Recombinant microorganism for producing valine and its construction method and application

Abstract

The present application relates to the field of microbial technology, and particularly relates to a recombinant microorganism for producing valine, a construction method and application thereof. Compared with a starting strain, in the recombinant microorganism, the promoter of the gene brnFE is mutated into a DNA molecule as shown in SEQ ID NO. 1, and / or the endoplasmic reticulum protein encoded by the gene brnQ in the recombinant microorganism is mutated to form a mutant, the mutant contains the amino acid sequence of the endoplasmic reticulum protein encoded by the wild-type gene brnQ of the starting strain as a reference sequence, and the alanine at the 112th position is replaced by threonine or serine or tyrosine. The recombinant microorganism of the present application is an L-valine high-yield strain, can effectively accumulate L-valine, improve the yield of L-valine, lay a foundation for the industrial production of L-valine, and has a wide industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a recombinant microorganism for producing L-valine, a construction method and application thereof. BACKGROUND

[0002] L-valine, also known as L-alpha-amino isovaleric acid, has a molecular formula of C5H 11 NO2 and a relative molecular mass of 117.15. L-valine is a white crystal or crystalline powder, odorless, bitter, and has a solubility of 88.5 g / L at 25℃ and 96.2 g / L at 50℃ in water, and is insoluble in cold ethanol, diethyl ether, and acetone. L-valine has an isoelectric point of 5.96 and a melting point of 315℃.

[0003] L-valine is one of the eight essential amino acids for human body and one of the three branched-chain amino acids (including valine, leucine, and isoleucine). Due to its special structure and function, L-valine plays an important role in human life metabolism. L-valine can be widely used in the pharmaceutical industry, food industry, and feed industry. In the pharmaceutical industry, L-valine can be used as a main component of amino acid infusion and comprehensive amino acid preparation, and can be used to treat liver failure and central nervous system dysfunction. In the food industry, L-valine can be used as a food additive, nutritional supplement, and flavoring agent. L-valine can also be used as an amino acid functional beverage and an athlete beverage, which has the effects of muscle formation, liver function strengthening, and muscle fatigue reduction. In the feed industry, L-valine has an important role in promoting the secretion of milk from animal mammary tissue.

[0004] Currently, there are three methods for producing L-valine: extraction, chemical synthesis, and microbial fermentation. The extraction and chemical synthesis methods are difficult to realize industrial production due to limited raw material sources, high production cost, and environmental pollution. The microbial fermentation method for producing L-valine 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 L-valine. However, the fermentation performance of the current L-valine strain is still poor, and the conversion rate of L-valine is still low, which cannot meet the needs of large-scale industrial production. SUMMARY

[0005] The present application aims to overcome the defects of the prior art and provide a mutant capable of producing high yield of valine, a corynebacterium, and a construction method and application thereof.

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

[0007] A recombinant microorganism, wherein, compared with a starting strain, a promoter of a gene brnFE is mutated into a DNA molecule as shown in SEQ ID NO. 1, and / or, an endoplasmic reticulum protein encoded by a gene brnQ in the recombinant microorganism is mutated to form a mutant, which contains, with reference to an amino acid sequence of the endoplasmic reticulum protein encoded by a wild-type gene brnQ of the starting strain, a mutation that an alanine at position 112 is replaced by threonine or serine or tyrosine.

[0008] The present application is characterized in that: 1. a point mutation is made to the gene brnQ, so that the amino acid at position 112 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. Thus, the pathway of the valine endoplasmic reticulum system is weakened, which helps to increase the yield of valine and avoid the reuse of valine by the strain; and / or 2. a specific point mutation is introduced into the promoter region of the gene brnFE of the branched-chain amino acid efflux protein, so as to improve the valine efflux efficiency, so as to finally obtain a genetically engineered strain for high-yield L-valine.

[0009] In the present application, the mutant has an amino acid sequence as shown in any one of SEQ ID NO. 2-4.

[0010] The starting strain is a corynebacterium or a brevibacterium.

[0011] Preferably, the corynebacterium is Corynebacterium glutamicum or Corynebacterium pekinense, and the brevibacterium is Breviabacterium flavum.

[0012] More preferably, the starting strain is a corynebacterium capable of accumulating valine.

[0013] The present application also provides a protein mutant, which is an endoplasmic reticulum protein mutant encoded by the gene brnQ, and the specific amino acid sequence is as described above.

[0014] Those skilled in the art should understand that, the tagged protein or the fusion protein formed by fusing the protein with other proteins at the N-terminus or C-terminus of the above-mentioned protein mutant sequence is also within the protection scope of the present application without changing the activity of the above-mentioned mutant protein itself.

[0015] The present application further provides a nucleic acid encoding the above-mentioned protein mutant, preferably, having a nucleotide sequence as shown in any one of SEQ ID NO. 5-7.

[0016] Based on the amino acid sequence of the protein mutant provided above, a person skilled in the art can obtain the sequence of the nucleic acid encoding it. Based on the degeneracy of codons, there are more than one nucleic acid sequences encoding the amino acid sequence above, and all nucleic acids capable of encoding the protein mutant above are within the protection scope of the present application.

[0017] The present application also provides a DNA molecule, which is a promoter mutant of the gene brnFE, and has the nucleotide sequence shown in SEQ ID NO. 1.

[0018] The present application also provides a biological material containing the nucleic acid and / or DNA molecule described above, which is an expression cassette, a vector or a host cell.

[0019] The expression cassette is a recombinant nucleic acid molecule obtained by connecting elements for driving the transcription and expression of the nucleic acid and / or DNA molecule upstream or downstream thereof.

[0020] The vector can be an expression vector or a cloning vector, including but not limited to a plasmid vector, a bacteriophage vector, a transposon, etc.

[0021] The host cell includes but is not limited to a microbial cell.

[0022] The present application also provides the use of the recombinant microorganism or the protein mutant or the nucleic acid or the DNA molecule or the biological material described above in improving the production of branched chain amino acids by microorganisms, the branched chain amino acids being valine, leucine or isoleucine, preferably valine.

[0023] The present application also provides a method for constructing a recombinant microorganism, which comprises: mutating the promoter of the gene brnFE in the starting strain to the DNA molecule shown in SEQ ID NO. 1, and / or mutating the gene brnQ in the starting strain to a gene encoding the protein mutant described above.

[0024] The present application also provides a method for producing valine, which comprises the steps of inoculating the recombinant microorganism described above into a seed culture medium for seed culture, and then transferring the seed culture into a fermentation culture medium for fermentation culture.

[0025] Preferably, the seed culture medium comprises the following components: soybean meal extract 10-20 g / L, glucose 15-25 g / L, ammonium sulfate 6-8 g / L, magnesium sulfate 0.4-0.6 g / L, potassium dihydrogen phosphate 0.9-1.1 g / L, dipotassium hydrogen phosphate 0.9-1.1 g / L, urea 1.8-2.2 g / L, the balance being water, pH 7.2-7.5.

[0026] The fermentation medium comprises the following components: soybean meal extract 10-20 g / L, glucose 65-75 g / L, ammonium sulfate 15-25 g / L, magnesium sulfate 0.4-0.6 g / L, potassium dihydrogen phosphate 0.9-1.1 g / L, dipotassium hydrogen phosphate 0.9-1.1 g / L, urea 1.8-2.2 g / L, calcium carbonate 35-45 g / L, VB3 14-16 mg / L, V H 45-55 μg / L, VB1·HCl 90-110 μg / L, the rest being water, pH 7.2-7.5.

[0027] More preferably, the seed culture medium comprises the following components: soybean meal extract 15 g / L, glucose 20 g / L, ammonium sulfate 7 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 2 g / L, the rest being water, pH 7.2.

[0028] The fermentation medium comprises the following components: soybean meal extract 15 g / L, glucose 60 g / L, ammonium sulfate 20 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 2 g / L, calcium carbonate 40 g / L, VB3 15 mg / L, V H 50 μg / L, VB1·HCl 100 μg / L, the rest being water, pH 7.2.

[0029] In the present application, the seed culture is cultured at 30°C with 220 rpm / min oscillation for 10-12 h. The fermentation culture is cultured at 30°C with 220 rpm / min oscillation for 48 h.

[0030] The present application has the following advantages:

[0031] The present application provides a construction method and application of the Corynebacterium glutamicum with high yield of valine. The gene brnQA112T, A112S and A112Y point mutation gene fragment and / or the mutant promoter of the gene brnFE are prepared, linked with a carrier to obtain a site mutation recombination carrier, and transformed into the Corynebacterium glutamicum to obtain the target site mutation Corynebacterium glutamicum. Experiments show that the Corynebacterium glutamicum is an L-valine high-yield strain, can effectively accumulate L-valine, and improve the yield of L-valine, which lays a foundation for the industrial production of L-valine and has a wide industrial application prospect. When the two mutation combinations are used, the above effects are more optimal.

[0032] In addition, these mutations can be used in other host bacteria such as Corynebacterium glutamicum and Escherichia coli, and can also be applied to the production of branched chain amino acids such as leucine and isoleucine. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Since the plasmid pK18mobsacB-brnQ(M1) / (M2) / (M3) is only different at the mutation point, the schematic diagram of pK18mobsacB-brnQ(M) is given to introduce the series of plasmids.

[0034] Figure 1 The schematic diagram of the recombinant plasmid pK18mobsacB-PbrnFE(M)-brnFE is shown in Figure 2.

[0035] Figure 2 The schematic diagram of the recombinant plasmid pK18mobsacB-brnQ(M) is shown in Figure 3. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present application, but not to limit the scope of the present application. The present application discloses a corynebacterium with high valine yield, and a construction method and application thereof. Those skilled in the art can refer to the content herein to make appropriate improvements to the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The present application has been described by preferred embodiments, and those skilled in the art can obviously make changes or appropriate changes and combinations to the methods described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0037] In order to further understand the present application, the present application will be described in detail in conjunction with specific embodiments. Unless otherwise specified, the reagents involved in the embodiments of the present application are all commercially available products, which can be purchased through commercial channels. Among them, the starting strain MHZ-1012-3 in the embodiments of the present application is corynebacterium glutamicum, the formula of the ordinary liquid brain heart infusion medium is 3.7% brain heart infusion powder solution, and the formula of the ordinary solid brain heart infusion medium is 3.7% brain heart infusion powder solution and 1.8% agar powder. If the specific technology or condition is not specified in the embodiments, it is carried out according to the technology or condition described in the literature in the art, or according to the product instruction. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased through a regular channel.

[0038] The primer names and sequences SEQ ID NO. 8-31 involved in the following examples are shown in Table 1.

[0039] Table 1 primer sequences

[0040]

[0041]

[0042] The strains involved in the following examples are as follows:

[0043] The starting strain MHZ-1012-3 is a valine-producing strain, which is obtained after mutating the first alanine (A) in the isopropyl malate synthase gene leuA of the starting strain MHZ-1012-2 to glycine (G). For specific construction methods, see Chinese Patent CN110982772A. MHZ-1012-2 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 30, 2016, at the address of No. 1, Beichen West Road, Haidian District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number of CGMCC No. 13406. Classification and naming: Corynebacterium glutamicum.

[0044] The Corynebacterium glutamicum MHZ-1012-2 (CGMCC No. 13406) mentioned in the present application has been disclosed in Chinese Patent Publication No. CN106520655A.

[0045] Construction of plasmid pK18mobsacB-PbrnFE(M)-brnFE and construction of recombinant strain MHZ-1012-4 of Example 1

[0046] The original brnFE promoter sequence is shown as SEQ ID NO. 32. The brnFE mutant promoter sequence is shown as SEQ ID NO. 1.

[0047] The specific construction process is as follows:

[0048] Using Phusion ultra-fidelity polymerase (New England BioLabs), the genome of Corynebacterium glutamicum MHZ-1012-3 as a template, PV370-UP-1F / PV371-DOWN-1R as primers, recombinant fragment UP-1 was prepared, PV372-UP-2F / PV373-DOWN-2R as primers, recombinant fragment DOWN-1 was prepared, plasmid pk18-mob-sacB as a template, PV374-UP-3F / PV375-DOWN-3R as primers, the obtained fragment pk18-1 was purified by agarose gel recovery kit (Tiangen), and then the reaction was carried out according to the Gibson assembly kit configuration system. The reaction system is as follows Table 2:

[0049] Table 2 Gibson assembly reaction system-1

[0050] Components UP-1 DOWN-1 pk18-1 CE Buffer CE Exnase Sterile water Volume / μL 1 1 2 4 2 9

[0051] After the system was configured, 10 μL of the transformed Trans1T1 competent cells (TransGen Biotech) were added and incubated at 37°C for 30 min. Kanamycin-resistant colonies were picked and identified by sequencing with P82 / P85 primers (Invitrogen). The positive colonies were further identified by EcoRI / SalI enzyme digestion. Finally, the plasmid was sent to GENEWIZ for sequencing. The plasmid obtained by sequencing was named pK18mobsacB-PbrnFE(M)-brnFE. The schematic diagram of the plasmid pK18mobsacB-PbrnFE(M)-brnFE is shown in Figure 1 .

[0052] The pK18mobsacB-PbrnFE(M)-brnFE was transformed into Corynebacterium glutamicum MHZ-1012-3, and the recombinants were selected on a selection medium containing 15 mg / L of kanamycin. The culture temperature was 30°C, and the culture was inverted. The screened transformants were cultured overnight in a general liquid brain heart infusion medium at 30°C with shaking at 220 rpm. During the culture, 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 dilutions were spread on a general solid brain heart infusion medium containing 10% sucrose and incubated at 30°C for 48 h. The screened strains were further phenotypically verified. The KanS recombinants were selected and verified by PV376-ID-F / PV-TEST-brnFE-R to verify the point mutation recombinants. By exploring the annealing temperature, the recombinants containing point mutations were obtained. The positive recombinants obtained were amplified and sequenced by PV-TEST-brnFE-F / PV-TEST-brnFE-R, and the obtained mutant strain was verified and named MHZ-1012-4.

[0053] Example 2 Fermentation of L-valine by the genetically engineered L-valine-producing strain MHZ-1012-4

[0054] 1. Culture medium

[0055] Seed medium: soybean meal extract 15 g / L, glucose 20 g / L, ammonium sulfate 7 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 2 g / L, and the balance was water, pH 7.2.

[0056] Fermentation medium: soybean meal extract 15 g / L, glucose 60 g / L, ammonium sulfate 20 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 2 g / L, calcium carbonate 40 g / L, VB3 15 mg / L, V H 50 μg / L, VB1·HCl 100 μg / L, the rest is water, pH 7.2.

[0057] 2. MHZ-1012-4 shake flask fermentation to produce L-valine

[0058] (1) Seed culture: 1 ring of MHZ-1012-3 and MHZ-1012-4 slant seed was inoculated into a 500 mL flask containing 50 mL of seed culture medium, and was cultured at 30°C and 220 r / min for 10-12 h;

[0059] (2) Fermentation culture: 5 mL of seed liquid was inoculated into a 500 mL flask containing 50 mL of fermentation medium, and was cultured at 30°C and 220 r / min for 48 h.

[0060] (3) 1 mL of fermentation broth was centrifuged (12000 rpm, 2 min), and the supernatant was collected. The L-valine in the fermentation broth of the engineering bacteria MHZ-1012-4 and the starting bacteria MHZ-1012-3 (control bacteria) was detected by HPLC, and the concentration was as shown in Table 3.

[0061] Table 3 L-valine concentration in fermentation broth

[0062] Strain L-val (g / L) Acid production improvement rate % Conversion rate g / g MHZ-1012-3 7.6 -- 0.124 MHZ-1012-4 8.9 17.1％ 0.139

[0063] The results show that the accumulation amount of L-valine of the starting strain MHZ-1012-3 is only 7.6 g / L, while the L-valine yield of the engineering bacteria MHZ-1012-4 described in the application is 8.9 g / L, which is increased by 1.3 g / L compared with the yield of the starting strain, and is increased by 17.1%, and the conversion rate of valine reaches 0.139 g / g (0.139 g of valine is produced per gram of glucose); it can be seen that the promoter mutation of brnFE enhances the export capacity of valine, so that the cell can more efficiently export the produced valine to the outside of the cell, thereby increasing the detectable product L-valine.

[0064] Example 3 Construction of plasmid pK18mobsacB-brnQ(M) and construction of recombinant strain MHZ-1012-5-1 / 2 / 3

[0065] The nucleic acid sequence of the CDS region of the mutated brnQ is shown as SEQ ID NO. 5-7, and the amino acid sequence of the CDS region of the mutated brnQ is shown as SEQ ID NO. 2-4.

[0066] The mutation A112T was introduced into the brnQ gene, and the specific construction process was as follows:

[0067] The recombinant fragment UP-2 was prepared by using Phusion ultra-fidelity polymerase (New England BioLabs) with the genome of C. glutamicum MHZ-1012-3 as a template and PV377-UP-1F / PV378-DOWN-1R as primers, the recombinant fragment DOWN-2 was prepared by using PV379-UP-2F / PV380-DOWN-2R as primers, and the fragment pk18-2 was prepared by using plasmid pk18-mob-sacB as a template and PV381-UP-3F / PV382-DOWN-3R as primers, and then the fragment pk18-2 was purified by an agarose gel recovery kit (Tiangen), and then a Gibson assembly kit was configured to perform a reaction, and the reaction system was as shown in Table 4:

[0068] Table 4 Gibson assembly reaction system-2

[0069] Components UP-2 DOWN-2 pk18-2 CE Buffer CE Exnase Sterile water Volume / μL 1 1 2 4 2 9

[0070] After the system was configured, 10 μL of Trans1T1 competent cells (TransGen Biotech) were taken, kanamycin-resistant clones were picked, and the inserted fragment was identified to be correct by sequencing (Invitrogen) with P82 / P85 primers, and further XbaI / NheI enzyme digestion identified that a positive clone of the fragment inserted into pK18mobsacB was obtained, and finally the plasmid was sent to Jinweizhi Sequencing Company for sequencing, and the obtained sequencing-corrected plasmid was named as pK18mobsacB-brnQ(M1). pK18mobsacB-brnQ(M1) was transferred into C. glutamicum MHZ-1012-3, and the exchange recombinants were selected on a selection medium containing 15 mg / L of kanamycin. The culture temperature was 30°C, and the culture was inverted. The screened transformants were cultured overnight in a general liquid brain heart infusion medium, and the culture temperature was 30°C, and the culture was shaken at 220 rpm. During the 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 Serial dilution to 10 -4), dilute liquid coating on the ordinary solid medium containing 10% sucrose brain heart infusion medium, 30°C static culture for 48h. The selected strains were further phenotypic verification, selection of KanS recombinants PV383-ID-F / PV-TEST-brnQ-R verification point mutation recombinants, by exploring the annealing temperature, obtain recombinants containing point mutations, the positive recombinants obtained by PV-TEST-brnQ-F / PV-TEST-brnQ-R amplification sequencing, verification obtained for the purpose of mutant strains, and named MHZ-1012-5-1.

[0071] Example 3.1: the same method as described in example 3, the construction of the introduction of mutation A112S and A112Y in brnQ gene, the difference is only in the mutant mutation brnQ(A112S) used primer for replacing 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 mutation brnQ(A112S) used primer for replacing PV378-DOWN-1R and PV379-UP-2F in example 3 with PV378-DOWN-1R-2 and PV379-UP-2F-2, respectively, to construct the vector pK18mobsacB-brnQ(M3) finally constructed engineering strains were named MHZ-1012-5-2 and MHZ-1012-5-3, respectively.

[0072] The schematic diagram of plasmid pK18mobsacB-brnQ(M)(pK18mobsacB-brnQ(M1), pK18mobsacB-brnQ(M2), pK18mobsacB-brnQ(M3)) is shown in Figure 2 .

[0073] Example 4 L-valine gene engineering bacteria MHZ-1012-5-1, MHZ-1012-5-2 and MHZ-1012-5-3 fermentation production of L-valine

[0074] 1、Culture medium

[0075] Seed medium: soybean meal extract 15g / L, glucose 20g / L, ammonium sulfate 7g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 1g / L, dipotassium hydrogen phosphate 1g / L, urea 2g / L, the rest is water, pH 7.2.

[0076] Fermentation medium: soybean meal extract 15 g / L, glucose 60 g / L, ammonium sulfate 20 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 2 g / L, calcium carbonate 40 g / L, VB3 15 mg / L, V H 50 μg / L, VB1-HCl 100 μg / L, the rest being water, pH 7.2.

[0077] 2. MHZ-1012-5-1 / 2 / 3 shake flask fermentation for producing L-valine

[0078] (1) Seed culture: 1 ring of MHZ-1012-3, MHZ-1012-5-1 / 2 / 3 slant seed was inoculated into a 500 mL flask containing 50 mL seed culture medium, and cultured at 30°C, 220 r / min for 10-12 h;

[0079] (2) Fermentation culture: 5 mL of seed liquid was inoculated into a 500 mL flask containing 50 mL fermentation medium, and cultured at 30°C, 220 r / min for 48 h.

[0080] (3) 1 mL of fermentation broth was centrifuged (12000 rpm, 2 min), and the supernatant was collected. The L-valine in the fermentation broth of the engineering bacteria MHZ-1012-5-1 / 2 / 3 and the starting bacteria MHZ-1012-3 (control bacteria) was detected by HPLC, and the concentration was shown in Table 5.

[0081] Table 5 L-valine concentration in fermentation broth

[0082] Strain L-val (g / L) Acid production improvement rate % Conversion rate 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

[0083] The results showed that the valine yield of the target engineering bacteria MHZ-1012-5-1 was 8.5 g / L, which was 0.9 g / L higher than that of the starting strain MHZ-1012-4, an increase of 11.8%, with the largest increase and the conversion rate also increased from 0.124 g / g to 0.135 g / g; the yield of strains MHZ-1012-5-2 and MHZ-1012-5-3 also increased to 8.2 g / L and 8.4 g / L after replacing Ser and Tyr, respectively, and the conversion rate also increased to different extents, as shown in Table 5. It can be seen that the mutant of brnQ reduces the internal transport and consumption of valine, so that the cell can more efficiently accumulate valine, and the detectable product L-valine is increased.

[0084] Example 5 Construction of recombinant strain MHZ-1012-6

[0085] The specific construction process is as follows:

[0086] The pK18mobsacB-brnQ (Ml) was transformed into C. glutamicum MHZ-1012-4, and the recombinants were selected on the selection medium containing 15 mg / L of kanamycin. The temperature of the culture was 30°C, and the culture was inverted. The screened transformants were cultured in the ordinary liquid brain heart infusion medium at 30°C with 220 rpm of shaking. During the culture, the transformants were recombined for the second time, and the vector sequence was removed from the genome by gene exchange. The culture was serially diluted (10 -2 to 10 -4 ), and the dilutions were spread on the ordinary solid brain heart infusion medium containing 10% sucrose and cultured at 30°C for 48 h. The screened strains were further verified for the phenotype, and the recombinants with KanS were selected to verify the point mutation recombinants using PV383-ID-F / PV-TEST-brnQ-R. The annealing temperature was explored to obtain the recombinants containing the point mutation. The obtained positive recombinants were amplified and sequenced using PV-TEST-brnQ-F / PV-TEST-brnQ-R to verify the obtained mutant strains, which were named as MHZ-1012-6.

[0087] Example 6 Fermentation of L-valine by the genetically engineered L-valine-producing strain MHZ-1012-6

[0088] 1. Culture medium

[0089] Seed culture medium: soybean extract 15 g / L, glucose 20 g / L, ammonium sulfate 7 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 2 g / L, and the balance was water, pH 7.2.

[0090] Fermentation medium: soybean extract 15 g / L, glucose 60 g / L, ammonium sulfate 20 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 2 g / L, calcium carbonate 40 g / L, VB3 15 mg / L, VB2 50 μg / L, VB1·HCl 100 μg / L, and the balance was water, pH 7.2. H 50μg / L, VB1·HCl 100μg / L, and the balance was water, pH 7.2.

[0091] 2. Fermentation of L-valine by MHZ-1012-6 in a flask

[0092] (1) Seed culture: 1 loop of the slant seed of MHZ-1012-3, MHZ-1012-4, MHZ-1012-5-1, and MHZ-1012-6 was inoculated into a 500 mL flask containing 50 mL of the seed culture medium, and the flask was cultured at 30°C with 220 r / min of shaking for 10-12 h.

[0093] (2) Fermentation culture: 5 mL seed liquid was inoculated into a 500 mL triangular flask containing 50 mL fermentation medium, and cultured at 30°C, 220 r / min for 48 h.

[0094] (3) 1 mL fermentation liquid was centrifuged (12000 rpm, 2 min), and the supernatant was collected. The concentration of L-valine in the fermentation liquid of the engineering bacteria was detected by HPLC, as shown in Table 6.

[0095] Table 6 L-valine concentration in fermentation liquid

[0096] Strain L-val (g / L) Acid production improvement rate % Conversion rate 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

[0097] The results show that the valine yield of the target engineering bacteria MHZ-1012-6 is 11.5 g / L, which is increased by 3.9 g / L compared with the starting strain MHZ-1012-3, and is increased by 51.3%. At the same time, the conversion rate is increased from 0.124 g / g before modification to 0.178 g / g, which is increased by 43.5%. Therefore, after introducing the mutation site A112T of brnQ and the point mutation of the promoter region of the gene brnFE of the outward transport protein, the inward transport of valine is successfully reduced and the outward transport efficiency is increased, so that the cell can more efficiently accumulate valine, and the detectable product L-valine is increased.

[0098] Although the present application has been described in detail with general description and specific embodiments above, 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 the present application. SEQUENCE LISTING <110> Meihua (Shanghai) Biotech Co., Ltd. <120> Recombinant microorganism for producing valine and construction method and application thereof <130> KHP211117899.9 <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 101 <212> DNA <213> Artificial Sequence <400> 1 aatagcctag ttgaggtgtg caaactagca acaaaactat ccggcaattg tgtgatgatt 60 gtagtgtgca aaaaacgcaa gagattcatt caagcctgga g 101 <210> 2 <211> 426 <212> PRT <213> 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 Phe Pro Ile Ala Ala Tyr Leu Ser Ile Gly Ala Phe Tyr Ala Leu ProLeu Ser Trp Asn Pro Asn Gly lie Ala Asp Lys Leu Gly Lys Trp Leu 130 135 140 Thr Pro Ala Leu Leu Thr Leu lie 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 Gin 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 lie Val Val lie Ser Ala Phe Lys Tyr 195 200 205 Gln Lys Val Asn Lys Val Arg Thr Ala Thr Val Val Ser Ala Phe lie 210 215 220 Ala Gly lie Leu Leu Ala Leu Val Tyr Leu Gly Leu Gly Ser lie Gly 225 230 235 240 Gln Val Val Asn Gly Glu Phe Ala Asp Gly Thr Ala lie Leu Asn Tyr 245 250 255 Ala Ala Leu Ser Thr Met Gly Gin Ala Gly Arg lie Met Phe Val Ala 260 265 270 Ile Leu lie Leu Ala Cys Met Thr Thr Ala Val Gly Leu lie 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 <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 Gin 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 Gin 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 <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 <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 agtaggcgt atgcgcagca gcctgcgggg 540 gcgggtttgc ttgagggcta catgacgatg gatgcgattg ctgcgttggc gtttggcatc 600. gtggtgattt ctgcgttcaa gtaccaaaag gttaacaagg tccgcacggc aactgtcgtg tcggcgttca ttgccggat tttgttggcg ctggtttatc ttggtttggg ctcaatcggt 720 caagtagtaa acggtgagtt cgctgatggc accgcaattt tgaactacgc tgcactgtcc acgatgggtc aggctggtcg catcatgttc gtggccattt tgatccttgc atgtatgacc 840 accgcagttg gtctgatcag tgcgacgtct gagtttttca attcgctgct gccaggtgtc 900 aagtaccacg tctgggccac tgttttcgcg ctgatttcct ttggcgttgc cacgatggga 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 <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 agtaggcgt atgcgcagca gcctgcgggg 540 gcgggtttgc ttgagggcta catgacgatg gatgcgattg ctgcgttggc gtttggcatc 600. gtggtgattt ctgcgttcaa gtaccaaaag gttaacaagg tccgcacggc aactgtcgtg tcggcgttca ttgccggat tttgttggcg ctggtttatc ttggtttggg ctcaatcggt 720 caagtagtaa acggtgagtt cgctgatggc accgcaattt tgaactacgc tgcactgtcc acgatgggtc aggctggtcg catcatgttc gtggccattt tgatccttgc atgtatgacc 840 accgcagttg gtctgatcag tgcgacgtct gagtttttca attcgctgct gccaggtgtc 900 aagtaccacg tctgggccac tgttttcgcg ctgatttcct ttggcgttgc cacgatggga 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 <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 agtaggcgt atgcgcagca gcctgcgggg 540 gcgggtttgc ttgagggcta catgacgatg gatgcgattg ctgcgttggc gtttggcatc 600. gtggtgattt ctgcgttcaa gtaccaaaag gttaacaagg tccgcacggc aactgtcgtg tcggcgttca ttgccggat tttgttggcg ctggtttatc ttggtttggg ctcaatcggt 720 caagtagtaa acggtgagtt cgctgatggc accgcaattt tgaactacgc tgcactgtcc acgatgggtc aggctggtcg catcatgttc gtggccattt tgatccttgc atgtatgacc 840 accgcagttg gtctgatcag tgcgacgtct gagtttttca attcgctgct gccaggtgtc 900 aagtaccacg tctgggccac tgttttcgcg ctgatttcct ttggcgttgc cacgatggga 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 <400> 8 attcgagctc ggtacccggg gatcctggaa ccaatggcgg tgga 44 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <400> 9 ttgccggata gttttgttgc tagtttgcac acctcaacta 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <400> 10 tagttgaggt gtgcaaacta gcaacaaaac tatccggcaa 40 <210> 11 <211> 52 <212> DNA <213> Artificial Sequence <400> 11 cttgcatgcc tgcaggtcga ctctagaaaa tcagttgtca tttagcagcc tt 52 <210> 12 <211> 44 <212> DNA <213> Artificial Sequence <400> 12 tccaccgcca ttggttccag gatccccggg taccgagctc gaat 44 <210> 13 <211> 52 <212> DNA <213> Artificial Sequence <400> 13 aaggctgcta aatgacaact gattttctag agtcgacctg caggcatgca ag 52 <210> 14 <211> twenty one <212> DNA <213> Artificial Sequence <400> 14 ctagttgagg tgtgcaaact g 21 <210> 15 <211> twenty three <212> DNA <213> Artificial Sequence <400> 15 ctcgtatgtt gtgtggaatt gtg 23 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 cgccctgagt gcttgcggca 20 <210> 17 <211> twenty three <212> DNA <213> Artificial Sequence <400> 17 atgggatcag tccttcacta gat 23 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <400> 18 catcgccatt ttgcccacaa attgt 25 <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <400> 19 ggatcctcta gacatgtggt tgctgctggt gtttctga 38 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <400> 20 cggcggttgg cgtcgataat acgctttat cgggcttgt 39 <210> twenty one <211> 39 <212> DNA <213> Artificial Sequence <400> twenty one acaagcccga ataaagcgta ttatcgacgc caaccgccg 39 <210> twenty two <211> 39 <212> DNA <213> Artificial Sequence <400> twenty two acaagcccga ataaagcgaa ttatcgacgc caaccgccg 39 <210> twenty three <211> 39 <212> DNA <213> Artificial Sequence <400> twenty three cggcggttgg cgtcgataat tcgctttatt cgggcttgt 39 <210> twenty four <211> 39 <212> DNA <213> Artificial Sequence <400> twenty four acaagcccga ataaagataa ttatcgacgc caaccgccg 39 <210> 25 <211> 39 <212> DNA <213> Artificial Sequence <400> 25 cggcggttgg cgtcgataat tatctttatt cgggcttgt 39 <210> 26 <211> 41 <212> DNA <213> Artificial Sequence <400> 26 gcttgcatgc ctgcagaaat gccgaatagg taggtccact t 41 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <400> 27 acctattcgg catttctgca ggcatgcaag cttggcact 39 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <400> 28 gcagcaacca catgtctaga ggatccccgg gtaccgagct 40 <210> 29 <211> twenty four <212> DNA <213> Artificial Sequence <400> 29 gacggcggtt ggcgtcgata atac 24 <210> 30 <211> twenty four <212> DNA <213> Artificial Sequence <400> 30 cattgatgca catgagtacg attt 24 <210> 31 <211> twenty one <212> DNA <213> Artificial Sequence <400> 31 tgcaaagact cctcgcaatt a 21 <210> 32 <211> 101 <212> DNA <213> Artificial Sequence <400> 32 aatagcctag ttgaggtgtg caaactggca acaaaactat ccggcaattg tgtgatgatt 60 gtagtgtgca aaaaacgcaa gagattcatt caagcctgga g 101

Claims

1. A recombinant microorganism, characterized in that, Compared with the starting strain, the recombinant microorganism has the following characteristics: the promoter of the gene brnFE is mutated to a DNA molecule as shown in SEQ ID NO.1; and / or, the transport protein encoded by the gene brnQ in the recombinant microorganism is mutated to form a mutant. The mutant contains a mutation in which the amino acid sequence of the transport protein encoded by the wild-type gene brnQ in the starting strain is used as a reference sequence, and the alanine at position 112 is replaced by threonine, serine, or tyrosine. The amino acid sequence of the mutant is shown in any one of SEQ ID NO.2-4. The starting strain is Corynebacterium glutamicum, which is capable of accumulating valine. Corynebacterium glutamicum ).

2. The application of the recombinant microorganism of claim 1 in increasing the yield of branched-chain amino acids in microorganisms, wherein the branched-chain amino acid is valine.

3. The method for constructing recombinant microorganisms according to claim 1, characterized in that, include: The promoter of the gene brnFE in the starting strain is mutated to a DNA molecule as shown in SEQ ID NO.1, and / or the gene brnQ in the starting strain is mutated to a gene encoding an amino acid sequence as shown in any of SEQ ID NO.2-4.

4. A method for producing valine, characterized in that, The method includes the steps of inoculating the recombinant microorganism of claim 1 into a seed culture medium for seed culture, and then transferring the seed culture into a fermentation culture medium for fermentation culture.

5. The method according to claim 4, characterized in that, The seed culture medium comprises the following components: soybean meal extract 10-20 g / L, glucose 15-25 g / L, ammonium sulfate 6-8 g / L, magnesium sulfate 0.4-0.6 g / L, potassium dihydrogen phosphate 0.9-1.1 g / L, dipotassium hydrogen phosphate 0.9-1.1 g / L, urea 1.8-2.2 g / L, with the balance being water, pH 7.2-7.5; The fermentation medium comprises the following components: soybean meal extract 10-20 g / L, glucose 15-25 g / L, ammonium sulfate 6-8 g / L, magnesium sulfate 0.4-0.6 g / L, potassium dihydrogen phosphate 0.9-1.1 g / L, dipotassium hydrogen phosphate 0.9-1.1 g / L, urea 1.8-2.2 g / L, vitamin B3 14-16 μg / L, vitamin B1·HCl 90-110 μg / L, with the balance being water, and a pH of 7.2-7.5.

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