Novel method for producing glutamine

Abstract

Modified bacteria that produce glutamine are provided wherein a heterologous polynucleotide of glutamine export protein (rarD) and a modification that increases glutamine synthase glnA activity are included in the genome as compared to the pre-modified bacteria. In addition, methods of producing glutamine using the modified bacteria are also provided.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a method for producing glutamine by using bacterial fermentation. BACKGROUND

[0002] Glutamine is a non-essential amino acid. Its chemical name is 2-amino-4- carbamoyl butyric acid. Glutamine is a coded amino acid in protein synthesis, which can promote the synthesis of protein and inhibit the decomposition of protein, and can be used for the treatment of gastric and duodenal ulcers, and plays an important role in the pharmaceutical industry.

[0003] At present, Corynebacterium glutamicum is mainly used as a production strain for the fermentation production of glutamine. Corynebacterium glutamicum is a heterotrophic aerobic bacterium, which is gram-positive, has the characteristics of fast growth, non-pathogenicity, and weak degradation ability to its own metabolites. The fermentation performance of the strain for producing glutamine is still poor, contains by-product isoleucine, and the conversion rate of glutamine is not ideal. The demand for glutamine in industry is extremely high, and the existing strains cannot meet the demand of large-scale industrial production. Therefore, it is particularly important to use genetic engineering means to improve the yield and conversion rate of glutamine.

[0004] Currently, there are many reports on methods to improve glutamine production. The mutation of serine at position 84 of inositol 3-phosphate synthase to other amino acids helps to enhance the ability of C. glutamicum to produce glutamine (CN113201524B); the mutation of amino acid at position 184 of CEY17_05975 protein to other amino acids can improve the efficiency of the microorganism in producing glutamine (CN117624316A); the mutation of threonine at position 65 of CEY17_04535 protein to other amino acids except threonine can significantly improve the glutamine production capacity of the strain (CN117946228A); the mutation of arginine at position 2916 of CEY17_04555 to other amino acids can make the recombinant microorganism have stronger ability to produce L-glutamine (CN118126146A); the mutation of alanine at position 139 of CEY17_13360 to other amino acids except alanine can make the recombinant microorganism have stronger ability to produce L-glutamine (CN118515735A); the point mutation of DNA gyrase gyrA (Gly821Ser Asp830Asn) in Escherichia coli can reduce the DNA supercoiling structure, which is conducive to the accumulation of glutamine (Hayashi, Mikiro, and K. Tabata. "Metabolic Engineering for l-Glutamine Overproduction by Using DNA Gyrase Mutations in Escherichia coli." Applied & Environmental Microbiology 79.9 (2013)); the mutation of the 405th amino acid of glnA from tyrosine (Y) to phenylalanine (F), i.e., the mutation of TAC to TTC, can eliminate the adenylation of glutamine synthase, effectively improving the yield of glutamine (CN1614008A); the modification of glsA to reduce the activity of glutamine enzyme in cells can effectively improve the yield of glutamine (US7943364B2); the simultaneous inactivation of glnE and glnB in E. coli JM101 can increase the content of glutamine from 0 to 620 mg / L (US20080038786A1).The production of glutamine in E. coli GJLE1 increased from 122.2 mg / L to 497.4 mg / L after the introduction of a plasmid carrying the expression of E. coli-derived rarD. In addition to transporting glutamine to the outside of the cell, rarD can also transport serine, threonine, and phenylalanine (US8623619B2); the activity of glutamine synthetase is enhanced by modification, and further modification of the activity of intracellular glutamate dehydrogenase (encoded by gdh) in corynebacterium bacteria increases the production of L-glutamine (CN100457894C); the glutamine synthase encoding gene of C. glutamicum is inhibited by repressor proteins or transcriptional regulators, resulting in a decrease in transcription level and a sharp decrease in enzyme activity, which causes the substrate glutamate to be underutilized. Among them, RosR (Cg1324) is a hydrogen peroxide-sensitive MarR-type transcriptional regulator (2010, Michael Bott, a Hydrogen Peroxide-sensitive MarR-type Transcriptional Regulator of Corynebacterium glutamicum), RosR can bind to the promoter region of the glutamine synthase encoding gene glnA, inhibit glnA transcription, and inactivation of rosR promotes the accumulation of glutamine (2022, Xiangfei Li, MarR-type transcription factor RosR regulates glutamate metabolism network and promotes accumulation of L-glutamate in Corynebacterium glutamicum G01); the 405th amino acid of glutamine synthase from C. glutamicum is mutated from tyrosine to phenylalanine, which eliminates the adenylation modification effect (CN100392075C); contains the vgb and glnA genes of the glutamine synthase of the wild-type C. glutamicum 14067, which can produce high-yield glutamine at low DO levels and low costs (CN1614008A). Y405F

[0005] Currently, glutamine-producing strains have accumulated mixed acids and glutamic acid, indicating that the terminal / transport is not strong enough, which limits the high-yield glutamine of the strain. The present application mainly aims at the existing mixed acid glutamic acid, promotes the conversion of glutamic acid to glutamine, and transports the synthesized glutamine to the outside of the cell. SUMMARY

[0006] ​The present disclosure provides a modified bacteria producing glutamine, wherein the genome of the bacteria comprises a heterologous polynucleotide of glutamine efflux protein (rarD) and a modification of increased glutamine synthase (glnA) activity compared to the bacteria before the modification.

[0007] In one embodiment, the modified bacteria produces glutamine at a higher yield than the bacteria before the modification.

[0008] In one embodiment, the bacteria is a Corynebacterium bacteria, preferably Corynebacterium glutamicum.

[0009] In one embodiment, the glutamine efflux protein (rarD) is derived from Escherichia coli.

[0010] In one embodiment, the modification of increased glutamine synthase (glnA) activity is a Y405F amino acid substitution in the glnA (glnA Y405F ), preferably the glnA is derived from Corynebacterium glutamicum or Saccharomyces cerevisiae.

[0011] In one embodiment, the modified bacteria comprises more than one copy of the glutamine efflux protein (rarD) gene.

[0012] In one embodiment, the modified bacteria comprises more than one copy of the glnA Y405F gene.

[0013] In one embodiment, the modified bacteria, wherein the insertion site of the glutamine efflux protein (rarD) gene is within the acetyltransferase-encoding gene CEY17_08220 ORF.

[0014] In one embodiment, the modified bacteria, wherein the insertion site of the glnA Y405F gene is within the glutaminase-encoding gene glsA ORF.

[0015] In one embodiment, the modified bacteria, the promoter of the glutamine efflux protein (rarD) gene is the Psod promoter.

[0016] In one embodiment, the modified bacteria, the insertion site of the glnA Y405F gene is within the glutaminase-encoding gene glsA ORF.

[0017] In one embodiment, the modified bacteria, wherein the Psod promoter is from Corynebacterium glutamicum.

[0018] In another aspect, there is provided use of the modified bacteria described herein in increasing glutamine production.

[0019] In another aspect, there is provided a method of producing glutamine, comprising culturing the modified bacteria described herein in a culture medium and isolating glutamine.

[0020] In another aspect, there is provided a bioreactor comprising the modified bacteria described herein.

[0021] Advantages

[0022] The genetically engineered bacteria provided herein have a high glutamine conversion rate. Strain QS10 was obtained by introducing the E. coli MG1655-derived rarD into QS09, which promotes glutamine efflux and increases the accumulation of glutamate, the precursor of glutamine. The acid production was increased from 23.9 g / L to 24.2 g / L, and the conversion rate was increased from 26.3% to 26.6%. Strain QS11 was obtained by introducing the E. coli MG1655-derived glnA into QS10, which promotes the conversion of glutamate to glutamine. The acid production was increased from 24.2 g / L to 25.5 g / L, and the conversion rate was increased from 26.6% to 28.1%. Y405F DETAILED DESCRIPTION

[0023] The following description of the present disclosure is merely intended to illustrate various different embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0024] In order to better enable persons skilled in the art to understand the present disclosure scheme, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, not all of the embodiments.

[0025] Table 1. Instruments used in the present application

[0026] Instrument name Manufacturer Model PCR instrument Bio-Rad S1000 Electrotransformation instrument Bio-Rad Gene Pulser Xcell Constant temperature incubator Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd. SPX-250BSH-II Constant temperature shaker Taicang Experimental Equipment Factory DHZ-DA Pulsating vacuum sterilization cabinet Jiangsu Shennong Sterilization Equipment Co., Ltd. YG-0.36 SBA enzyme electrode analyzer Shandong Academy of Sciences SBA-40C Ultraviolet-visible spectrophotometer Tianmei (China) Scientific Instrument Co., Ltd. UH5300

[0027] Table 2. Reagents used in the present application

[0028] Reagent name Manufacturer Part number Proteose peptone Oxoid, UK LP0042B NaCl National Pharmaceutical Group Chemical Reagent Co., Ltd. 10019318 Yeast extract Oxoid, UK LP0021B Sorbitol Shenguo Bioengineering Co., Ltd. A610491-0500 Sucrose National Pharmaceutical Group Chemical Reagent Co., Ltd. 10021418 Brain heart infusion Oxoid, UK CM1135B Agar Beijing Aobosan Biotechnology Co., Ltd. 01-023 Glucose National Pharmaceutical Group Chemical Reagent Co., Ltd. 10010518 Urea National Pharmaceutical Group Chemical Reagent Co., Ltd. 10023218 KH2PO4 National Pharmaceutical Group Chemical Reagent Co., Ltd. 10017618 MgSO4.7H2O National Pharmaceutical Group Chemical Reagent Co., Ltd. 10013018 Corn syrup dry powder Baolingbao Biological Co., Ltd. (NH4)2SO4 National Pharmaceutical Group Chemical Reagent Co., Ltd. 10002918 CaCO3 Tianjin Damao Chemical Reagent Factory 1653

[0029] Table 3. Primer sequence information

[0030] ​

[0031]

[0032]

[0033]

[0034] In this paper, two glutamine-synthesizing bacteria (QS09, o-QS04) were used as the starting bacteria, and rarD and glnA Y405F were introduced, which improved the glutamine conversion rate and proved that the introduction of rarD and glnA Y405F in different strains had unexpected effects.

[0035] 1. Introduction of rarD and glnA in QS09 Y405F

[0036] This disclosure first constructed a glutamine-producing strain QS09, specifically using Corynebacterium glutamicum ATCC14067 as the starting bacteria to introduce ino-1 S84A , CEY17_06485 A386T , CEY17_05975 V184I , CEY17_04535 T65I , CEY17_04555 R2916C , CEY17_13360 A139T , gyrA A466V、 , ΔglsA, glnA Y405F The glutamine yield reached 19.2 g / L. To further improve the glutamine yield, rarD and glnA Y405F were introduced, obtaining strain QS12, with a 2.6% increase in conversion rate, which had unexpected effects.

[0037] 2. Introduction of rarD and glnA in o-QS04 Y405F

[0038] To further confirm that the combination of rarD and glnA Y405F copies is effective in different glutamine-synthesizing strains, another glutamine-producing strain o-QS04 was constructed, rarD and glnA Y405F copies were introduced, obtaining strain o-QS07, with a 1.3% increase in conversion rate, which had unexpected effects.

[0039] The genotypes of the strains are shown in Table 4:

[0040] Table 4. Genotypes of strains

[0041]

[0042]

[0043] Table 5. Sequence information

[0044]

[0045]

[0046]

[0047]

[0048] Example 1: Construction of QS09 starting strain 1.1 Construction of ATCC 14067→QS01 strain and performance verification

[0049] a) Plasmid construction

[0050] The upstream homology arm UP (594bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ28-UP-F / PQ29-UP-R as primers. The downstream homology arm DN (555bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ30-DN-F / PQ31-DN-R as primers. The overlap fragment (1120bp) was obtained by amplifying UP and DN using PQ28-UP-F / PQ31-DN-R as primers. The overlap fragment and pK18mobsacB were digested with XbaI and PstI at 37°C for 1 hour. The fragment was directly purified, and the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 hour. The gel was recovered. Subsequently, the vector was ligated and transformed, and colony PCR was performed using primers P82 / P85, with a length of 1.4kb. The correct transformants were inoculated in test tubes with LBK50, and plasmids were extracted and sent for sequencing.

[0051] b) Strain construction

[0052] Plasmid electrotransformation of C. glutamicum ATCC 14067, spread on LBHIS K15 plates, primary selection on LB K25S, LB K25 plates, the latter not growing phenotype correct, identified with PQ28-UP-F / P85, P82 / PQ31-DN-R, positive control plasmid, negative control ATCC 14067 genome, correct length 1.3 kb, 1.2 kb. Primary recombinants spread on LB tubes overnight, diluted 10, 100, 1000 times and spread on LB K25, LB K25S, LBS plates, secondary selection on LB K25, LB plates, the former not growing phenotype correct. Suitable annealing temperature for colony PCR identification with PQ32-id-f / PQ31-DN-R, positive control plasmid, negative control C. glutamicum ATCC 14067 genome, correct secondary recombinants identified with this annealing temperature, amplified with primers PQ33-ID-F / PQ34-ID-R and sequenced, length 1.4 kb, correct strain noted as QS01.

[0053] c) Performance verification

[0054] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance, and the method for verifying the glutamine yield in fermentation was as follows:

[0055] The strain in a glycerol tube frozen at -80°C was inoculated in BHI slant medium for activation, and after 24 hours of culture at 33°C, a bacterial lawn was grown. The bacterial lawn was picked from the freshly activated slant and inoculated in the following seed culture medium, and after being cultured at 33°C, 100 rpm to the middle or late logarithmic growth phase, the culture time was 5 hours, a seed liquid was prepared. The above seed liquid was inoculated into a 500 ml flask containing 20 ml of fermentation medium at a 10% inoculation amount, and was cultured at 33°C, 150 rpm for 48 hours. After the glucose was completely consumed, the concentration of glutamine accumulated in the medium was determined by HPLC method.

[0056] The formula of the culture medium was as follows:

[0057] LB medium: 10 g / L of proteose peptone, 10 g / L of NaCl, 5 g / L of yeast extract, 1.8% of agar, sterilized at 121°C, 0.1 MPa for 20 minutes; LB K25 was LB plus kanamycin 25 μg / mL.

[0058] LBHIS medium: 5 g / L of proteose peptone, 5 g / L of NaCl, 2.5 g / L of yeast extract, 18.5 g / L of brain heart infusion, 91 g / L of sorbitol, 1.8% of agar, sterilized at 121°C, 0.1 MPa for 20 minutes; LBHIS K15 was LBHIS plus kanamycin 15 μg / mL.

[0059] LBS medium: Tryptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, sucrose 0.1 g / L, agar 1.8%, sterilized at 121°C 0.1 MPa for 20 min; LBK25S is LBS plus kanamycin 25 μg / mL.

[0060] BHI slant medium: Brain Heart Infusion 37 g / L, agar 1.8%, sterilized at 121°C 0.1 MPa for 20 min;

[0061] Seed medium: Glucose 25 g / L, urea 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.4 g / L, corn syrup dry powder 15 g / L, pH 7.0;

[0062] Fermentation medium: Glucose 90.9 g / L, (NH4)2SO4 50 g / L, KH2PO4 2.5 g / L, corn syrup dry powder 2 g / L, CaCO3 40 g / L, pH 7.0.

[0063] Table 6. Glutamine content of C. glutamicum QS01

[0064] Strain Genotype Growth (OD562nm) gln (g / L) Conversion rate (%) Conversion rate increase (%) ATCC 14067 Wild type 62.3 0.4 0.45 - QS01 ino-1S84A 60.7 1.1 1.22 0.77

[0065] Strain QS01 was obtained by introducing ino-1 S84A amino acid mutation into C. glutamicum ATCC 14067, as shown in Table 6. The glutamine yield of the obtained strain QS01 was increased from 0.4 g / L to 1.1 g / L, and the conversion rate was increased by 0.77%.

[0066] 1.2 Construction of QS01→QS02 strain and performance verification

[0067] a) Plasmid construction

[0068] The genomic DNA of C. glutamicum ATCC 14067 was used as template and the primers PQ35-UP-F / PQ36-UP-R were used to amplify the UP (543 bp). The genomic DNA of C. glutamicum ATCC 14067 was used as template and the primers PQ37-DN-F / PQ38-DN-R were used to amplify the DN (548 bp). The UP and DN were used as template and the primers PQ35-UP-F / PQ38-DN-R were used to amplify the overlap fragment (1053 bp). The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour, the fragment was directly purified, the vector was dephosphorylated with 3 μL FastAP at 37°C for 1 hour, and then recovered by gel. Subsequently, the ligation, transformation, and colony PCR were performed using the primers P82 / P85, and the length was 1.3 kb. The correct transformants were inoculated into test tubes with LBK50, and the plasmid was sent for sequencing.

[0069] b) Strain construction

[0070] The plasmid was electroporated into C. glutamicum QS01, and then plated on LBHISK15 plates. Once the colonies were formed, they were inoculated on LBK25S and LBK25 plates. The colonies on the LBK25 plates were not grown, indicating that the correct recombinant strain was obtained. The colonies were identified using the primers PQ35-UP-F / P85, P82 / PQ38-DN-R, and the correct length was 1.1 kb and 1.2 kb. The correct recombinant strain was inoculated into test tubes with LB and incubated overnight. Then, it was diluted by 10, 100, and 1000 times and plated on LBK25, LBK25S, and LBS plates. The colonies on the LBS plates were grown, and then they were inoculated on LBK25 and LB plates. The colonies on the LB plates were grown, indicating that the correct recombinant strain was obtained. The appropriate annealing temperature was determined using the primers PQ39-id-f / PQ38-DN-R, and the correct length was 1.4 kb. The correct recombinant strain was identified using the primers PQ40-ID-F / PQ41-ID-R, and the correct strain was named QS02.

[0071] c) Performance verification

[0072] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield is shown in Example 1.1.

[0073] Table 7. Detection of glutamine content in C. glutamicum QS02

[0074]

[0075] The strain QS02 was constructed by introducing the CEY17_06485 gene into the strain QS01. A386TThe amino acid mutation is obtained as shown in Table 7, and the glutamine yield of the obtained strain QS02 is increased from 1.1 g / L to 1.7 g / L, and the conversion rate is increased by 0.65%.

[0076] 1.3 Construction of QS02→QS03 strain and performance verification

[0077] a) Plasmid construction

[0078] The upstream homology arm UP (555 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ42-UP-F / PQ43-UP-R as primers. The downstream homology arm DN (500 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ44-DN-F / PQ45-DN-R as primers. The overlap fragment (1029 bp) was obtained by amplifying UP and DN using PQ42-UP-F / PQ45-DN-R as primers. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour, and the fragment was directly purified. The vector was dephosphorylated by adding 3 μL of FastAP at 37°C for 1 hour, and the gel was recovered. Subsequently, the vector was ligated and transformed, and colony PCR was performed using primers P82 / P85, with a length of 1.3 kb. The correct transformants were inoculated into LBK50 test tubes, and the plasmid was sent for testing.

[0079] b) Strain construction

[0080] The plasmid was electroporated into Corynebacterium glutamicum QS02, and LBHISK15 plates were coated. LBK25S and LBK25 plates were inoculated at one time, and the latter was not grown. The correct phenotype was identified using PQ42-UP-F / P85, P82 / PQ45-DN-R, positive control plasmid, and negative control Corynebacterium glutamicum ATCC 14067 genome. The correct length was 1.1 kb and 1.2 kb. The recombinants were inoculated into LB test tubes overnight, and then diluted by 10, 100, and 1000 times to coat LBK25, LBK25S, and LBS plates. The second time, the recombinants were inoculated into LBK25 and LB plates, and the former was not grown. The correct phenotype was identified using PQ46-id-f / PQ45-DN-R, positive control plasmid, and negative control Corynebacterium glutamicum ATCC 14067 genome. The appropriate annealing temperature was determined by colony PCR. The correct second recombinants were amplified using primers PQ47-ID-F / PQ48-ID-R, with a length of 1.3 kb, and the correct strain was recorded as QS03.

[0081] c) Performance verification

[0082] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0083] Table 8. Glutamine content detection of C. glutamicum QS03

[0084]

[0085] Strain QS03 was constructed by introducing CEY17_05975 into strain QS02 V184I The glutamine production of strain QS03 was increased from 1.7 g / L to 2.6 g / L and the conversion rate was increased by 1.02% as shown in Table 8.

[0086] 1.4 Construction of QS03→QS04 and performance verification

[0087] a) Plasmid construction

[0088] The UP (505 bp) was amplified from the genome of C. glutamicum ATCC 14067 using primers PQ49-UP-F / PQ50-UP-R. The DN (542 bp) was amplified from the genome of C. glutamicum ATCC 14067 using primers PQ51-DN-F / PQ52-DN-R. The overlap fragment (1050 bp) was amplified using primers PQ49-UP-F / PQ52-DN-R with UP and DN as templates. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour. The fragment was directly purified, the vector was dephosphorylated with 3 μL FastAP at 37°C for 1 hour, and the gel was recovered. Then the vector was ligated and transformed, and the length of 1.3 kb was verified by colony PCR using primers P82 / P85. The correct transformants were inoculated in test tubes with LBK50, and the plasmid was sent for testing.

[0089] b) Strain construction

[0090] Plasmid electroporation of C. glutamicum QS03, spread on LBHISK15 plates, primary counter selection on LBK25S, LBK25 plates, the latter not growing phenotype correct, identified with PQ49-UP-F / P85, P82 / PQ52-DN-R, positive control plasmid, negative control ATCC 14067 genome, correct length 1.1 kb, 1.2 kb. Primary recombinants spread on LB tubes overnight, diluted 10, 100, 1000 fold and spread on LBK25, LBK25S, LBS plates, secondary counter selection on LBK25, LB plates, the former not growing phenotype correct. Annealing temperature for colony PCR identified with PQ55-id-f / PQ52-DN-R, positive control plasmid, negative control C. glutamicum ATCC 14067 genome, band length 689 bp, used for colony PCR with this annealing temperature. Correct secondary recombinants sequenced after amplification with primers PQ53-ID-F / PQ54-ID-R, length 1.3 kb, correct strain noted as QS04.

[0091] c) Performance verification

[0092] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance, and the method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0093] Table 9. Detection of glutamine content of C. glutamicum QS04

[0094]

[0095] Strain QS04 was obtained by introducing CEY17_04535 T65I amino acid mutation into strain QS03, as shown in Table 9, the glutamine yield of the obtained strain QS04 was increased from 2.6 g / L to 3 g / L, and the conversion rate was increased by 0.41%.

[0096] 1.5 Construction of QS04→QS05 strain and performance verification

[0097] a) Plasmid construction

[0098] The genomic DNA of C. glutamicum ATCC 14067 was used as template and the primers PQ56-UP-F / PQ57-UP-R were used to amplify the UP (522 bp). The genomic DNA of C. glutamicum ATCC 14067 was used as template and the primers PQ58-DN-F / PQ59-DN-R were used to amplify the DN (523 bp). The UP and DN were used as template and the primers PQ56-UP-F / PQ59-DN-R were used to amplify the overlap fragment (1020 bp). The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour, the fragment was directly purified, the vector was dephosphorylated with 3 μL FastAP at 37°C for 1 hour, and then recovered by gel. Subsequently, the ligation, transformation, and colony PCR were performed using the primers P82 / P85, and the length was 1.3 kb. The correct transformants were inoculated into test tubes with LBK50, and the plasmid was sent for sequencing.

[0099] b) Strain construction

[0100] The plasmid was electroporated into C. glutamicum QS04, and then plated on LBHISK15 plates. Once the colonies were formed, they were inoculated on LBK25S and LBK25 plates. The colonies on the LBK25 plates were not grown, and the colonies on the LBK25S plates were grown. The correct colonies were identified using the primers PQ56-UP-F / P85, P82 / PQ59-DN-R, and the correct length was 1.1 kb and 1.2 kb. The correct colonies were inoculated into test tubes with LB and incubated overnight. The colonies were then inoculated on LBK25, LBK25S, and LBS plates at dilutions of 10, 100, and 1000 times. The colonies on the LBS plates were grown, and the colonies were then inoculated on LBK25 and LB plates. The colonies on the LBK25 plates were not grown, and the colonies on the LB plates were grown. The appropriate annealing temperature was determined using the primers PQ62-id-f / PQ59-DN-R, and the correct colonies were identified using the annealing temperature. The correct colonies were identified using the primers PQ60-ID-F / PQ61-ID-R, and the length was 1.2 kb. The correct strain was named QS05.

[0101] c) Performance verification

[0102] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0103] Table 10. Detection of glutamine content in C. glutamicum QS05

[0104]

[0105] The strain QS05 was constructed by introducing the CEY17_04555 gene into the strain QS04. R2916CThe amino acid mutation is obtained as shown in Table 10, and the glutamine yield of the obtained strain QS05 is increased from 3 g / L to 3.8 g / L, and the conversion rate is increased by 0.88%.

[0106] 1.6 Construction of QS05→QS06 strain and performance verification

[0107] a) Plasmid construction

[0108] The upstream homology arm UP (539 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ63-UP-F / PQ64-UP-R as primers. The downstream homology arm DN (542 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ65-DN-F / PQ66-DN-R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 h, and the vector was recovered by gel recovery. The digested vector, UP, and DN were assembled seamlessly at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.3 kb. The correct transformant was inoculated into a test tube containing LBK50, and the plasmid was sent for testing.

[0109] b) Strain construction

[0110] The plasmid was electroporated into Corynebacterium glutamicum QS05, and then plated on LBHISK15 plates. Once the LBK25S and LBK25 plates were inoculated, the latter was not grown, and the phenotype was correct. Primers PQ63-UP-F / P85, P82 / PQ66-DN-R were used for identification, and the positive control plasmid and the negative control Corynebacterium glutamicum ATCC 14067 genome had correct lengths of 1.1 kb and 1.2 kb, respectively. The primary recombinants were inoculated into an LB test tube overnight, and then diluted by 10, 100, and 1000 times to inoculate LBK25, LBK25S, and LBS plates. The LBS plate was grown, and then the secondary recombinants were inoculated into LBK25 and LB plates. The former was not grown, and the latter was grown, and the phenotype was correct. The appropriate annealing temperature was determined using primers PQ67-id-f / PQ66-DN-R, and the positive control plasmid and the negative control Corynebacterium glutamicum ATCC 14067 genome were used for identification. The correct secondary recombinants were amplified using primers PQ68-ID-F / PQ69-ID-R, and then sequenced. The length was 1.3 kb, and the correct strain was recorded as QS06.

[0111] c) Performance verification

[0112] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0113] Table 11. Detection of glutamine content of Corynebacterium glutamicum QS06

[0114]

[0115] Strain QS06 was obtained by introducing the CEY17_13360 A139T amino acid mutation into strain QS05. As shown in Table 11, the glutamine yield of the obtained strain QS06 was increased from 3.8 g / L to 5.2 g / L, and the conversion rate was increased by 1.54%.

[0116] 1.7 Strain construction and performance verification of QS06→QS07

[0117] a) Plasmid construction

[0118] The upstream homology arm UP (477 bp) was amplified from the genome of Corynebacterium glutamicum ATCC 14067 using PQ70-UP-F / PQ71-UP-R as primers. The downstream homology arm DN (550 bp) was amplified from the genome of Corynebacterium glutamicum ATCC 14067 using PQ72-DN-F / PQ73-DN-R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 h, and the vector was recovered by gel recovery. The digested vector, UP, and DN were assembled seamlessly at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.3 kb. The correct transformant was inoculated into a test tube of LBK50, and the plasmid was sent for testing.

[0119] b) Strain construction

[0120] The plasmid was electroporated into Corynebacterium glutamicum QS06, and then plated on LBHISK15 plates. Once the LBK25S and LBK25 plates were inoculated, the latter was not grown, and the phenotype was correct. Primers PQ70-UP-F / P85, P82 / PQ73-DN-R were used for identification, and the correct length was 1.1 kb and 1.2 kb. The primary recombinants were inoculated into a test tube of LB overnight, and then diluted by 10, 100, and 1000 times to inoculate LBK25, LBK25S, and LBS plates. The secondary recombinants that grew on the LBS plates were inoculated into LBK25 and LB plates, and the former did not grow, and the latter grew, which was the correct phenotype. The appropriate annealing temperature was determined by using primers PQ74-id-f / PQ73-DN-R, and the correct secondary recombinants were identified by colony PCR using the annealing temperature. The length was 1.2 kb, and the correct strain was QS07.

[0121] c) Performance verification

[0122] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0123] Table 12. Glutamine content detection of C. glutamicum QS07

[0124]

[0125] Strain QS07 was obtained by introducing gyrA A466V amino acid mutation into strain QS06, as shown in Table 12. The glutamine yield of the obtained strain QS07 was increased from 5.2 g / L to 8 g / L, and the conversion rate was increased by 3.08%.

[0126] 1.8 Strain construction and performance verification of QS07→QS08

[0127] a) Plasmid construction

[0128] The upstream homology arm UP (530 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ01-UP-F / PQ02-UP-R as primers. The downstream homology arm DN (550 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ03-DN-F / PQ04-DN-R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 h. The vector was recovered by gel recovery. The digested vector, UP, and DN were assembled at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.4 kb. The correct transformant was inoculated into a test tube of LBK50, and the plasmid was sent for sequencing.

[0129] b) Strain construction

[0130] The plasmid was electroporated into C. glutamicum QS07, and then plated on LBHISK15 plates. Once the LBK25S and LBK25 plates were inoculated, the latter was not grown, and the phenotype was correct. The correct length was 1.3 kb and 1.2 kb, which was identified by PQ01-UP-F / P85, P82 / PQ04-DN-R, positive control plasmid, and negative control ATCC14067 genome. The primary recombinants were inoculated into a test tube of LB overnight, and then diluted by 10, 100, and 1000 times to plate on LBK25, LBK25S, and LBS plates. The secondary recombinants were inoculated on LBK25 and LB plates, and the former was not grown, and the phenotype was correct. The correct length was 1.3 kb, which was identified by PQ05-ID-F / PQ06-ID-R, and then sequenced by amplification with the primers. The correct strain was recorded as QS08.

[0131] c) Performance verification

[0132] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine production in fermentation is shown in Example 1.1.

[0133] Table 13. Glutamine content detection of C. glutamicum QS08

[0134]

[0135] Strain QS08 was obtained by inactivating glsA based on strain QS07. As shown in Table 13, the glutamine yield of the obtained strain QS08 was increased from 8 g / L to 12.4 g / L, and the conversion rate was increased by 4.8%.

[0136] 1.9 Strain construction and performance verification of QS08→QS09

[0137] a) Plasmid construction

[0138] The upstream homology arm UP (526 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ07-UP-F / PQ08-UP-R as primers. The downstream homology arm DN (502 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ09-DN-F / PQ10-DN-R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and 3 μL of FastAP was added for dephosphorylation at 37°C for 1 h. The vector was recovered by gel recovery. The digested vector, UP, and DN were assembled at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.3 kb. The correct transformant was placed in a test tube with LBK50, and the plasmid was sent for testing.

[0139] b) Strain construction

[0140] Plasmid electroporation of C. glutamicum QS08, spread on LBHISK15 plate, primary counter selection on LBK25S, LBK25 plate, the latter not growing phenotype correct, identified with PQ07-UP-F / P85, P82 / PQ10-DN-R, positive control plasmid, negative control ATCC 14067 genome, correct length 1.1 kb, 1.2 kb. Primary recombinants spread on LB tube overnight, dilution 10, 100, 1000 times spread on LBK25, LBK25S, LBS plate, secondary counter selection on LBK25, LB plate, the former not growing phenotype correct. Use PQ13-id-f / PQ10-DN-R to find the suitable annealing temperature, positive control plasmid, negative control C. glutamicum ATCC 14067 genome, band length 551 bp, use this annealing temperature to identify the colony PCR, correct secondary recombinants amplified with primer PQ11-ID-F / PQ12-ID-R and sequenced, length 1.1 kb, correct strain recorded as QS09.

[0141] c) Performance verification

[0142] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance, and the method for verifying the glutamine yield in fermentation was as shown in Example 1.1.

[0143] Table 14. Detection of glutamine content of C. glutamicum QS09

[0144]

[0145] Strain QS09 was obtained by introducing glnA Y405F amino acid mutation into strain QS08, as shown in Table 14, the glutamine yield of the obtained strain QS09 was increased from 12.4 g / L to 19.2 g / L, and the conversion rate was increased by 7.5%.

[0146] Example 2: Construction of starting strain QS09→QS10 and performance verification

[0147] 2.1 Plasmid construction

[0148] The upstream homology arm (563 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using primers PQ265-UP-1f / PQ266-UP-1r. The promoter Psod (215 bp) was amplified from the Corynebacterium glutamicum ATCC 13032 (type strain, available from Bomeiweibio) genome using primers PQ267-Psod-2f / PQ279-Psod-2r. The rarD (913 bp) was amplified from the Escherichia coli MG1655 (type strain, available from Hubei Aipti Bioengineering Effective Company) genome using primers PQ280-rarD-3f / PQ281-rarD-3r. The downstream homology arm (536 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using primers PQ282-DN-4f / PQ272-DN-4r. Subsequently, the 1-2 fragment (751 bp) was amplified by fusion using the upstream homology arm UP and the promoter Psod as templates and primers PQ265-UP-1f / PQ279-Psod-2r. The 3-4 fragment (1427 bp) was amplified by fusion using the rarD and the downstream homology arm DN as templates and primers PQ280-rarD-3f / PQ272-DN-4r. Finally, the 1-4 fragment (2157 bp) was amplified using the 1-2 and 3-4 fragments as templates and primers PQ265-UP-1f / PQ272-DN-4r. The 1-4 fragment and the pK18mobsacB were digested with EcoRI and NheI at 37°C for 1 hour. The fragment was directly purified from the product, and the vector was dephosphorylated by adding 3 μL of FastAP at 37°C for 1 hour. The gel was then recovered. Subsequently, the enzyme was connected, transformed, and verified by colony PCR using primers P82 / P85, with a length of 2.3 kb. The correct transformant was placed in a test tube LBK50, and the plasmid was sent for measurement.

[0149] 2.2 Strain construction

[0150] The plasmid was electroporated into Corynebacterium glutamicum QS09, which was coated on LBHISK15 plates. The primary recombinants were spotted on LBK25S and LBK25 plates, and the latter was not grown. The correct phenotype was identified using primers PQ265-UP-1f / P85, P82 / PQ272-DN-4r, positive control plasmid, and negative control ATCC 14067 genome. The correct length was 2.3 kb and 1.2 kb. The primary recombinants were placed in an antibiotic-free LB test tube overnight, and then diluted 10, 100, and 1000 times and coated on LBK25, LBK25S, and LBS plates. The secondary recombinants were spotted on LBK25 and LB plates, and the former was not grown. The correct phenotype was identified using primers PQ273-ID-F / PQ274-ID-R, and the correct strain was recorded as QS10 after amplification and sequencing using the primers.

[0151] 2.3 Performance verification

[0152] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0153] Table 15. Glutamine content detection of C. glutamicum QS10

[0154]

[0155] Strain QS10 was obtained by inserting a single copy of the rarD gene from E. coli MG1655 into strain QS09. As shown in Table 15, the glutamine yield of the obtained strain QS10 increased from 19.2 g / L to 19.8 g / L, and the conversion rate increased by 0.7%.

[0156] Example 3: Construction of QS09→QS11 strain and performance verification

[0157] 3.1 Construction of plasmids used

[0158] The upstream homology arm UP (514 bp) was amplified from the genome of C. glutamicum QS09 using PQ320-UP-1F / PQ321-UP-1R as primers. The promoter Psod (244 bp) was amplified from the genome of C. glutamicum ATCC 13032 using PQ322-Psod-2F / PQ323-Psod-2R as primers. The glnA Y405F (1447 bp) was amplified from the genome of C. glutamicum QS09 using PQ324-glnA-3F / PQ325-glnA-3R as primers. The downstream homology arm DN (539 bp) was amplified from the genome of C. glutamicum QS09 using PQ326-DN-4F / PQ327-DN-4R as primers. Subsequently, the 1-2 fragment (733 bp) was obtained by fusion amplification using the upstream homology arm UP and the promoter Psod as templates and PQ320-UP-1F / PQ323-Psod-2R as primers. The glnA Y405FUsing downstream homologous arm DN as a template, and with PQ324-glnA-3F / PQ327-DN-4R as primers, fragment 3-4 (1956bp) was amplified. Finally, using fragments 1-2 and 3-4 as templates, and with PQ320-UP-1F / PQ327-DN-4R as primers, fragment 1-4 (2832bp) was amplified. Fragments 1-4 and pK18mobsacB were then digested with XbaI and SalI at 37℃ for 1 hour. The fragments were directly purified, and the vector was dephosphorylated with 3μL of FastAP (Thermo Scientific FastAP thermosensitive alkaline phosphatase, catalog number EF0654), incubated at 37℃ for 1 hour, and recovered by gel electrophoresis. Subsequently, enzyme ligation and transformation were performed, and colony PCR was performed using primers P82 / P85 to verify the colony length (3.1kb). Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0159] 3.2 Strain Construction

[0160] The plasmid was electroporated onto QS09 plates and plated on LBHISK15 plates. The first recombinant was compared to LBK25S and LBK25 plates; the latter showed longer growth than the former, indicating a correct phenotype. Identification was performed using PQ320-UP-1F / P85 and P82 / PQ327-DN-4R. Positive control plasmid and negative control QS09 genome were used, with correct lengths of 3kb and 2.9kb respectively. The first recombinant was inoculated overnight into antibiotic-free LB tubes and diluted 10, 100, and 1000 times before being plated on LBK25, LBK25S, and LBS plates. Secondary growth on LBS plates was compared to LBK25 and LB plates; the former showed no growth, while the latter showed growth, indicating a correct phenotype. The secondary recombinant was identified using primers PQ328-ID-F / PQ329-ID-R, with a correct band length of 3kb. Amplification with these primers followed by sequencing revealed a correct strain, designated QS11.

[0161] 3.3 Performance Verification

[0162] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield through fermentation is described in Example 1.1.

[0163] Table 16. Detection of glutamine content in Corynebacterium glutamicum QS11

[0164]

[0165] Strain QS11 is based on strain QS09 with its own glnA inserted at ΔglsA. Y405F As shown in Table 16, the obtained strain QS11 showed an increase in glutamine production from 19.2 g / L to 19.6 g / L, with a 0.6% increase in conversion rate. Example 4: Construction and Performance Verification of the QS10→QS12 Strain

[0166] 4.1 Plasmid construction

[0167] The upstream homology arm UP (514 bp) was amplified from the Corynebacterium glutamicum QS09 genome using primers PQ320-UP-1F / PQ321-UP-1R. The promoter Psod (244 bp) was amplified from the Corynebacterium glutamicum ATCC 13032 genome using primers PQ322-Psod-2F / PQ323-Psod-2R. The glnAY405F (1447 bp) was amplified from the Corynebacterium glutamicum QS09 genome using primers PQ324-glnA-3F / PQ325-glnA-3R. The downstream homology arm DN (539 bp) was amplified from the Corynebacterium glutamicum QS09 genome using primers PQ326-DN-4F / PQ327-DN-4R. Then, the 1-2 fragment (733 bp) was amplified by fusion using UP and Psod as templates and primers PQ320-UP-1F / PQ323-Psod-2R. The 3-4 fragment (1956 bp) was amplified using glnAY405F and DN as templates and primers PQ324-glnA-3F / PQ327-DN-4R. Finally, the 1-4 fragment (2832 bp) was amplified using the 1-2 and 3-4 fragments as templates and primers PQ320-UP-1F / PQ327-DN-4R. The 1-4 fragment and pK18mobsacB were digested with XbaI and Sail, respectively, at 37°C for 1 hour. The fragment was directly purified from the product, and the vector was dephosphorylated by adding 3 μL of FastAP at 37°C for 1 hour. The gel was recovered. Then, the vector was ligated and transformed, and the length of 3.1 kb was verified by colony PCR using primers P82 / P85. The correct transformants were inoculated into LBK50 test tubes, and the plasmid was sent for sequencing.

[0168] 4.2 Strain construction

[0169] The plasmid was electroporated into Corynebacterium glutamicum QS10, and LBHISK15 plates were coated. LBK25S and LBK25 plates were inoculated, and the latter was not grown. The correct phenotype was identified using primers PQ320-UP-1F / P85, P82 / PQ327-DN-4R, the positive control plasmid, and the negative control QS09 genome. The correct length was 3 kb and 2.9 kb. The primary recombinants were inoculated into LB test tubes overnight, and LBK25, LBK25S, and LBS plates were coated at dilutions of 10, 100, and 1000 times. The secondary recombinants were inoculated into LBK25 and LB plates, and the former was not grown. The correct phenotype was identified using primers PQ328-ID-F / PQ329-ID-R, and the correct length was 3 kb. Then, the correct strain was identified by sequencing using the primers. The correct strain was designated QS12.

[0170] 4.3 Performance verification

[0171] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0172] Table 17. Glutamine content detection of C. glutamicum QS10-12

[0173]

[0174] Strain QS12 was obtained by inserting a single copy of the rarD gene from E. coli MG1655 at ΔglsA and inserting the glnA gene from C. glutamicum QS09 into strain QS09. Y405F

[0175] In combination with Examples 2, 3 and 4, the conversion rate of strain QS10 obtained by introducing rarD into QS09 was increased by 0.7%, the conversion rate of strain obtained by introducing glnA into QS09 was increased by 0.6%, and the conversion rate of strain o-QS04 obtained by simultaneously introducing rarD and glnA into QS09 was increased by 2.6%. Y405F Y405F The acid production was increased from 19.2 g / L to 21.5 g / L, and the conversion rate was increased by 2.6%, which was unexpectedly effective (Table 17).

[0176] To further confirm that the combination of rarD and glnA Y405F is effective in different glutamine synthesis strains, another glutamine production strain o-QS04 was constructed, and the introduction of rarD, glnA Y405F , and the combination of rarD and glnA Y405F was verified, respectively.

[0177] Example 5: Construction of C. glutamicum ATCC 14067→o-QS04 and performance verification

[0178] 5.1 Construction of C. glutamicum ATCC 14067→o-QS01 and performance verification

[0179] a) Plasmid construction

[0180] ​​The upstream homology arm UP (530 bp) was amplified from the genome of C. glutamicum ATCC 14067 using primers PQ01-UP-F / PQ02-UP-R. The downstream homology arm DN (550 bp) was amplified from the genome of C. glutamicum ATCC 14067 using primers PQ03-DN-F / PQ04-DN-R. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated with 3 μL FastAP at 37°C for 1 h, and the vector was recovered by gel recovery. The digested vector, UP, and DN were assembled at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length of the correct transformant was 1.4 kb. The correct transformant was inoculated into a test tube containing LBK50, and the plasmid was sent for sequencing.

[0181] b) Strain construction

[0182] The plasmid was electroporated into C. glutamicum ATCC 14067, and then plated on LBHISK15 plates. The recombinants were inoculated on LBK25S plates and LBK25 plates, and the recombinants that did not grow on the LBK25 plates were inoculated on LBK25S plates. The recombinants were identified using primers PQ01-UP-F / P85, P82 / PQ04-DN-R, and the correct length was 1.3 kb and 1.2 kb. The recombinants were inoculated into a test tube containing LB and incubated overnight. The recombinants were inoculated on LBK25, LBK25S, and LBS plates at dilutions of 10, 100, and 1000 times, respectively. The recombinants that grew on the LBS plates were inoculated on LBK25 and LB plates, and the recombinants that did not grow on the LBK25 plates were inoculated on the LB plates. The recombinants were identified using primers PQ05-ID-F / PQ06-ID-R, and the correct length was 1.3 kb. The recombinants were then amplified and sequenced using the primers, and the correct strain was designated o-QS01.

[0183] c) Performance verification

[0184] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0185] Table 18. Detection of glutamine content in C. glutamicum o-QS01

[0186]

[0187] The strain o-QS01 was obtained by inactivating glsA in C. glutamicum ATCC 14067. As shown in Table 18, the glutamine yield of the obtained strain o-QS01 was increased from 0.4 g / L to 0.9 g / L, and the conversion rate was increased by 0.55%.

[0188] 5.2 Strain construction and performance verification of o-QS01→o-QS02

[0189] a) Plasmid construction

[0190] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (526 bp) was amplified using primers PQ07-UP-F / PQ08-UP-R. The downstream homologous arm DN (502 bp) was amplified using primers PQ09-DN-F / PQ10-DN-R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h. The vector was dephosphorylated with 3 μL of FastAP and incubated at 37°C for 1 h. The vector was then recovered from the gel. The digested vector, UP, and DN were seamlessly assembled and incubated at 37°C for 30 minutes before transformation. Colony PCR was then performed using primers P82 / P85 to verify the colony length (1.3 kb). Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0191] b) Strain construction

[0192] Plasmids were electroporated into Corynebacterium glutamicum o-QS01 and plated on LBHISK15 plates. A second plating was performed on LBK25S and LBK25 plates; the latter grew longer than the former, indicating a correct phenotype. Identification was performed using PQ07-UP-F / P85 and P82 / PQ10-DN-R plasmids. Positive control plasmids and negative control ATCC 14067 genomes were used, with correct lengths of 1.1kb and 1.2kb respectively. The recombinants were inoculated into LB tubes overnight and diluted 10, 100, and 1000 times before being plated on LBK25, LBK25S, and LBS plates. A second plating was performed on LBK25 and LB plates; the former did not grow, while the latter grew, indicating a correct phenotype. The appropriate annealing temperature was determined using PQ13-id-f / PQ10-DN-R. A positive control plasmid and a negative control Corynebacterium glutamicum ATCC14067 genome were used. Colony PCR was performed at this annealing temperature. The correct secondary recombinant was amplified with primers PQ11-ID-F / PQ12-ID-R and sequenced. The length was 1.1kb. The correct strain was recorded as o-QS02.

[0193] c) Performance Verification

[0194] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield through fermentation is described in Example 1.1.

[0195] Table 19. Detection of glutamine content in Corynebacterium glutamicum o-QS02

[0196]

[0197] Strain o-QS02 is based on strain o-QS01 with the introduction of glnA. Y405FThe amino acid mutation is obtained as shown in Table 19, and the obtained strain o-QS02 glutamine yield is increased from 0.9 g / L to 1.1 g / L, and the conversion rate is increased by 0.21%.

[0198] 5.3 Construction of o-QS02→o-QS03 strain and performance verification

[0199] a) Plasmid construction

[0200] The upstream homology arm UP (536 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ14-UP-F / PQ15-UP-R as primers. The downstream homology arm DN (536 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ16-DN-F / PQ17-DN-R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 h, and the vector was recovered by gel recovery. The digested vector, UP, and DN were assembled seamlessly at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.3 kb. The correct transformant was inoculated into a test tube containing LBK50, and the plasmid was sent for testing.

[0201] b) Strain construction

[0202] The plasmid was electroporated into Corynebacterium glutamicum o-QS02, and then plated on LBHISK15 plates. LBK25S and LBK25 plates were inoculated once, and the latter was not grown. The correct phenotype was identified using PQ14-UP-F / P85, P82 / PQ17-DN-R, and the correct length was 1.1 kb and 1.6 kb. The positive control plasmid and the negative control ATCC 14067 genome were used as controls. The recombinants were inoculated into LB test tubes overnight, and then diluted by 10, 100, and 1000 times and plated on LBK25, LBK25S, and LBS plates. The LBS plate was grown, and then inoculated on LBK25 and LB plates. The former was not grown, and the latter was grown. The correct phenotype was identified using primers PQ18-ID-F / PQ19-ID-R, and the correct band length was 1.2 kb. Then, the correct strain was identified by sequencing using the primers for amplification, and the correct strain was designated as o-QS03.

[0203] c) Performance verification

[0204] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0205] Table 20. Detection of glutamine content of Corynebacterium glutamicum o-QS03

[0206]

[0207]

[0208] Strain o-QS03 was obtained by inactivating glnE based on strain o-QS02, as shown in Table 20, the glutamine yield of the obtained strain o-QS03 increased from 1.1 g / L to 1.7 g / L, and the conversion rate increased by 0.67%.

[0209] 5.4 Strain construction and performance verification of o-QS03→o-QS04

[0210] a) Plasmid construction

[0211] The upstream homology arm UP (520 bp) was amplified using the genome of Corynebacterium glutamicum ATCC 14067 as a template and the primers PQ20-UP-F / PQ21-UP-R. The promoter Psod (192 bp) was amplified using the genome of Corynebacterium glutamicum ATCC 13032 as a template and the primers PQ22-Psod-F / PQ23-Psod-R. The downstream homology arm DN (526 bp) was amplified using the genome of Corynebacterium glutamicum ATCC 14067 as a template and the primers PQ24-DN-F / PQ25-DN-R. The UP-Psod (692 bp) was amplified by fusion using the UP and Psod fragments as templates and the primers PQ20-UP-F / PQ23-Psod-R.

[0212] The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, 3 μL FastAP was added to dephosphorylate the vector, which was then placed at 37°C for 1 h, and the vector was recovered by gel recovery. The digested vector, UP-Psod, and DN were assembled seamlessly at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using the primers P82 / P85, and the length was 1.5 kb. The correct transformants were transferred to test tubes containing LBK50, and the plasmid was sent for testing.

[0213] b) Strain construction

[0214] Plasmid electrotransformation of C. glutamicum o-QS03, spread on LBHISK15 plates, once against LBK25S, LBK25 plates, the latter longer than the former phenotype correct, identified with PQ20-UP-F / P85, P82 / PQ25-DN-R, positive control plasmid, negative control ATCC 14067 genome, correct length 1.4 kb, 1.1 kb. Once recombinants spread on LB tubes overnight, dilute 10, 100, 1000 times spread on LBK25, LBK25S, LBS plates, LBS plates grown against LBK25, LB plates, the former not grown, the latter grown phenotype correct. Secondary recombinants identified with primers PQ26-ID-F / PQ27-ID-R, correct band length 1.4 kb, then amplified with this primer and sequenced, correct strain noted as o-QS04.

[0215] c) Performance verification

[0216] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance, and the method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0217] Table 21. Detection of glutamine content of C. glutamicum o-QS04

[0218]

[0219] Strain o-QS04 was obtained by inserting Psod promoter in front of gdh gene based on strain o-QS03, as shown in Table 21, the glutamine yield of the obtained strain o-QS04 was increased from 1.7 g / L to 2.4 g / L, and the conversion rate was increased by 0.72%.

[0220] Example 6: Construction of Strain o-QS04→o-QS05 and Performance Verification

[0221] 6.1 Plasmid construction

[0222] The genomic DNA of C. glutamicum ATCC 14067 was used as template and the primers of PQ265-UP-1f / PQ266-UP-1r were used to amplify the upstream homology arm UP (563 bp). The genomic DNA of C. glutamicum ATCC 13032 was used as template and the primers of PQ267-Psod-2f / PQ279-Psod-2r were used to amplify the promoter Psod (215 bp). The genomic DNA of E. coli MG1655 was used as template and the primers of PQ280-rarD-3f / PQ281-rarD-3r were used to amplify the rarD (913 bp). The genomic DNA of C. glutamicum ATCC 14067 was used as template and the primers of PQ282-DN-4f / PQ272-DN-4r were used to amplify the downstream homology arm DN (536 bp). Then, the 1-2 fragment (751 bp) was amplified by using UP and Psod as templates and the primers of PQ265-UP-1f / PQ279-Psod-2r. The 3-4 fragment (1427 bp) was amplified by using rarD and DN as templates and the primers of PQ280-rarD-3f / PQ272-DN-4r. Finally, the 1-4 fragment (2157 bp) was amplified by using the 1-2 and 3-4 fragments as templates and the primers of PQ265-UP-1f / PQ272-DN-4r. The 1-4 fragment and pK18mobsacB were digested with EcoRI and NheI at 37 °C for 1 h. The fragment was directly purified, the vector was dephosphorylated by adding 3 μL FastAP at 37 °C for 1 h, and the gel was recovered. Then, the vector was ligated and transformed, and the length of 2.3 kb was verified by colony PCR using primers P82 / P85. The correct transformant was inoculated into a test tube containing LBK50, and the plasmid was sent for sequencing.

[0223] 6.2 Strain construction

[0224] The plasmid was electroporated into C. glutamicum o-QS04, which was spread on LBHISK15 plates. LBK25S and LBK25 plates were inoculated with the primary recombinants, and the latter was not grown. The correct phenotype was identified by using primers PQ265-UP-1f / P85, P82 / PQ272-DN-4r, the positive control plasmid, and the negative control ATCC 14067 genome. The correct length was 2.3 kb and 1.2 kb. The primary recombinants were inoculated into test tubes containing LB overnight, and LBK25, LBK25S, and LBS plates were inoculated with 10-, 100-, and 1000-fold dilutions of the primary recombinants. The secondary recombinants were inoculated into LBK25 and LB plates, and the former was not grown. The correct phenotype was identified by using primers PQ273-ID-F / PQ274-ID-R, and the correct length was 2.3 kb. Then, the correct strain was sequenced by using the primers for amplification. The correct strain was designated o-QS05.

[0225] 6.3 Performance verification

[0226] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield through fermentation is described in Example 1.1.

[0227] Table 22. Detection of glutamine content in Corynebacterium glutamicum o-QS05

[0228]

[0229] The strain o-QS05 was obtained by inserting a single copy of the rarD gene from E. coli MG1655 into the strain o-QS04. As shown in Table 22, the glutamine yield of the obtained strain o-QS05 increased from 2.4 g / L to 2.9 g / L, and the conversion rate increased by 0.6%.

[0230] Example 7: Construction and performance verification of the o-QS04→o-QS06 strain

[0231] 7.1 Plasmid Construction

[0232] Using the *Corynebacterium glutamicum* o-QS04 genome as a template, the upstream homologous arm UP (514 bp) was amplified using primers PQ320-UP-1F / PQ321-UP-1R. Using the *Corynebacterium glutamicum* ATCC 13032 genome as a template, the promoter Psod (244 bp) was amplified using primers PQ322-Psod-2F / PQ323-Psod-2R. Using the *Corynebacterium glutamicum* o-QS04 genome as a template, glnA was amplified using primers PQ324-glnA-3F / PQ325-glnA-3R. Y405F (1447bp). Using the Corynebacterium glutamicum o-QS04 genome as a template, and PQ326-DN-4F / PQ327-DN-4R as primers, the downstream homologous arm DN (539bp) was amplified. Subsequently, using UP and Psod as templates, and PQ320-UP-1F / PQ323-Psod-2R as primers, fragments 1-2 (733bp) were fused and amplified. Using glnAY405F and DN as templates, and PQ324-glnA-3F / PQ327-DN-4R as primers, fragments 3-4 (1956bp) were amplified. Finally, using fragments 1-2 and 3-4 as templates, and PQ320-UP-1F / PQ327-DN-4R as primers, fragments 1-4 (2832bp) were amplified. Finally, fragments 1-4 and pK18mobsacB were digested with XbaI and SalI at 37°C for 1 hour. The fragment products were purified directly, and the vector was dephosphorylated with 3 μL of FastAP, incubated at 37°C for 1 hour, and then recovered by gel extraction. Subsequently, enzyme ligation and transformation were performed, and colony PCR was performed using primers P82 / P85 to verify the colony length, which was 3.1 kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assay.

[0233] 7.2 Strain Construction

[0234] Plasmids were electroporated into Corynebacterium glutamicum o-QS04 and plated on LBHISK15 plates. A second plasmid was used to compare the plasmid growth on LBK25S and LBK25 plates; the latter showed longer growth than the former, indicating a correct phenotype. Identification was performed using PQ320-UP-1F / P85 and P82 / PQ327-DN-4R. The positive control plasmid and negative control o-QS04 genome showed correct lengths of 3kb and 2.9kb, respectively. The recombinant was inoculated into LB tubes overnight and diluted 10, 100, and 1000 times before being plated on LBK25, LBK25S, and LBS plates. A second plasmid grown on LBS was compared to LBK25 and LB plates; the former showed no growth, while the latter showed growth, indicating a correct phenotype. The second recombinant was identified using primers PQ328-ID-F / PQ329-ID-R, showing a correct band length of 3kb. Amplification with these primers followed by sequencing revealed a correct strain, designated o-QS06.

[0235] 7.3 Performance Verification

[0236] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield through fermentation is described in Example 1.1.

[0237] Table 23. Detection of glutamine content in Corynebacterium glutamicum o-QS06

[0238]

[0239] Strain o-QS06 is based on strain o-QS04 with its own glnA inserted at ΔglsA. Y405F As shown in Table 23, the glutamine yield of the obtained strain o-QS06 increased from 2.4 g / L to 2.6 g / L, and the conversion rate increased by 0.3%.

[0240] Example 8: Construction and performance verification of o-QS05→o-QS07 strain

[0241] 8.1 Plasmid Construction

[0242] The genomic DNA of C. glutamicum o-QS04 was used as the template, and the primers PQ320-UP-1F / PQ321-UP-1R were used to amplify the upstream homology arm UP (514 bp). The genomic DNA of C. glutamicum ATCC 13032 was used as the template, and the primers PQ322-Psod-2F / PQ323-Psod-2R were used to amplify the promoter Psod (244 bp). The genomic DNA of C. glutamicum o-QS04 was used as the template, and the primers PQ324-glnA-3F / PQ325-glnA-3R were used to amplify glnAY405F (1447 bp). The genomic DNA of C. glutamicum o-QS04 was used as the template, and the primers PQ326-DN-4F / PQ327-DN-4R were used to amplify the downstream homology arm DN (539 bp). Subsequently, the 1-2 fragment (733 bp) was obtained by fusion amplification using UP and Psod as the templates and the primers PQ320-UP-1F / PQ323-Psod-2R. The 3-4 fragment (1956 bp) was obtained by amplification using glnAY405F and DN as the templates and the primers PQ324-glnA-3F / PQ327-DN-4R. Finally, the 1-4 fragment (2832 bp) was obtained by amplification using the 1-2 and 3-4 fragments as the templates and the primers PQ320-UP-1F / PQ327-DN-4R. The 1-4 fragment and pK18mobsacB were digested with XbaI and SalI at 37°C for 1 hour, the fragment was directly purified, 3 μL of FastAP dephosphorylation was added to the vector, which was placed at 37°C for 1 hour, and the gel was recovered. Subsequently, the vector was ligated and transformed, and colony PCR was performed using the primers P82 / P85, the length was 3.1 kb, and the correct transformants were inoculated into test tubes containing LBK50, and the plasmid was sent for sequencing.

[0243] 8.2 Strain construction

[0244] The plasmid was electroporated into C. glutamicum o-QS05, which was inoculated onto LBHISK15 plates, and then inoculated onto LBK25S and LBK25 plates. The correct phenotype was obtained when the growth on the LBK25 plate was not as good as that on the LBK25S plate. The recombinant strain was identified using the primers PQ320-UP-1F / P85, P82 / PQ327-DN-4R, the positive control plasmid, and the negative control o-QS04 genome. The correct lengths were 3 kb and 2.9 kb. The recombinant strain was inoculated into test tubes containing LB and incubated overnight. The culture was diluted by 10, 100, and 1000 times, and then inoculated onto LBK25, LBK25S, and LBS plates. The correct phenotype was obtained when the growth on the LB plate was better than that on the LBK25 plate. The recombinant strain was identified using the primers PQ328-ID-F / PQ329-ID-R, and the correct length was 3 kb. The correct strain was named o-QS07.

[0245] 8.3 Performance verification

[0246] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0247] Table 24. Glutamine content detection of C. glutamicum o-QS05-07

[0248]

[0249] Strain o-QS07 is a single copy of the insertion of the MG1655-derived rarD gene in strain o-QS04, and the insertion of the self glnA gene at ΔglsA Y405F The glutamine yield of the new strain obtained is increased from 2.4 g / L to 3.5 g / L, and the conversion rate is increased by 1.3%, which is better than the sum of o-QS05 and o-QS06.

[0250] In summary, Examples 6, 7, and 8, the introduction of rarD in o-QS04, the conversion rate of o-QS05 is increased by 0.6%, and the introduction of glnA in o-QS04 Y405F The conversion rate of the strain is increased by 0.3%, and the rarD and glnA are introduced at the same time Y405F The glutamic acid yield is increased from 2.4 g / L to 3.5 g / L, the conversion rate is increased by 1.3%, and the effect is unexpected (Table 24).

[0251] In summary, Examples 2, 3, 4, 6, 7, and 8, it is proved that the combination of rarD and glnA in different starting strains Y405F has unexpected effects. Therefore, the combination is applied to other glutamine-producing strains and has unexpected effects.

[0252] Incorporated by reference

[0253] The entire contents of each patent and scientific document referred to herein are incorporated by reference for all purposes.

[0254] Equivalents

[0255] The present disclosure can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments should be considered in all aspects as illustrative and not restrictive. Thus, the scope of the disclosure is indicated by the appended claims rather than by the description preceding them, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. Modified bacteria that produce glutamine, wherein the genome contains a heterologous polynucleotide of glutamine efflux protein (rarD) and increased glutamine synthase (glnA) activity compared to the unmodified bacteria.

2. The modified bacteria of claim 1, wherein the modified bacteria produce glutamine in higher yield than the unmodified bacteria.

3. The modified bacteria as described in claim 1, wherein the bacteria are Corynebacterium, preferably Corynebacterium glutamicum.

4. The modified bacteria of claim 1, wherein the glutamine efflux protein (rarD) is derived from Escherichia coli.

5. The modified bacteria of claim 1, wherein the modification increasing glutamine synthase (glnA) activity is a substitution of the amino acid Y405F in glnA. Y405F Preferably, the glnA is derived from Corynebacterium glutamicum or Saccharomyces cerevisiae.

6. The modified bacteria as described in any one of claims 1 to 5, comprising one or more copies of the glutamine efflux protein (rarD) gene.

7. The modified bacteria of claim 6, wherein the glutamine efflux protein (rarD) gene insertion site is located within the acetyltransferase encoding gene CEY17_08220ORF.

8. The modified bacteria of claim 5, wherein it comprises one or more glnA molecules. Y405F . gene copy.

9. The modified bacteria of claim 8, wherein glnA Y405F The gene insertion site is within the glsA ORF gene encoding glutaminase.

10. The modified bacteria according to any one of claims 1 to 9, wherein the promoter of the glutamine efflux protein (rarD) gene is the Psod promoter.

11. The modified bacteria according to any one of claims 5 to 9, wherein the glnA Y405F The gene promoter is the Psod promoter.

12. The modified bacteria of claim 10 or 11, wherein the Psod promoter is derived from Corynebacterium glutamicum.

13. Use of the modified bacteria as described in any one of claims 1 to 12 in the production of glutamine.

14. A method for producing glutamine, comprising culturing the modified bacteria as described in any one of claims 1 to 12 in a culture medium and isolating glutamine.

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