttca attcgctgct gccaggtgtc 900

aagtaccacg tctgggccac tgttttcgcg ctgatttcct ttggcgttgc cacgatggga 960

ttggatacgg tgttggccgt tgcggctcca gtgattagtt tcatttaccc atcggccatc 1020

accttggtgt tcttgtcgct catcgagccc ctgctgttcc gtctcaagtg gacctaccta 1080

ttcggcattt ggactgcagt tgtgtgggcg ctgttcatgt ctatccctgc gctgaatcca 1140

ttcatcgaat gggcgccgct gcacagcatg tctttgggtt gggttgtccc agttctcgtg 1200

gcctctgcca tcggtttggc tattgattgg aacaagaaag gtgcccagtc tgttgcagag 1260

aaggaatcca tttccgtcta a 1281

<210> 7

<211> 1281

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgagtaaaa agtctgtcct gattacttct ttgatgctgt tttccatgtt cttcggagct 60

ggaaacctca tcttcccgcc gatgcttgga ttgtcggcag gaaccaacta tctaccagct 120

atcttaggat ttctagcaac gagtgttctg ctcccggtgc tggcgattat cgcggtggtg 180

ttgtcgggag aaaatgtcaa ggacatggct tctcgtggcg gtaagatctt tggcctggtg 240

tttcctattg ctgcctattt gtccatcggt gcgttttacg cgctgccgag gactggggcg 300

gtgagctatt cgacggcggt tggcgtcgat aattaccttt attcgggctt gtttaacttt 360

gtgttttttg cggtggcact ggcgttgtcg tggaatccga atggcattgc agacaagttg 420

ggtaagtggc tcacgccagc gttgctcacg ttgattgtgg tgctggtggt gttgtcggta 480

gccaagttgg atggcacgcc aggtgagcca agtagtgcgt atgcgcagca gcctgcgggg 540

gcgggtttgc ttgagggcta catgacgatg gatgcgattg ctgcgttggc gtttggcatc 600

gtggtgattt ctgcgttcaa gtaccaaaag gttaacaagg tccgcacggc aactgtcgtg 660

tcggcgttca ttgccggaat tttgttggcg ctggtttatc ttggtttggg ctcaatcggt 720

caagtagtaa acggtgagtt cgctgatggc accgcaattt tgaactacgc tgcactgtcc 780

acgatgggtc aggctggtcg catcatgttc gtggccattt tgatccttgc atgtatgacc 840

accgcagttg gtctgatcag tgcgacgtct gagtttttca attcgctgct gccaggtgtc 900

aagtaccacg tctgggccac tgttttcgcg ctgatttcct ttggcgttgc cacgatggga 960

ttggatacgg tgttggccgt tgcggctcca gtgattagtt tcatttaccc atcggccatc 1020

accttggtgt tcttgtcgct catcgagccc ctgctgttcc gtctcaagtg gacctaccta 1080

ttcggcattt ggactgcagt tgtgtgggcg ctgttcatgt ctatccctgc gctgaatcca 1140

ttcatcgaat gggcgccgct gcacagcatg tctttgggtt gggttgtccc agttctcgtg 1200

gcctctgcca tcggtttggc tattgattgg aacaagaaag gtgcccagtc tgttgcagag 1260

aaggaatcca tttccgtcta a 1281

<210> 8

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

attcgagctc ggtacccggg gatcctggaa ccaatggcgg tgga 44

<210> 9

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttgccggata gttttgttgc tagtttgcac acctcaacta 40

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tagttgaggt gtgcaaacta gcaacaaaac tatccggcaa 40

<210> 11

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cttgcatgcc tgcaggtcga ctctagaaaa tcagttgtca tttagcagcc tt 52

<210> 12

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tccaccgcca ttggttccag gatccccggg taccgagctc gaat 44

<210> 13

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aaggctgcta aatgacaact gattttctag agtcgacctg caggcatgca ag 52

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctagttgagg tgtgcaaact g 21

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ctcgtatgtt gtgtggaatt gtg 23

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgccctgagt gcttgcggca 20

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgggatcag tccttcacta gat 23

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

catcgccatt ttgcccacaa attgt 25

<210> 19

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggatcctcta gacatgtggt tgctgctggt gtttctga 38

<210> 20

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cggcggttgg cgtcgataat acgctttatt cgggcttgt 39

<210> 21

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

acaagcccga ataaagcgta ttatcgacgc caaccgccg 39

<210> 22

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acaagcccga ataaagcgaa ttatcgacgc caaccgccg 39

<210> 23

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cggcggttgg cgtcgataat tcgctttatt cgggcttgt 39

<210> 24

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

acaagcccga ataaagataa ttatcgacgc caaccgccg 39

<210> 25

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cggcggttgg cgtcgataat tatctttatt cgggcttgt 39

<210> 26

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gcttgcatgc ctgcagaaat gccgaatagg taggtccact t 41

<210> 27

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

acctattcgg catttctgca ggcatgcaag cttggcact 39

<210> 28

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gcagcaacca catgtctaga ggatccccgg gtaccgagct 40

<210> 29

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gacggcggtt ggcgtcgata atac 24

<210> 30

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cattgatgca catgagtacg attt 24

<210> 31

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tgcaaagact cctcgcaatt a 21

<210> 32

<211> 101

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

aatagcctag ttgaggtgtg caaactggca acaaaactat ccggcaattg tgtgatgatt 60

gtagtgtgca aaaaacgcaa gagattcatt caagcctgga g 101

Claims (10)

1. A recombinant microorganism, characterized in that, compared with the original strain, the promoter of the brnFE gene in the recombinant microorganism is mutated into the DNA molecule shown in SEQ ID NO.1, and/or the internal transport protein coded by the brnQ gene in the recombinant microorganism is mutated to form a mutant, wherein the mutant contains the mutation that the amino acid sequence of the internal transport protein coded by the wild type gene brnQ of the original strain is taken as a reference sequence, and the 112 th alanine is replaced by threonine, serine or tyrosine.

2. The recombinant microorganism according to claim 1, wherein the mutant has an amino acid sequence as set forth in any of SEQ ID nos. 2 to 4.

3. The recombinant microorganism according to claim 1 or 2, wherein the starting strain is a corynebacterium or brevibacterium bacterium;

preferably, the Corynebacterium genus bacterium is Corynebacterium glutamicum (Corynebacterium glutamicum) or Corynebacterium beijing (Corynebacterium pekinense); the brevibacterium is brevibacterium flavum (brevibacterium flavum);

more preferably, the starting strain is a bacterium of the genus Corynebacterium which is capable of accumulating valine.

4. A protein mutant encoded by the brnQ gene, wherein the specific amino acid sequence is as defined in any one of claims 1 to 3.

5. Nucleic acid encoding a mutant protein according to claim 4, preferably having the nucleotide sequence shown in any one of SEQ ID No.5 to 7.

6. A DNA molecule, which is a promoter mutant of brnFE gene and has a nucleotide sequence shown as SEQ ID NO. 1.

7. A biological material comprising a nucleic acid according to claim 5 and/or a DNA molecule according to claim 6, said biological material being an expression cassette, a vector or a host cell.

8. Use of a recombinant microorganism of any one of claims 1 to 3 or a protein mutant of claim 4 or a nucleic acid of claim 5 or a DNA molecule of claim 6 or a biological material of claim 7 for increasing the production of a branched-chain amino acid, valine, leucine or isoleucine, preferably valine, in a microorganism.

9. The method for constructing a recombinant microorganism according to any one of claims 1 to 3, comprising: mutating the promoter of brnFE gene in the original strain to DNA molecule shown as SEQ ID NO.1, and/or mutating the brnQ gene in the original strain to gene encoding the protein mutant as claimed in claim 4.

10. A method for producing valine, comprising the steps of inoculating the recombinant microorganism according to any one of claims 1 to 3 to a seed culture medium for seed culture, and then transferring the seed culture to a fermentation medium for fermentation culture;

preferably, the seed culture medium comprises the following components: 10-20g/L of soybean meal extract, 15-25g/L of glucose, 6-8g/L of ammonium sulfate, 0.4-0.6g/L of magnesium sulfate, 0.9-1.1g/L of monopotassium phosphate, 0.9-1.1g/L of dipotassium phosphate, 1.8-2.2g/L of urea and the balance of water, wherein the pH value is 7.2-7.5;

the fermentation medium comprises the following components: 10-20g/L of soybean meal extract, 65-75g/L of glucose, 15-25g/L of ammonium sulfate, 0.4-0.6g/L of magnesium sulfate, 0.9-1.1g/L of monopotassium phosphate, 0.9-1.1g/L of dipotassium phosphate, 1.8-2.2g/L of urea, 35-45g/L of calcium carbonate, 14-16mg/L of VB3 and V _H 45-55 mug/L, VB1 & HCl 90-110 mug/L and the balance of water, and the pH value is 7.2-7.5.

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