CN114277003B

CN114277003B - Glutamine synthase mutant and application thereof

Info

Publication number: CN114277003B
Application number: CN202111530525.2A
Authority: CN
Inventors: 栾明月; 吴涛; 姚嘉琪; 赵津津; 李岩
Original assignee: Langfang Meihua Bio Technology Development Co Ltd
Current assignee: Langfang Meihua Bio Technology Development Co Ltd
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2023-06-06
Anticipated expiration: 2041-12-14
Also published as: CN114277003A

Abstract

The invention relates to the technical field of recombinant microorganisms, in particular to a glutamine synthase mutant and application thereof. The invention strengthens the heterologous glutamine synthase by an error-prone PCR method and promotes the production of glutamine or glutamine derivatives. Specifically, the glutamine synthase mutants are obtained by codon optimization of glutamine synthase encoding genes of different sources and then constructing a mutant library by an error-prone PCR method. Fermentation experiments prove that the strain containing the glutamine synthase mutant provided by the invention is more beneficial to the production of glutamine; the yield of the glutamine can reach 32.7-33.5g/L, and the acid production is improved by 5.7-12.4%.

Description

Glutamine synthase mutant and application thereof

Technical Field

The invention relates to the technical field of recombinant microorganisms, in particular to a glutamine synthase mutant and application thereof.

Background

Glutamine is a non-essential amino acid. The chemical name is 2-amino-4-carbamoyl butyric acid. Glutamine is an encoded amino acid in protein synthesis, can promote protein synthesis and inhibit protein decomposition, can be used for treating gastric and duodenal ulcers, and has an important role in the pharmaceutical industry.

At present, the most commonly used production method of glutamine is a fermentation method, and corynebacterium glutamicum (Corynebacterium glutamicum) is mainly used as a production bacterium for fermenting and producing the glutamine. Corynebacterium glutamicum is heterotrophic aerobic bacteria, is a gram-positive bacterium, and has the characteristics of high growth speed, non-pathogenicity and weak degradation capability on self metabolites. The fermentation method has the advantages of wide raw material sources, low production cost, controllable product quality, single product and the like. The glutamine synthase of corynebacterium glutamicum itself may undergo adenylation, resulting in a drastic decrease in enzyme activity, resulting in insufficient utilization of substrate glutamate. Therefore, finding a highly active glutaminase, which promotes the conversion of glutamate to glutamine, is of great importance.

Disclosure of Invention

The invention aims to strengthen heterologous glutamine synthase by an error-prone PCR method and promote the production of glutamine or glutamine derivatives.

The technical scheme provided by the invention is as follows: the coding gene of the glutamine synthase from lactobacillus acidophilus is subjected to codon optimization, then a mutant library is constructed by an error-prone PCR method, the screened plasmid is transformed into corynebacterium glutamicum MHZ-0513-3, the glutamine yield is compared by shake flask fermentation, the plasmid pXMJ19-L.a-glnA2 is selected, sequencing results show that the mutation sites of the amino acid sequence are F106L and Y305F respectively, meanwhile, the coding gene of the lactobacillus acidophilus from lactobacillus acidophilus reported in patent CN201711031742.0 is constructed into an expression plasmid pXMJ19-L.a-glnA0, and the expression plasmid is transformed into corynebacterium glutamicum MHZ-0513-3, so that the glutamine synthase mutant is more favorable for glutamine production by comparison.

The coding gene of the glutamine synthase from the corynebacterium crenatum is subjected to codon optimization, then a mutant library is constructed by an error-prone PCR method, the screened plasmid is transformed into corynebacterium glutamicum MHZ-0513-3, the glutamine yield is compared by shaking flask fermentation, the plasmid pXMJ19-C.c-glnA5 is selected, the sequencing result shows that the mutation site of the amino acid sequence is F211L, Q257T, F379S, meanwhile, the glutamine synthase from the corynebacterium crenatum reported by the patent CN201610574794.1 is imported into corynebacterium glutamicum MHZ-0513-3, and the glutamine synthase mutant is more favorable for glutamine production compared and found.

The coding gene of the glutamine synthase from bacillus subtilis is subjected to codon optimization, then a mutant library is constructed by an error-prone PCR method, and plasmids obtained through screening are introduced into the corynebacterium glutamicum MHZ-0513-3, so that the production of glutamine is facilitated compared with the fact that the glutamine synthase mutant containing the point mutations L299P and E378G is found.

Based on the above technical scheme, in a first aspect, the invention provides a glutamine synthase mutant, wherein the glutamine synthase mutant contains point mutations F106L and Y305F.

Alternatively, the glutamine synthase mutants provided by the present invention contain point mutations F211L, Q257T and F379S.

Alternatively, the glutamine synthase mutants provided by the present invention contain point mutations L299P and E378G.

The amino acid sequence of the glutamine synthase mutant provided by the invention is shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.

In a second aspect, the present invention provides a gene encoding a mutant glutamine synthase, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5.

In a third aspect, the present invention provides a biological material comprising the coding gene described above, said biological material being an expression cassette, a vector or a host cell.

In a fourth aspect, the present invention provides a recombinant microorganism expressing the above-described glutamine synthase mutant or comprising the above-described encoding gene.

The recombinant microorganism provided by the invention is corynebacterium glutamicum or escherichia coli.

The use of the above mutants or the above coding genes or the above biological materials or the above recombinant microorganisms for increasing glutamine production is also claimed, as will be appreciated by the person skilled in the art.

Specifically, the invention provides a method for improving the glutamine yield, which comprises the steps of activating the recombinant microorganism and inoculating the recombinant microorganism into a fermentation medium for fermentation production.

The invention has the beneficial effects that:

(1) The invention strengthens heterologous glutamine synthase by error-prone PCR to obtain a glutamine synthase mutant containing a plurality of point mutations.

(2) The strain containing the glutamine synthase mutant provided by the invention has the capability of high-yield glutamine and derivatives thereof.

(3) Corynebacterium glutamicum containing glutamine synthase point mutations F106L and Y305F has increased glutamine yield to 33.5g/L and increased acid production by 12.4%;

corynebacterium glutamicum containing glutamine synthase point mutations F211L, Q257T and F379S, the glutamine yield is improved to 33.2g/L, and the acid production is improved by 5.7%;

the corynebacterium glutamicum containing glutamine synthase point mutations L299P and E378G improves the glutamine yield to 32.7G/L and improves the acid production by 10.4 percent.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

The primers and sequences according to the present invention are shown in Table 1.

TABLE 1 primer sequences

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The starting strain MHZ-0513-3 used in the examples was disclosed in CN106701649A, which was classified and named Corynebacterium glutamicum (Corynebacterium glutamicum), and was deposited in China general microbiological culture Collection center (China Committee) at 11 and 30 days of 2016: the collection number of the microbiological institute of China is CGMCC No.13405, and the collection number of the microbiological institute of China is China, national institute of sciences, no.1, no.3, north Chen West Lu, the Korean region of Beijing city.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.

EXAMPLE 1 Lactobacillus acidophilus-derived Glutamine synthase mutant library

To ensure the normal expression of the Lactobacillus acidophilus-derived glutamine synthase in Corynebacterium glutamicum, the glutamine synthase was codon optimized, followed by gene synthesis at Jin Weizhi to obtain plasmid pUC-L.a-glnA, which was then used to construct a library of glutamine synthase mutants by error-prone PCR.

The error-prone PCR system adopted by the invention is as follows: mu.L of 10 Xerror-prone PCR buffer (100 mmol/L Tris-HCl pH 8.3, 500mmol/L KCl,1% Triton,20 mmol/LMgCl) was added per 50. Mu.L of system ₂ mu.L of 10 XdNTP mixture (1 mmol/L dGTP,1mmol/L dATP,5mmol/L dCTP,5mmol/L dTTP); primer L.a-glnA-F/L.a-glnA-R50 pmol each, DNA template (pUC-L.a-glnA) 10ng, 5. Mu.L of 5mmol/L Mn ²⁺ 15U/. Mu.L Taq DNA polymerase 2.5. Mu.L, 5. Mu.L Mg 25mmol/L ²⁺ The sterilized ultrapure water was added to a total volume of 50. Mu.L.

The error-prone PCR procedure was 95℃for 5min;94℃1min,55℃1min,72℃1min 30s,30 cycles; and at 72℃for 10min. And (3) carrying out the next round of error-prone PCR by taking the first obtained PCR glue recovery product as a template, and repeating the 5 rounds of error-prone PCR to finally obtain the L.a-glnA mutant fragment.

Construction of L.a-glnA expression plasmid: the resulting L.a-glnA mutant fragment was purified by agarose gel recovery kit (Tiangen) and then digested with XmaI while pXMJ19 was digested with XmaI and dephosphorylated with FastAP, the fragment was ligated with vector using T4DNA ligase (TransGen Biotech), trans1T1 competent cells (TransGen Biotech) were transformed, the Canada resistant clone was picked up, xmaI restriction identified to give a positive clone of fragment insertion into pXMJ19, and further the correct inserted fragment was identified by sequencing with P1/P2 primers (Invitrogen). The resulting plasmid was designated pXMJ19-L.a-glnAX, X=1, 2,3 … n. The control plasmid was pXMJ19-L.a-glnA, i.e., plasmid insert L.a-glnA was codon optimized, but not mutated.

EXAMPLE 2 construction of the MHZ-0513-3/pXMJ19-L.a-glnA mutant Strain and Glutamine production Properties

The plasmid pXMJ19-L.a-glnAX was electrotransferred to MHZ-0513-3 to obtain strain MHZ-0513-3/pXMJ19-L.a-glnAX, followed by shaking fermentation to examine the glutamine yield. Through multiple experiments, a strain MHZ-0513-3/pXMJ19-L.a-glnA2 with higher glutamine yield is selected from the strain, the sequencing result of a glutamine synthase coding gene on an expression plasmid is shown as a sequence 1 (SEQ ID No. 1), the amino acid sequence is shown as a sequence 2 (SEQ ID No. 2), and 2 amino acids are subjected to point mutation in the sequencing, namely F106L and Y305F.

Meanwhile, the glutamine synthase from lactobacillus acidophilus reported in patent CN201711031742.0 is introduced into corynebacterium glutamicum MHZ-0513-3, and the mutant strain MHZ-0513-3/pXMJ19-L.a-glnA0 is obtained by the same construction method.

The strain MHZ-0513-3/pXMJ19-L.a-glnA, MHZ-0513-3/pXMJ19-L.a-glnA2 and MHZ-0513-3/pXMJ19-L.a-glnA0 were subjected to shaking fermentation, and the glutamine content was detected.

Method for verifying glutamine yield by fermentation: inoculating the strain frozen in-80 ℃ glycerol pipe into the slant culture medium for activation, culturing at 33 ℃ for 24 hours, growing a lawn, picking the lawn from the freshly activated slant, inoculating into the seed culture medium, culturing at 33 ℃ for 5 hours under 100rpm in a shaking way until the middle and later period of logarithmic growth, obtaining seed liquid, inoculating the seed liquid into a 500ml shaking bottle filled with 20ml fermentation culture medium with 10% inoculum size, and culturing at 33 ℃ for 48 hours under 150rpm in a shaking way. The results are shown in Table 3 (OD ₅₆₂ The turbidity of the culture solution at 562nm and expressed the cell amount, and Gln (g/L) expressed the amount of accumulated L-glutamine.

The formula of the culture medium is as follows:

slant culture medium: 37g/L brain heart infusion, 1.8% agar, and sterilizing at 121deg.C under 0.1MPa for 20min;

the seed culture medium is as follows: glucose 50g/L, urea 5g/L, KH ₂ PO ₄ 2.0g/L，MgSO ₄ ·7H ₂ O1.0 g/L, corn steep liquor 30g/L, pH 7.0;

the fermentation medium is as follows: glucose 90g/L, (NH 4) ₂ SO ₄ 40g/L，KH ₂ PO ₄ 2.0g/L，MgSO ₄ ·7H ₂ O1.0 g/L, corn steep liquor 10g/L, caCO ₃ 50g/L，pH 7.0。

Table 2 glutamine content assay of mutant strains

The shake flask fermentation result shows that MHZ-0513-3/pXMJ19-L.a-glnA and MHZ-0513-3/pXMJ19-L.a-glnA0 glutamine production are basically consistent, namely, codon optimized L.a-glnA and lactobacillus acidophilus-derived glutamine synthase reported in patent CN201711031742.0 are basically consistent in the glutamine production of the strain, but MHZ-0513-3/pXMJ19-L.a-glnA2 glutamine production is improved to 33.5g/L, acid production is improved by 12.4%, namely, the L.a-glnA2 point mutations F106L and Y305F are beneficial to glutamine production, and the glutamine synthase is superior to that reported in patent CN 201711031742.0.

EXAMPLE 3 construction of a library of Corynebacterium crenatum-derived glutamine synthase mutants

To ensure the normal expression of the glutamine synthase from Corynebacterium crenatum in Corynebacterium glutamicum, the glutamine synthase was codon optimized, followed by gene synthesis at Jin Weizhi to obtain plasmid pUC-C.c-glnA, and then an error-prone PCR method was used to construct a glutamine synthase mutant library. The primers used were C.c-glnA-F/C.c-glnA-R in the same manner as above.

Construction of C.c-glnA expression plasmid: the construction method was the same as above, and the resulting plasmid was designated pXMJ19-C.c-glnAX, X=1, 2,3 … n, and the control plasmid was pXMJ19-C.c-glnA, i.e., plasmid insert C.c-glnA was codon-optimized, but not mutated.

EXAMPLE 4 construction of the MHZ-0513-3/pXMJ19-C.c-glnA mutant Strain and Glutamine production Properties

The plasmid pXMJ19-C.c-glnAX was electrotransferred to MHZ-0513-3 to obtain strain MHZ-0513-3/pXMJ19-C.c-glnAX, followed by shaking fermentation to examine the glutamine yield. Through multiple experiments, a strain MHZ-0513-3/pXMJ19-C.c-glnA5 with higher glutamine yield is selected from the strain, the sequencing result of a glutamine synthase coding gene on an expression plasmid is shown as a sequence 3 (SEQ ID No. 3), the amino acid sequence is shown as a sequence 4 (SEQ ID No. 4), and the sequencing discovers point mutations F211L, Q257T and F379S. Meanwhile, the glutamine synthase from corynebacterium crenatum reported in patent CN201610574794.1 is introduced into corynebacterium glutamicum MHZ-0513-3, and the mutant strain MHZ-0513-3-C.c-glnA0 is obtained by the same construction method.

The strain MHZ-0513-3/pXMJ19-C.c-glnA, MHZ-0513-3/pXMJ19-C.c-glnA5 and MHZ-0513-3-C.c-glnA0 were subjected to shaking fermentation, and the glutamine content was detected.

TABLE 3 detection of glutamine content in mutant strains

The shake flask fermentation result shows that the MHZ-0513-3/pXMJ19-C.c-glnA and MHZ-0513-3/pXMJ19-C.c-glnA0 glutamine yield are basically consistent, namely, the codon optimized C.c-glnA and the corynebacterium crenatum-derived glutamine synthase reported in patent CN201610574794.1 are basically consistent, but the MHZ-0513-3/pXMJ19-C.c-glnA5 glutamine yield is improved to 33.2g/L, the acid production is improved by 5.7%, namely, the C.c-glnA5 midpoint mutation F211L, Q257T, F379S is beneficial to glutamine production, and is superior to the glutamine synthase reported in patent CN 201610574794.1.

EXAMPLE 5 construction of Bacillus subtilis-derived Glutamine synthase mutant library

To ensure the proper expression of the Bacillus subtilis-derived glutamine synthase in Corynebacterium glutamicum, the glutamine synthase was codon optimized, followed by gene synthesis at Jin Weizhi to obtain plasmid pUC-B.s-glnA, and then an error-prone PCR method was used to construct a glutamine synthase mutant library. The primers used were B.s-glnA-F/B.s-glnA-R in the same manner as above.

Construction of B.s-glnA expression plasmid: the resulting plasmid was designated pXMJ19-B.s-glnAX, X=1, 2,3 … n, as a control plasmid, i.e., B.s-glnA, which was codon-optimized but not mutated, as described above.

EXAMPLE 6 construction of the MHZ-0513-3/pXMJ19-B.s-glnA mutant Strain and Glutamine production Properties

The plasmid pXMJ19-B.s-glnAX was electrotransferred to MHZ-0513-3 to obtain strain MHZ-0513-3/pXMJ19-B.s-glnAX, followed by shaking fermentation to examine the glutamine yield. Through multiple experiments, a strain MHZ-0513-3/pXMJ19-B.s-glnA11 with higher glutamine yield is selected from the strain, the sequence of a glutamine synthase coding gene on an expression plasmid is shown as a sequence 5 (SEQ ID No. 5), the amino acid sequence is shown as a sequence 6 (SEQ ID No. 6), and the sequencing discovers point mutations L299P and E378G.

The strain MHZ-0513-3/pXMJ19-B.s-glnA and MHZ-0513-3-B.s-glnA11 were subjected to shaking fermentation, and the glutamine content was detected.

Table 4 glutamine content assay of mutant strains

The shake flask fermentation result shows that compared with MHZ-0513-3/pXMJ19-B.s-glnA11 and MHZ-0513-3/pXMJ19-B.s-glnA, the glutamine yield is improved to 32.7G/L, the acid production is improved by 10.4%, namely, the B.s-glnA11 midpoint mutation L299P, E378G is beneficial to the production of glutamine.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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Gly Met His Ala His Gln Ser Leu Trp Lys Asp Gly Lys Pro Leu Phe

275 280 285

His Asp Glu Ser Gly Tyr Ala Gly Leu Ser Asp Ile Ala Arg Tyr Tyr

290 295 300

Ile Gly Gly Ile Leu His His Ala Gly Ala Val Leu Ala Phe Thr Asn

305 310 315 320

Ala Thr Leu Asn Ser Tyr His Arg Leu Val Pro Gly Phe Glu Ala Pro

325 330 335

Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Ala Val Arg Ile

340 345 350

Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Ile Glu Phe Arg Ala

355 360 365

Pro Asp Pro Ser Gly Asn Pro Tyr Leu Gly Ser Ala Ala Met Met Met

370 375 380

Ala Gly Leu Asp Gly Ile Lys Asn Arg Ile Glu Pro His Ala Pro Val

385 390 395 400

Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile Pro

405 410 415

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

420 425 430

Thr Asp Phe Leu Thr Glu Ser Asp Val Phe Thr Glu Asp Leu Ile Glu

435 440 445

Ala Tyr Ile Gln Tyr Lys Tyr Asp Asn Glu Ile Ser Pro Val Arg Leu

450 455 460

Arg Pro Thr Pro Gln Glu Phe Glu Leu Tyr Phe Asp Cys

465 470 475

<210> 5

<211> 1335

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atggcaaagt acacccgcga agatatcgaa aagctggtga aggaagaaaa cgtgaagtac 60

atccgcctgc agttcaccga tatcctgggc accatcaaga acgtggaaat ccctgtgtct 120

cagctgggca aggcactgga taacaaggtg atgttcgatg gctcctccat cgaaggcttc 180

gtgcgcatcg aagaatccga tatgtacctg taccctgatc tgaacacctt cgtgatcttc 240

ccatggaccg cagaaaaggg caaggtggca cgcttcatct gcgatatcta caaccctgat 300

ggcaccccat tcgaaggcga tccacgcaac aacctgaagc gcatcctgaa ggaaatggaa 360

gatctgggct tctccgattt caacctgggc cctgaacctg aattcttcct gttcaagctg 420

gatgaaaagg gcgaaccaac cctggaactg aacgataagg gcggctactt cgatctggca 480

ccaaccgatc tgggcgaaaa ctgccgccgc gatatcgtgc tggaactgga agaaatgggc 540

ttcgaaatcg aagcatccca ccacgaagtg gcacctggtc agcacgaaat cgatttcaag 600

tacgccggcg cagtgcgctc ctgcgatgat attcagacct tcaagctggt ggtgaagacc 660

atcgcacgca agcacggcct gcacgcaacc ttcatgccaa agccactgtt cggcgtgaac 720

ggctccggca tgcactgcaa cctgtccctg ttcaagaacg gcgtgaacgc attcttcgat 780

gaaaacgcag atctgcagct gtccgaaacc gcaaagcact tcatcgccgg catcgtgaag 840

cacgcaactt ccttcaccgc agtgaccaac ccaaccgtga actcctacaa gcgcccggtg 900

cctggctacg aagcaccatg ctacgtggca tggtccgcac agaaccgctc cccactgatc 960

cgcatccctg catcccgcgg catctccacc cgcgtggaag tgcgctccgt ggatcctgca 1020

gcaaacccat acctggcact gtccgtgctg ctggcagccg gcctggatgg catcaagaac 1080

aagctggaag cacctgcacc aatcgatcgc aacatctacg tgatgtccaa gggagaacgc 1140

atggaaaacg gcatcgtgga tctgcctgca accctggcag aagcactgga agaattcaag 1200

tccaacgaag tgatggtgaa ggcactgggc gaacacctgt tcgaacactt catcgaagca 1260

aaggaaatcg aatgggatat gttccgcacc caagtgcacc catgggaacg cgaacagtac 1320

atgtctcagt actaa 1335

<210> 6

<211> 444

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Met Ala Lys Tyr Thr Arg Glu Asp Ile Glu Lys Leu Val Lys Glu Glu

1 5 10 15

Asn Val Lys Tyr Ile Arg Leu Gln Phe Thr Asp Ile Leu Gly Thr Ile

20 25 30

Lys Asn Val Glu Ile Pro Val Ser Gln Leu Gly Lys Ala Leu Asp Asn

35 40 45

Lys Val Met Phe Asp Gly Ser Ser Ile Glu Gly Phe Val Arg Ile Glu

50 55 60

Glu Ser Asp Met Tyr Leu Tyr Pro Asp Leu Asn Thr Phe Val Ile Phe

65 70 75 80

Pro Trp Thr Ala Glu Lys Gly Lys Val Ala Arg Phe Ile Cys Asp Ile

85 90 95

Tyr Asn Pro Asp Gly Thr Pro Phe Glu Gly Asp Pro Arg Asn Asn Leu

100 105 110

Lys Arg Ile Leu Lys Glu Met Glu Asp Leu Gly Phe Ser Asp Phe Asn

115 120 125

Leu Gly Pro Glu Pro Glu Phe Phe Leu Phe Lys Leu Asp Glu Lys Gly

130 135 140

Glu Pro Thr Leu Glu Leu Asn Asp Lys Gly Gly Tyr Phe Asp Leu Ala

145 150 155 160

Pro Thr Asp Leu Gly Glu Asn Cys Arg Arg Asp Ile Val Leu Glu Leu

165 170 175

Glu Glu Met Gly Phe Glu Ile Glu Ala Ser His His Glu Val Ala Pro

180 185 190

Gly Gln His Glu Ile Asp Phe Lys Tyr Ala Gly Ala Val Arg Ser Cys

195 200 205

Asp Asp Ile Gln Thr Phe Lys Leu Val Val Lys Thr Ile Ala Arg Lys

210 215 220

His Gly Leu His Ala Thr Phe Met Pro Lys Pro Leu Phe Gly Val Asn

225 230 235 240

Gly Ser Gly Met His Cys Asn Leu Ser Leu Phe Lys Asn Gly Val Asn

245 250 255

Ala Phe Phe Asp Glu Asn Ala Asp Leu Gln Leu Ser Glu Thr Ala Lys

260 265 270

His Phe Ile Ala Gly Ile Val Lys His Ala Thr Ser Phe Thr Ala Val

275 280 285

Thr Asn Pro Thr Val Asn Ser Tyr Lys Arg Pro Val Pro Gly Tyr Glu

290 295 300

Ala Pro Cys Tyr Val Ala Trp Ser Ala Gln Asn Arg Ser Pro Leu Ile

305 310 315 320

Arg Ile Pro Ala Ser Arg Gly Ile Ser Thr Arg Val Glu Val Arg Ser

325 330 335

Val Asp Pro Ala Ala Asn Pro Tyr Leu Ala Leu Ser Val Leu Leu Ala

340 345 350

Ala Gly Leu Asp Gly Ile Lys Asn Lys Leu Glu Ala Pro Ala Pro Ile

355 360 365

Asp Arg Asn Ile Tyr Val Met Ser Lys Gly Glu Arg Met Glu Asn Gly

370 375 380

Ile Val Asp Leu Pro Ala Thr Leu Ala Glu Ala Leu Glu Glu Phe Lys

385 390 395 400

Ser Asn Glu Val Met Val Lys Ala Leu Gly Glu His Leu Phe Glu His

405 410 415

Phe Ile Glu Ala Lys Glu Ile Glu Trp Asp Met Phe Arg Thr Gln Val

420 425 430

His Pro Trp Glu Arg Glu Gln Tyr Met Ser Gln Tyr

435 440

<210> 7

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tccccccggg atgtccaagc agtacaccgc ag 32

<210> 8

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tccccccggg ttaccagttc atgtagcgct 30

<210> 9

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tccccccggg gtggcattcg aaacccctga aga 33

<210> 10

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tccccccggg ttagcaatcg aagtacagtt c 31

<210> 11

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tccccccggg atggcaaagt acacccgcga aga 33

<210> 12

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tccccccggg ttagtactga gacatgtact gt 32

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tgtgagcgga taacaatttc a 21

<210> 14

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ttctgattta atctgtatca ggctga 26

Claims

1. The glutamine synthase mutant is characterized in that the amino acid sequence of the glutamine synthase mutant is shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.

2. The coding gene of the glutamine synthase mutant as defined in claim 1, wherein the nucleotide sequence of the coding gene is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5.

3. A biological material comprising the coding gene of claim 2, wherein the biological material is an expression cassette, a vector or a host cell.

4. A recombinant microorganism expressing the glutamine synthase mutant of claim 1 or comprising the encoding gene of claim 2.

5. The recombinant microorganism according to claim 4, wherein the recombinant microorganism is Corynebacterium glutamicum or Escherichia coli.

6. Use of a mutant according to claim 1 or a coding gene according to claim 2 or a biomaterial according to claim 3 or a recombinant microorganism according to any one of claims 4 to 5 for increasing the production of glutamine or a derivative thereof.

7. A method for increasing glutamine production, characterized in that the recombinant microorganism according to any one of claims 4 to 5 is inoculated into a fermentation medium after activation, and fermentation production is performed.