CN114277003A

CN114277003A - Glutamine synthase mutant and application thereof

Info

Publication number: CN114277003A
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: 2022-04-05
Anticipated expiration: 2041-12-14
Also published as: CN114277003B

Abstract

The invention relates to the technical field of recombinant microorganisms, in particular to a glutamine synthase mutant and application thereof. The invention enhances the heterologous glutamine synthase by an error-prone PCR method and promotes the production of glutamine or glutamine derivatives. Specifically, glutamine synthase mutants are obtained by codon optimization of glutamine synthase encoding genes from different sources and then construction of mutant libraries by error-prone PCR. Fermentation experiments prove that the strain containing the glutamine synthase mutant provided by the invention is more beneficial to the production of glutamine; the output of 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-carbamoylbutyric acid. Glutamine is a coded amino acid in protein synthesis, can promote protein synthesis and inhibit protein decomposition, can be used for treating gastric and duodenal ulcer, and has important effect in the pharmaceutical industry.

At present, the most common production method of glutamine is fermentation method, mainly using Corynebacterium glutamicum (Corynebacterium glutamicum) as production bacteria to produce glutamine by fermentation. Corynebacterium glutamicum is a heterotrophic aerobic strain, a gram-positive strain, and has the characteristics of rapid growth, non-pathogenic property, and weak degradation capability on its own metabolites. The fermentation method has the advantages of wide raw material source, low production cost, controllable product quality, single product and the like. The glutamine synthase of the corynebacterium glutamicum can generate adenylylation phenomenon, so that the enzyme activity is reduced rapidly, and the substrate glutamic acid cannot be fully utilized. Therefore, the search for high-activity glutaminase has important significance for promoting the conversion of glutamic acid into glutamine.

Disclosure of Invention

The object of the present invention is to enhance the production of glutamine or glutamine derivatives by an error-prone PCR method to enhance heterologous glutamine synthases.

The technical scheme provided by the invention is as follows: carrying out codon optimization on a glutamine synthase coding gene from lactobacillus acidophilus, constructing a mutant library by an error-prone PCR method, screening the obtained plasmid, transforming the plasmid into Corynebacterium glutamicum MHZ-0513-3, comparing the glutamine yield by shake flask fermentation, selecting a plasmid pXMJ19-L.a-glnA2, and sequencing results show that the amino acid sequence mutation sites are F106L and Y305F respectively, and constructing an expression plasmid pXMJ19-L.a-glnA0 from the glutamine synthase coding gene from lactobacillus acidophilus reported in patent CN201711031742.0, transforming the expression plasmid into Corynebacterium glutamicum MHZ-0513-3, and comparing to find that a glutamine synthase mutant is more beneficial to the production of glutamine.

Carrying out codon optimization on a gene coding for glutamine synthase from corynebacterium crenatum, then constructing a mutant library by an error-prone PCR method, screening the obtained plasmid and transforming the plasmid into corynebacterium glutamicum MHZ-0513-3, comparing the glutamine yield by shake flask fermentation, selecting a plasmid pXMJ19-C.c-glnA5, and sequencing results show that the amino acid sequence mutation sites are F211L, Q257T and F379S, and simultaneously introducing the glutamine synthase from corynebacterium crenatum reported in patent CN201610574794.1 into the corynebacterium glutamicum MHZ-0513-3, so that the glutamine synthase mutant is more beneficial to the production of glutamine through comparison.

The encoding gene of glutamine synthase from bacillus subtilis is subjected to codon optimization, then a mutant library is constructed by an error-prone PCR method, and the screened plasmid is introduced into Corynebacterium glutamicum MHZ-0513-3, which is more beneficial to the production of glutamine compared with the glutamine synthase mutant containing point mutations L299P and E378G.

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

Alternatively, the glutamine synthase mutant provided by the invention contains point mutations F211L, Q257T and F379S.

Alternatively, the glutamine synthase mutant provided by the invention contains 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 invention provides a coding gene of a glutamine synthase mutant, the nucleotide sequence of the coding 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 above-described encoding gene, wherein the biological material is an expression cassette, a vector or a host cell.

In a fourth aspect, the present invention provides a recombinant microorganism expressing the above glutamine synthase mutant or containing the above coding gene in the recombinant microorganism.

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

The present invention also claims the use of the above mentioned mutants or the above mentioned coding genes or the above mentioned biological material or the above mentioned recombinant microorganisms for increasing glutamine production as understood by the person skilled in the art.

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

The invention has the beneficial effects that:

(1) the invention uses error-prone PCR to strengthen heterologous glutamine synthase to obtain glutamine synthase mutant containing multiple point mutations.

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

(3) The yield of glutamine of the corynebacterium glutamicum containing the glutamine synthase point mutations F106L and Y305F is improved to 33.5g/L, and the acid production is improved by 12.4%;

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

the corynebacterium glutamicum containing the glutamine synthase point mutations L299P and E378G has the advantages that the glutamine yield is improved to 32.7g/L, and the acid production is improved by 10.4%.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

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

TABLE 1 primer sequences

The starting strain MHZ-0513-3 used in the examples was disclosed in CN106701649A, which was classified and named as Corynebacterium glutamicum (Corynebacterium glutamicum), and was deposited in the general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 30/11/2016, with the addresses: the microbial research institute of China academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, and the preservation number is CGMCC No. 13405.

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.

Example 1 Lactobacillus acidophilus-derived glutamine synthase mutant library

To ensure that Lactobacillus acidophilus-derived glutamine synthase was normally expressed in Corynebacterium glutamicum, glutamine synthase was codon-optimized, followed by gene synthesis in Kinzhi to obtain plasmid pUC-L.a-glnA, and then a glutamine synthase mutant library was constructed using an error-prone PCR method.

The error-prone PCR system adopted by the invention is as follows: add 5. mu.L of 10 × error-prone PCR buffer (100mmol/L Tris-HCl pH 8.3, 500mmol/L KCl, 1% Triton, 20 mmol/LMgCl) per 50. mu.L system₂5. mu.L of a 10 XdNTP mixture (1mmol/L dGTP, 1mmol/L dATP, 5mmol/L dCTP, 5mmol/L dTTP); 50pmol each of the primers L.a-glnA-F/L.a-glnA-R, 10ng of DNA template (pUC-L.a-glnA), 5. mu.L of 5mmol/L Mn²⁺15U/. mu.L Taq DNA polymerase 2.5. mu.L, 5. mu.L Mg 25mmol/L²⁺Sterilized ultrapure water was added to a total volume of 50. mu.L.

The error-prone PCR procedure is 95 ℃ for 5 min; 1min at 94 ℃, 1min at 55 ℃, 1min at 72 ℃ for 30s, and 30 cycles; 10min at 72 ℃. And (3) carrying out the next round of error-prone PCR by taking the recovered product of the PCR gel obtained for the first time as a template, and repeating the steps for 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), then digested with XmaI, while pXMJ19 was digested with XmaI and dephosphorylated with FastAP, the fragment was ligated to the vector with T4DNA ligase (TransGen Biotech), Trans1T1 competent cells (TransGen Biotech) were transformed, kanamycin resistant clones were picked, XmaI digestion was identified to give positive clones with the fragment inserted in pXMJ19, and the inserted fragment was further identified to be correct by sequencing with P1/P2 primer (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.the plasmid insert L.a-glnA was codon optimized but not mutated.

Example 2 MHZ-0513-3/pXMJ19-L.a-glnA mutant Strain construction and Glutamine production Performance

And (3) electrifying the plasmid pXMJ19-L.a-glnAX to MHZ-0513-3 to obtain a strain MHZ-0513-3/pXMJ19-L.a-glnAX, and then performing shake flask fermentation to detect the yield of the glutamine. Through multiple experiments, a strain MHZ-0513-3/pXMJ19-L.a-glnA2 with high glutamine yield is selected, the sequencing result of glutamine synthase coding genes on expression plasmids is shown in a sequence 1(SEQ ID No.1), the amino acid sequence is shown in a sequence 2(SEQ ID No.2), and sequencing has 2 amino acids with point mutation, namely F106L and Y305F.

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

The strains MHZ-0513-3/pXMJ19-L.a-glnA, MHZ-0513-3/pXMJ19-L.a-glnA2 and MHZ-0513-3/pXMJ19-L.a-glnA0 are subjected to shake flask fermentation to detect the glutamine content.

The method for verifying the glutamine yield by fermentation comprises the following steps: inoculating strain frozen in-80 deg.C glycerin pipe into the slant culture medium for activation, culturing at 33 deg.C for 24 hr to grow thallus Porphyrae, selecting thallus Porphyrae from the fresh activated slant, inoculating into the seed culture medium, culturing at 33 deg.C and 100rpm under shaking for 5 hr to logarithmic growth middle and late stage to obtain seed solution, inoculating 10% of strain in the culture medium, and culturing at room temperature and temperatureThe seed solution was inoculated into a 500ml shake flask containing 20ml of fermentation medium and cultured at 33 ℃ for 48 hours with shaking at 150 rpm. The results are shown in Table 3 (OD)₅₆₂The turbidity of the culture broth at 562nm and the amount of cells, Gln (g/L) the amount of accumulated L-glutamine).

The formula of the culture medium is as follows:

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

the seed culture medium is as follows: 50g/L glucose, 5g/L urea and KH₂PO₄ 2.0g/L，MgSO₄·7H₂O1.0 g/L, corn steep liquor 30g/L and pH 7.0;

the fermentation medium is as follows: glucose 90g/L (NH4)₂SO₄ 40g/L，KH₂PO₄ 2.0g/L，MgSO₄·7H₂O1.0 g/L, corn steep liquor 10g/L, CaCO₃ 50g/L，pH 7.0。

TABLE 2 detection of Glutamine content of mutant strains

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

Example 3 construction of a Corynebacterium crenatum-derived glutamine synthase mutant library

To ensure that a Corynebacterium crenatum-derived glutamine synthase was normally expressed in Corynebacterium glutamicum, the glutamine synthase was codon-optimized, followed by gene synthesis in Kinzhi to obtain a plasmid pUC-C.c-glnA, and then a glutamine synthase mutant library was constructed using an error-prone PCR method. The method was as above, and the primers used were C.c-glnA-F/C.c-glnA-R.

Construction of C.c-glnA expression plasmid: the construction method was as above, and the resulting plasmid was designated pXMJ19-C.c-glnAX, X1, 2, 3 … n, and the control plasmid was pXMJ19-C.c-glnA, i.e.the plasmid insert C.c-glnA was codon optimized but not mutated.

Example 4 MHZ-0513-3/pXMJ19-C.c-glnA mutant Strain construction and Glutamine production Performance

And (3) electrifying the plasmid pXMJ19-C.c-glnAX to MHZ-0513-3 to obtain a strain MHZ-0513-3/pXMJ19-C.c-glnAX, and then performing shake flask fermentation to detect the yield of the glutamine. Through multiple experiments, a strain MHZ-0513-3/pXMJ19-C.c-glnA5 with high glutamine yield is selected, the sequencing result of glutamine synthase coding genes on expression plasmids is shown in sequence 3(SEQ ID No.3), the amino acid sequence is shown in sequence 4(SEQ ID No.4), and point mutations F211L, Q257T and F379S are found through sequencing. Meanwhile, glutamine synthase from corynebacterium crenatum reported in patent CN201610574794.1 is introduced into corynebacterium glutamicum MHZ-0513-3, and the construction method is the same as the above, so that mutant MHZ-0513-3-C.c-glnA0 is obtained.

The strains MHZ-0513-3/pXMJ19-C.c-glnA, MHZ-0513-3/pXMJ19-C.c-glnA5 and MHZ-0513-3-C.c-glnA0 are subjected to shake flask fermentation, and the glutamine content is detected.

TABLE 3 detection of Glutamine content of mutant strains

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

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

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

Construction of B.s-glnA expression plasmid: the construction method is the same as above, and the obtained plasmid is named as pXMJ19-B.s-glnAX, wherein, the X is 1, 2, 3 … n, and pXMJ19-B.s-glnA is a control plasmid, namely B.s-glnA which is optimized by codons and is not mutated.

Example 6 MHZ-0513-3/pXMJ19-B.s-glnA mutant Strain construction and Glutamine production Performance

And (3) electrifying the plasmid pXMJ19-B.s-glnAX to MHZ-0513-3 to obtain a strain MHZ-0513-3/pXMJ19-B.s-glnAX, and then performing shake flask fermentation to detect the yield of the glutamine. Through multiple experiments, a strain MHZ-0513-3/pXMJ19-B.s-glnA11 with high glutamine yield is selected, the sequencing result of glutamine synthase coding genes on expression plasmids is shown in sequence 5(SEQ ID No.5), the amino acid sequence is shown in sequence 6(SEQ ID No.6), and point mutation L299P and E378G are discovered through sequencing.

The strains MHZ-0513-3/pXMJ19-B.s-glnA and MHZ-0513-3-B.s-glnA11 are subjected to shake flask fermentation, and the glutamine content is detected.

TABLE 4 detection of Glutamine content of mutant strains

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

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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Thr Ser Ala Thr Phe Met Pro Lys Pro Leu Ala Gly Asp Asn Gly Ser

260 265 270

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. A glutamine synthase mutant characterized in that said glutamine synthase mutant contains point mutations F106L and Y305F.

2. The glutamine synthase mutant according to claim 1, characterized in that said glutamine synthase mutant contains point mutations F211L, Q257T and F379S.

3. The glutamine synthase mutant according to claim 1, characterized in that said glutamine synthase mutant contains point mutations L299P and E378G.

4. The glutamine synthase mutant according to any one of claims 1 to 3, characterized in that the amino acid sequence of the glutamine synthase mutant is shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.

5. The glutamine synthase mutant coding gene according to any one of claims 1 to 4, characterized in that the nucleotide sequence of the coding gene is as shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5.

6. Biological material containing the coding gene according to claim 5, characterized in that the biological material is an expression cassette, a vector or a host cell.

7. A recombinant microorganism expressing the glutamine synthase mutant according to any one of claims 1 to 4 or containing the coding gene according to claim 5.

8. The recombinant microorganism according to claim 7, wherein the recombinant microorganism is Corynebacterium glutamicum or Escherichia coli.

9. Use of a mutant according to any one of claims 1 to 4 or a gene encoding according to claim 5 or a biological material according to claim 6 or a recombinant microorganism according to any one of claims 7 to 8 for increasing the production of glutamine or a derivative thereof.

10. A method for increasing glutamine production, comprising activating the recombinant microorganism according to any one of claims 7 to 8, inoculating the activated microorganism to a fermentation medium, and performing fermentation production.