Method for producing glutamine

Abstract

Provided is a modified bacterium for producing glutamine, wherein, compared with an unmodified bacterium, the genome contains a modification that increases the activity of glutamine synthetase glnA and a modification that decreases the activity of RosR. In addition, further provided is a method for producing glutamine by using the modified bacterium.

Description

A method for producing glutamine

[0001] priority

[0002] This application claims the rights and priority of Chinese application No. 2024118342223, filed on December 12, 2024. The entire contents of Chinese application No. 2024118342223 are incorporated herein by reference for all purposes. Technical Field

[0003] This disclosure relates to a method for producing glutamine by using bacterial fermentation. Background Technology

[0004] Glutamine is an amino acid that encodes proteins, promoting protein synthesis and inhibiting protein breakdown. It plays an important role in the pharmaceutical industry and is used to treat gastric and duodenal ulcers. Currently, glutamine is mainly produced through fermentation using *Corynebacterium glutamicum*. *Corynebacterium glutamicum* is a heterotrophic aerobic, Gram-positive bacterium characterized by rapid growth, non-pathogenicity, and weak degradation of its own metabolites. However, the fermentation performance of glutamine-producing strains remains poor, containing the byproduct isoleucine, and resulting in unsatisfactory glutamine conversion rates. Industrial demand for glutamine is extremely high, and existing strains cannot meet the needs of large-scale industrial production. Therefore, using genetic engineering techniques to improve glutamine yield and conversion rates is particularly important.

[0005] Numerous reports have mentioned methods to increase glutamine production. One method involves mutating the 405th amino acid of glnA from tyrosine (Y) to phenylalanine (F), i.e., from TAC to TTC, which can undo adenylation of glutamine synthase and effectively increase glutamine production (CN1614008A). Another method involves modifying glsA to reduce intracellular glutaminase activity, effectively increasing glutamine production (US7943364B2). A third method involves inhibiting the glutamine synthase-encoding gene in Corynebacterium glutamicum, which can lead to reduced transcription levels and a sharp decrease in enzyme activity, resulting in insufficient utilization of the substrate glutamate. 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, inhibiting glnA transcription. Inactivation of RosR promotes glutamine accumulation (2022, Xiangfei Li, MarR-type transcription factor RosR regulates glutamate metabolism network and promotes accumulation of Lglutamate in Corynebacterium glutamicum G01). The glutamine synthase from Corynebacterium glutamicum has a tyrosine mutation at amino acid position 405, replacing phenylalanine, thus removing the adenylate modification (CN100392075C). It contains the hemoglobin vgb of Corynebacterium glutamicum and the glutamine synthase gene glnA. Y405F Wild-type Corynebacterium glutamicum 14067 can produce high yields of glutamine at lower DO levels and lower costs (CN1614008A).

[0006] Currently, glutamine-producing strains accumulate heteroacid glutamic acid, indicating that the terminal / extracellular transport is not strong enough, which limits the high glutamine production of the strains. This invention mainly targets the existing heteroacid glutamic acid, promotes the conversion of glutamic acid to glutamine, and transports the synthesized glutamine to the extracellular space. Summary of the Invention

[0007] This disclosure provides a modified bacterium for producing glutamine, wherein, compared with the unmodified bacterium, its genome contains modifications that increase glutamine synthase (glnA) activity and decrease RosR activity.

[0008] In one specific embodiment, the modified bacteria that produce glutamine contain, compared with the unmodified bacteria, modifications in their genome that increase glutamine synthase (glnA) activity, decrease RosR activity, and decrease glsA activity, preferably, the modification that decreases glsA activity is the deletion or partial deletion of the glsA gene.

[0009] In one specific implementation, the modified bacteria produce glutamine in higher yields than the unmodified bacteria.

[0010] In one specific embodiment, the bacteria are Corynebacterium spp., preferably Corynebacterium glutamicum.

[0011] In one specific embodiment, the modification that increases the activity of glutamine synthase (glnA) involves replacing the tyrosine residue at position 405 of the glutamine synthase with a phenylalanine residue, i.e., glnA contains a Y405F amino acid substitution (glnA). Y405F Preferably, the glnA is derived from Corynebacterium glutamicum or Saccharomyces cerevisiae.

[0012] In one specific implementation, the modification that reduces RosR activity is a deletion or partial deletion of the nucleic acid sequence of the RosR gene.

[0013] In one specific embodiment, the modified bacteria comprises the nucleic acid sequence shown in SEQ ID NO: 85.

[0014] In one specific implementation, the modified bacteria contains one or more glnA molecules. Y405F . gene copy.

[0015] In one specific implementation, the modified bacteria, wherein glnA Y405F The gene insertion site is within the glsA ORF gene encoding glutaminase.

[0016] In one specific implementation, the modified bacteria, wherein the Psod promoter is derived from Corynebacterium glutamicum.

[0017] On the other hand, the use of the modified bacteria described in this disclosure in increasing glutamine production is provided.

[0018] On the other hand, a method for producing glutamine is provided, comprising culturing the modified bacteria described in this disclosure in a culture medium and isolating glutamine.

[0019] On the other hand, a bioreactor includes the modified bacteria described in this disclosure. Beneficial effects

[0020] Introducing glnA Y405F Both ΔrosR and ΔrosR improved glutamine conversion, confirming that the introduction of glnA into different strains... Y405F Both ΔrosR and ΔrosR have unexpected effects. Detailed Implementation

[0021] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this 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.

[0022] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0023] Table 1. Instruments used in this invention

[0024] Table 2. Reagents used in this invention

[0025] Table 3. Primer sequence information

[0026] The genotypes of the strains are shown in Table 4:

[0027] Table 4. Strain Genotypes

[0028] Table 5. Sequence Information

[0029] Example 1: Construction and performance verification of the QS08 starting strain

[0030] 1.1 ATCC14067→QS01(ino-1 S84A Strain construction and performance verification

[0031] a) Plasmid construction

[0032] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (594 bp) was amplified using primers PQ28-UP-F / PQ29-UP-R. The downstream homologous arm DN (555 bp) was amplified using the same genome as the template, with primers PQ30-DN-F / PQ31-DN-R. Using UP and DN as templates, the overlap fragment (1120 bp) was amplified using primers PQ28-UP-F / PQ31-DN-R. The overlap fragment and pK18mobsacB were digested with XbaI and PstI at 37°C for 1 hour. The direct product was purified, dephosphorylated with 3 μL of FastAP, incubated at 37°C for 1 hour, and then recovered via gel electrophoresis. Subsequently, enzyme ligation and transformation were performed, and colony PCR was conducted using primers P82 / P85 to verify the colony length, which was 1.4kb. Correct transformants were inoculated into LBK50 test tubes, and plasmids were extracted and sent for testing.

[0033] b) Strain construction

[0034] Plasmids were electroporated into Corynebacterium glutamicum ATCC14067 and plated on LBHISK15 plates. A second plasmid comparison was made between LBK25S and LBK25 plates; the latter grew longer than the former, indicating a correct phenotype. Identification was performed using PQ28-UP-F / P85 and P82 / PQ31-DN-R. The positive control plasmid and the negative control ATCC 14067 genome showed correct lengths of 1.3kb 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 comparison of LBS plate growth with LBK25 and LB plates showed that the former did not grow, while the latter showed a correct phenotype. The appropriate annealing temperature was determined using PQ32-id-f / PQ31-DN-R. A positive control plasmid and a negative control Corynebacterium glutamicum ATCC 14067 genome were used. Colony PCR was performed at this annealing temperature. The correct secondary recombinant was amplified with primers PQ33-ID-F / PQ34-ID-R and sequenced. The length was 1.4kb. The correct strain was recorded as QS01.

[0035] c) Performance Verification

[0036] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield through fermentation is as follows:

[0037] The strain, frozen in -80℃ glycerol tubes, was activated by inoculating it onto BHI slant medium. After culturing at 33℃ for 24 hours, bacterial growth occurred. Bacterial growth was picked from the freshly activated slant and inoculated into the seed culture medium described below. The culture was then incubated at 33℃ with shaking at 100 rpm until the mid-to-late logarithmic growth stage, for 5 hours to obtain the seed culture. A 10% inoculum of this seed culture was inoculated into a 500ml shake flask containing 20ml of fermentation medium and incubated at 33℃ with shaking at 150 rpm for 48 hours. After complete glucose consumption, the concentration of glutamine accumulated in the culture medium was determined by HPLC.

[0038] The culture medium formula is as follows:

[0039] LB medium: peptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, agar 1.8%, sterilized at 121℃ and 0.1 MPa for 20 minutes; LBK25 is LB with kanamycin 25 μg / mL.

[0040] LBHIS medium: peptone 5 g / L, NaCl 5 g / L, yeast extract 2.5 g / L, brain and heart extract 18.5 g / L, sorbitol 91 g / L, agar 1.8%, sterilized at 121℃ and 0.1 MPa for 20 minutes; LBHISK15 is LBHIS with kanamycin 15 μg / mL.

[0041] LBS medium: peptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, sucrose 0.1 g / L, agar 1.8%, sterilized at 121℃ and 0.1 MPa for 20 minutes; LBK25S is LBS with kanamycin 25 μg / mL.

[0042] BHI slant culture medium: brain heart extract 37g / L, agar 1.8%, sterilized at 121℃ and 0.1MPa for 20 minutes;

[0043] Seed culture medium: glucose 25 g / L, urea 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.4 g / L, corn steep liquor powder 15 g / L, pH 7.0;

[0044] Fermentation medium: glucose 90.9 g / L, (NH4)2SO4 50 g / L, KH2PO4 2.5 g / L, corn steep liquor powder 2 g / L, CaCO3 40 g / L, pH 7.0.

[0045] Table 6. Glutamine content detection in Corynebacterium glutamicum QS01

[0046] Strain QS01 is based on Corynebacterium glutamicum ATCC14067 with the introduction of ino-1.S84A As shown in Table 6, the amino acid mutations resulted in an increase in glutamine production from 0.4 g / L to 1.1 g / L in strain QS01, with a conversion rate increase of 0.77%.

[0047] 1.2 QS01→QS02(CEY17_06485 A386T Strain construction and performance verification

[0048] a) Plasmid construction

[0049] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (543 bp) was amplified using primers PQ35-UP-F / PQ36-UP-R. The downstream homologous arm DN (548 bp) was amplified using the same genome as the template, with primers PQ37-DN-F / PQ38-DN-R. Using UP and DN as templates, the overlap fragment (1053 bp) was amplified using primers PQ35-UP-F / PQ38-DN-R. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour. The direct product was purified, dephosphorylated with 3 μL of FastAP, incubated at 37°C for 1 hour, and then recovered via gel electrophoresis. Subsequently, enzyme ligation and transformation were performed, and colony PCR was conducted using primers P82 / P85. The colony length was 1.3kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0050] b) Strain construction

[0051] Plasmids were electroporated into *Corynebacterium glutamicum* 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 PQ35-UP-F / P85 and P82 / PQ38-DN-R. The positive control plasmid and negative control ATCC 14067 genome 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, then 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 showed a correct phenotype. The appropriate annealing temperature was determined using PQ39-id-f / PQ38-DN-R. The positive control plasmid and negative control *Corynebacterium glutamicum* ATCC 14067 genome were used for colony PCR identification at this annealing temperature. The correct secondary recombinant was amplified with primers PQ40-ID-F / PQ41-ID-R and sequenced. The length was 1.4kb. The correct strain was recorded as QS02.

[0052] c) Performance Verification

[0053] 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.

[0054] Table 7. Detection of glutamine content in Corynebacterium glutamicum QS02

[0055] Strain QS02 is based on strain QS01 with the introduction of CEY17_06485. A386T As shown in Table 7, the amino acid mutations resulted in an increase in glutamine production from 1.1 g / L to 1.7 g / L in strain QS02, with a conversion rate increase of 0.65%.

[0056] 1.3 QS02→QS03(CEY17_05975 V184I Strain construction and performance verification

[0057] a) Plasmid construction

[0058] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (555 bp) was amplified using primers PQ42-UP-F / PQ43-UP-R. The downstream homologous arm DN (500 bp) was amplified using the same genome as the template, with primers PQ44-DN-F / PQ45-DN-R. Using UP and DN as templates, the overlap fragment (1029 bp) was amplified using primers PQ42-UP-F / PQ45-DN-R. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour. The direct product was purified, dephosphorylated with 3 μL of FastAP, incubated at 37°C for 1 hour, and then recovered via gel electrophoresis. Subsequently, enzyme ligation and transformation were performed, and colony PCR was conducted using primers P82 / P85. The colony length was 1.3kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0059] b) Strain construction

[0060] The plasmid was electroporated into *Corynebacterium glutamicum* QS02 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 PQ42-UP-F / P85 and P82 / PQ45-DN-R. The positive control plasmid and negative control ATCC 14067 genome were used, with correct lengths of 1.1kb and 1.2kb respectively. The recombinant was inoculated into LB tubes overnight and diluted 10, 100, and 1000 times, then 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 showed a correct phenotype. The appropriate annealing temperature was determined using PQ46-id-f / PQ45-DN-R. The positive control plasmid and negative control *Corynebacterium glutamicum* ATCC 14067 genome were used for colony PCR identification at this annealing temperature. The correct secondary recombinant was amplified with primers PQ47-ID-F / PQ48-ID-R and sequenced. The length was 1.3kb. The correct strain was recorded as QS03.

[0061] c) Performance Verification

[0062] 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.

[0063] Table 8. Detection of glutamine content in Corynebacterium glutamicum QS03

[0064] Strain QS03 was developed by introducing CEY17_05975 into strain QS02. V184I As shown in Table 8, the amino acid mutations resulted in an increase in glutamine production from 1.7 g / L to 2.6 g / L in strain QS03, with a conversion rate increase of 1.02%.

[0065] 1.4 QS03→QS04(CEY17_04535 T65I Strain construction and performance verification

[0066] a) Plasmid construction

[0067] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (505 bp) was amplified using primers PQ49-UP-F / PQ50-UP-R. The downstream homologous arm DN (542 bp) was amplified using the same genome as the template, with primers PQ51-DN-F / PQ52-DN-R. Using UP and DN as templates, the overlap fragment (1050 bp) was amplified using primers PQ49-UP-F / PQ52-DN-R. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour. The direct product was purified, dephosphorylated with 3 μL of FastAP, incubated at 37°C for 1 hour, and then recovered via gel electrophoresis. Subsequently, enzyme ligation and transformation were performed, and colony PCR was conducted using primers P82 / P85. The colony length was 1.3kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0068] b) Strain construction

[0069] Plasmids were electroporated into *Corynebacterium glutamicum* QS03 and plated on LBHISK15 plates. A second plasmid comparison was performed on LBK25S and LBK25 plates; the latter showed longer growth than the former, indicating a correct phenotype. Identification was performed using PQ49-UP-F / P85 and P82 / PQ52-DN-R. The positive control plasmid and negative control ATCC 14067 genome showed correct lengths of 1.1kb and 1.2kb, respectively. The recombinant was inoculated into LB tubes overnight and diluted 10, 100, and 1000 times, then plated on LBK25, LBK25S, and LBS plates. A second comparison of LBS plate growth with LBK25 and LB plates showed that the former showed no growth, while the latter showed growth, indicating a correct phenotype. The optimal annealing temperature was determined using PQ55-id-f / PQ52-DN-R. The positive control plasmid and negative control *Corynebacterium glutamicum* ATCC 14067 genome showed a band length of 689bp. Colony PCR identification was performed using this annealing temperature. The correct secondary recombinant was amplified with primers PQ53-ID-F / PQ54-ID-R and sequenced. The length was 1.3kb. The correct strain was recorded as QS04.

[0070] c) Performance Verification

[0071] 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.

[0072] Table 9. Glutamine content detection in Corynebacterium glutamicum QS04

[0073] Strain QS04 is based on strain QS03 with the introduction of CEY17_04535. T65IAs shown in Table 9, the amino acid mutations resulted in an increase in glutamine production from 2.6 g / L to 3 g / L in strain QS04, with a conversion rate increase of 0.41%.

[0074] 1.5 QS04→QS05(CEY17_04555 R2916C Strain construction and performance verification

[0075] a) Plasmid construction

[0076] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (522 bp) was amplified using primers PQ56-UP-F / PQ57-UP-R. The downstream homologous arm DN (523 bp) was amplified using the same genome as the template, with primers PQ58-DN-F / PQ59-DN-R. Using UP and DN as templates, the overlap fragment (1020 bp) was amplified using primers PQ56-UP-F / PQ59-DN-R. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour. The direct product was purified, dephosphorylated with 3 μL of FastAP, incubated at 37°C for 1 hour, and then recovered via gel electrophoresis. Subsequently, enzyme ligation and transformation were performed, and colony PCR was conducted using primers P82 / P85. The colony length was 1.3kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0077] b) Strain construction

[0078] Plasmids were electroporated into *Corynebacterium glutamicum* QS04 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 PQ56-UP-F / P85 and P82 / PQ59-DN-R. A positive control plasmid and a negative control ATCC 14067 genome 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, then 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 showed a correct phenotype. The appropriate annealing temperature was determined using PQ62-id-f / PQ59-DN-R. A positive control plasmid and a negative control *Corynebacterium glutamicum* ATCC 14067 genome were used for colony PCR identification at this annealing temperature. The correct secondary recombinant was amplified with primers PQ60-ID-F / PQ61-ID-R and sequenced. The length was 1.2kb. The correct strain was recorded as QS05.

[0079] c) Performance Verification

[0080] 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.

[0081] Table 10. Detection of glutamine content in Corynebacterium glutamicum QS05

[0082] Strain QS05 is based on strain QS04 with the introduction of CEY17_04555. R2916C As shown in Table 10, the amino acid mutations resulted in an increase in glutamine production from 3 g / L to 3.8 g / L in strain QS05, with a conversion rate increase of 0.88%.

[0083] 1.6 QS05→QS06(CEY17_13360 A139T Strain construction and performance verification

[0084] a) Plasmid construction

[0085] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (539 bp) was amplified using primers PQ63-UP-F / PQ64-UP-R. The downstream homologous arm DN (542 bp) was amplified using primers PQ65-DN-F / PQ66-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 for 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.

[0086] b) Strain construction

[0087] Plasmids were electroporated into *Corynebacterium glutamicum* QS05 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 PQ63-UP-F / P85 and P82 / PQ66-DN-R. A positive control plasmid and a negative control ATCC 14067 genome 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, then 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 showed a correct phenotype. The appropriate annealing temperature was determined using PQ67-id-f / PQ66-DN-R. A positive control plasmid and a negative control *Corynebacterium glutamicum* ATCC 14067 genome were used for colony PCR identification at this annealing temperature. The correct secondary recombinant was amplified with primers PQ68-ID-F / PQ69-ID-R and sequenced. The length was 1.3kb. The correct strain was recorded as QS06.

[0088] c) Performance Verification

[0089] 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.

[0090] Table 11. Detection of glutamine content in Corynebacterium glutamicum QS06

[0091] 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 strain QS06 increased from 3.8 g / L to 5.2 g / L, and the conversion rate increased by 1.54%.

[0092] 1.7 QS06→QS07(gyrA A466V Strain construction and performance verification

[0093] a) Plasmid construction

[0094] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (477 bp) was amplified using primers PQ70-UP-F / PQ71-UP-R. The downstream homologous arm DN (550 bp) was amplified using primers PQ72-DN-F / PQ73-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.

[0095] b) Strain construction

[0096] Plasmids were electroporated into Corynebacterium glutamicum QS06 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 PQ70-UP-F / P85 and P82 / PQ73-DN-R. A positive control plasmid and a negative control ATCC 14067 genome 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 PQ74-id-f / PQ73-DN-R. A positive control plasmid and a negative control Corynebacterium glutamicum ATCC 14067 genome were used. Colony PCR was performed at this annealing temperature. The correct secondary recombinant was amplified with primers PQ75-ID-F / PQ76-ID-R and sequenced. The length was 1.2kb. The correct strain was recorded as QS07.

[0097] c) Performance Verification

[0098] 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.

[0099] Table 12. Detection of glutamine content in Corynebacterium glutamicum QS07

[0100] Strain QS07 is based on strain QS06 with the introduction of gyrA. A466V As shown in Table 12, the amino acid mutations resulted in an increase in glutamine production from 5.2 g / L to 8 g / L in strain QS07, with a conversion rate increase of 3.08%.

[0101] 1.8 Construction and performance verification of the QS07→QS08(ΔglsA) strain

[0102] a) Plasmid construction

[0103] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (530 bp) was amplified using primers PQ01-UP-F / PQ02-UP-R. The downstream homologous arm DN (550 bp) was amplified using primers PQ03-DN-F / PQ04-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 for transformation. Colony PCR was then performed using primers P82 / P85 to verify the colony length (1.4 kb). Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0104] b) Strain construction

[0105] Plasmids were electroporated into Corynebacterium glutamicum QS07 and plated on LBHISK15 plates. A second plasmid was used to cross-pollinate LBK25S and LBK25 plates; the latter showed longer growth than the former, indicating a correct phenotype. The plasmids were identified using PQ01-UP-F / P85 and P82 / PQ04-DN-R, serving as a positive control and an ATCC 14067 genome as a negative control, with correct lengths of 1.3kb 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 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 PQ05-ID-F / PQ06-ID-R, with a correct band length of 1.3kb. Amplification with these primers followed by sequencing revealed a correct strain, designated QS08.

[0106] c) Performance Verification

[0107] 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.

[0108] Table 13. Detection of glutamine content in Corynebacterium glutamicum QS08

[0109] Strain QS08 was obtained by inactivating glsA based on strain QS07. As shown in Table 13, the yield of glutamine in strain QS08 increased from 8 g / L to 12.4 g / L, and the conversion rate increased by 4.8%.

[0110] Example 2: QS08→QS17(Psod-glnA) Y405F Strain construction and performance verification

[0111] a) Plasmid construction

[0112] Using the *Corynebacterium glutamicum* QS08 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* QS11 (patent application number 2024113541837) genome as a template, glnA was amplified using primers PQ324-glnA-3F / PQ325-glnA-3R. Y405F (1447bp). Using the Corynebacterium glutamicum QS08 genome as a template, and with primers PQ326-DN-4F / PQ327-DN-4R, the downstream homologous arm DN (539bp) was amplified. Subsequently, using UP and Psod as templates, and with primers PQ320-UP-1F / PQ323-Psod-2R, fragments 1-2 (733bp) were fused and amplified. Y405F Using DN as a template and 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 PQ320-UP-1F / PQ327-DN-4R as primers, fragment 1-4 (2832bp) was amplified. Fragments 1-4 and pK18mobsacB were digested with XbaI and SalI at 37℃ for 1 hour, respectively. The fragment products were purified directly, and the vector was dephosphorylated with 3 μL of FastAP, incubated at 37℃ for 1 hour, and then recovered via gel extraction. 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.

[0113] b) Strain construction

[0114] Plasmids were electroporated into Corynebacterium glutamicum QS08 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. The plasmids were identified using PQ320-UP-1F / P85 and P82 / PQ327-DN-4R. A positive control plasmid and a negative control QS09 genome were used, with correct lengths of 3kb and 2.9kb 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 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, with a correct band length of 3kb. Amplification with these primers followed by sequencing revealed a correct strain, designated QS17.

[0115] c) Performance Verification

[0116] 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.

[0117] Table 14. Detection of glutamine content in Corynebacterium glutamicum Qs17

[0118] Strain QS17 is derived from strain QS08 by inserting Psod-glnA at the ΔglsA position. Y405F As shown in Table 14, the glutamine yield of the obtained strain QS17 increased from 12.4 g / L to 13.4 g / L, and the conversion rate increased by 1.1%.

[0119] Example 3: Construction and performance verification of QS08→QS18(ΔrosR) strain

[0120] a) Plasmid construction

[0121] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ445-UP-1F / PQ446-UP-1R. The downstream homologous arm (500 bp) was also amplified using primers PQ447-DN-2F / PQ448-DN-2R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h. The vector was then dephosphorylated with 3 μl of FastAP and incubated at 37°C for 1 h, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed at 37°C for 30 minutes. Colony PCR was then performed for verification. Correct transformants were inoculated into test tubes, plasmids were extracted, digested, and then sent for assays.

[0122] b) Strain construction

[0123] Plasmid QS08 was electroporated and plated on LBHISK15. One recombination was performed on LBK25S and LBK25. The phenotype was correct when the latter grew longer than the former. This was identified using PQ445-UP-1F / P85 and P82 / PQ448-DN-2R. A positive control plasmid and a negative control ATCC 14067 genome were used. One long and one short plasmid were correct. One plasmid from each of the first and third recombinations were selected and inoculated into antibiotic-free LB tubes overnight. The plasmids were diluted 10, 100, and 1000 times and plated on LBK25, LBK25S, and LBS plates. The plasmid that grew on the LBS plate was compared to LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ449-ID-F / PQ464-ID-R. The correct plasmid was 1280 bp long. Sequencing was then performed using this primer pair. The correct strain was designated QS18.

[0124] c) Performance Verification

[0125] 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.

[0126] Table 15. Detection of glutamine content in Corynebacterium glutamicum QS18

[0127] Strain QS18 was obtained by ΔrosR based on strain QS08. As shown in Table 15, the yield of glutamine in strain QS18 increased from 12.4 g / L to 13.1 g / L, and the conversion rate increased by 0.8%.

[0128] Example 4: QS17→QS19(Psod-glnA) Y405F Construction and performance validation of ΔrosR strains

[0129] a) Plasmid construction

[0130] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ445-UP-1F / PQ446-UP-1R. The downstream homologous arm (500 bp) was also amplified using primers PQ447-DN-2F / PQ448-DN-2R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h. The vector was then dephosphorylated with 3 μl of FastAP and incubated at 37°C for 1 h, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed at 37°C for 30 minutes. Colony PCR was then performed for verification. Correct transformants were inoculated into test tubes, plasmids were extracted, digested, and then sent for assays.

[0131] b) Strain construction

[0132] Plasmid QS17 was electroporated and plated on LBHISK15. One recombination was performed on LBK25S and LBK25. The phenotype was correct when the latter grew longer than the former. This was identified using PQ445-UP-1F / P85 and P82 / PQ448-DN-2R. A positive control plasmid and a negative control ATCC 14067 genome were used. One long and one short plasmid were correct. One plasmid from each of the one- and three-digit recombinations were selected and inoculated into antibiotic-free LB tubes overnight. The plasmids were diluted 10, 100, and 1000 times and plated on LBK25, LBK25S, and LBS plates. The plasmid that grew on the LBS plate was compared to LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ449-ID-F / PQ464-ID-R. The correct plasmid was 1280 bp long. Sequencing was then performed using this primer pair. The correct strain was designated QS19.

[0133] c) Performance Verification

[0134] 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.

[0135] Table 16. Detection of glutamine content in Corynebacterium glutamicum QS08, QS17, QS18, and QS19

[0136] Strain QS19 is based on strain QS08 with the insertion of Psod-glnA. Y405F Obtained from ΔrosR.

[0137] Combining embodiments 2, 3, and 4, QS08 single-point introduction of glnA Y405F The transformation rate of strain QS17 was increased by 1.1%, and the transformation rate of strain QS18 obtained from QS08 using ΔrosR was increased by 0.8%. glnA was also introduced into QS08. Y405F The acid production of ΔrosR increased from 12.4 g / L to 14.5 g / L, and the conversion rate increased by 2.4%, which was an unexpected effect (Table 18).

[0138] To further confirm glnA Y405F The combination of glnA and ΔrosR was effective in different glutamine-producing strains. Another glutamine-producing strain, o-QS01, was constructed, and the effects of introducing glnA were verified. Y405F ,ΔrosR,glnA Y405F Combined with ΔrosR.

[0139] Example 5: Construction and performance verification of Corynebacterium glutamicum ATCC 14067→o-QS01 starting strain

[0140] 5.1 Construction and performance verification of ATCC 14067→o-QS01(ΔglsA) strain

[0141] a) Plasmid construction

[0142] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (530 bp) was amplified using primers PQ01-UP-F / PQ02-UP-R. The downstream homologous arm DN (550 bp) was amplified using primers PQ03-DN-F / PQ04-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 for transformation. Colony PCR was then performed using primers P82 / P85 to verify the colony length (1.4 kb). Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0143] b) Strain construction

[0144] The plasmid was electroporated into Corynebacterium glutamicum ATCC 14067 and plated on LBHISK15 plates. It was then compared to LBK25S and LBK25 plates; the latter showed longer growth than the former, indicating a correct phenotype. Identification was performed using PQ01-UP-F / P85 and P82 / PQ04-DN-R. The positive control plasmid and negative control ATCC 14067 genome showed correct lengths of 1.3kb and 1.2kb, 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. The LBS plate showed growth compared to LBK25 and LB plates; the former showed no growth, while the latter showed growth, indicating a correct phenotype. The recombinant was then identified using primers PQ05-ID-F / PQ06-ID-R, showing a correct band length of 1.3kb. Amplification with these primers followed by sequencing revealed a correct strain, designated o-QS01.

[0145] c) Performance Verification

[0146] 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.

[0147] Table 17. Detection of glutamine content in Corynebacterium glutamicum o-QS01

[0148] The strain o-QS01 was obtained by inactivating glsA on the basis of Corynebacterium glutamicum ATCC14067. As shown in Table 17, the glutamine yield of the obtained strain o-QS01 increased from 0.4 g / L to 0.9 g / L, and the conversion rate increased by 0.55%.

[0149] Example 6: o-QS01→o-QS11(Psod-glnA) Y405F Strain construction and performance verification

[0150] a) Plasmid construction

[0151] Using the *Corynebacterium glutamicum* o-QS01 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* QS11 (patent application number 2024113541837) genome as a template, glnA was amplified using primers PQ324-glnA-3F / PQ325-glnA-3R. Y405F (1447bp). Using the Corynebacterium glutamicum o-QS01 genome as a template, and with primers PQ326-DN-4F / PQ327-DN-4R, the downstream homologous arm DN (539bp) was amplified. Subsequently, using UP and Psod as templates, and with primers PQ320-UP-1F / PQ323-Psod-2R, fragments 1-2 (733bp) were fused and amplified. Y405F Using DN as a template and 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 PQ320-UP-1F / PQ327-DN-4R as primers, fragment 1-4 (2832bp) was amplified. Fragments 1-4 and pK18mobsacB were digested with XbaI and SalI at 37℃ for 1 hour, respectively. The fragment products were purified directly, and the vector was dephosphorylated with 3 μL of FastAP, incubated at 37℃ for 1 hour, and then recovered via gel extraction. 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.

[0152] b) Strain construction

[0153] Plasmids were electroporated into *Corynebacterium glutamicum* o-QS01 and plated on LBHISK15 plates. A second plasmid was used to compare the plasmid growth on LBK25S and LBK25 plates; the latter showed a 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-QS01 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-QS11.

[0154] c) Performance Verification

[0155] 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.

[0156] Table 18. Detection of glutamine content in Corynebacterium glutamicum o-QS11

[0157] strain o-QS11 is based on strain o-QS01 with the insertion of glnA. Y405F As shown in Table 18, the glutamine yield of strain o-QS11 increased from 0.9 g / L to 1.4 g / L, and the conversion rate increased by 0.5%.

[0158] Example 7: Construction and performance verification of o-QS01→o-QS12(ΔrosR) strain

[0159] a) Plasmid construction

[0160] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ445-UP-1F / PQ446-UP-1R. The downstream homologous arm (500 bp) was also amplified using primers PQ447-DN-2F / PQ448-DN-2R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h. The vector was then dephosphorylated with 3 μl of FastAP and incubated at 37°C for 1 h, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed at 37°C for 30 minutes. Colony PCR was then performed for verification. Correct transformants were inoculated into test tubes, plasmids were extracted, digested, and then sent for assays.

[0161] b) Strain construction

[0162] Plasmid o-QS01 was electroporated and plated on LBHISK15. One recombination was performed on LBK25S and LBK25. The phenotype was correct when the latter grew longer than the former. This was identified using PQ445-UP-1F / P85 and P82 / PQ448-DN-2R. A positive control plasmid and a negative control ATCC 14067 genome were used. One long and one short plasmid were correct. One plasmid from each of the one- and three-digit recombinations were selected and inoculated into antibiotic-free LB tubes overnight. The plasmids were diluted 10, 100, and 1000 times and plated on LBK25, LBK25S, and LBS plates. The plasmid that grew on the LBS plate was plated on LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ449-ID-F / PQ464-ID-R. The correct plasmid was 1280 bp long. Sequencing was then performed using this primer pair. The correct strain was recorded as o-QS12.

[0163] c) Performance Verification

[0164] 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.

[0165] Table 19. Detection of glutamine content in Corynebacterium glutamicum o-QS12

[0166] The strain o-QS12 was obtained by ΔrosR based on the strain o-QS01. As shown in Table 19, the yield of glutamine in the obtained strain o-QS12 increased from 0.9 g / L to 1.6 g / L, and the conversion rate increased by 0.8%.

[0167] Example 8: 0-QS11→o-QS13(Psod-glnA) Y405F Construction and performance validation of ΔrosR strains

[0168] a) Plasmid construction

[0169] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ445-UP-1F / PQ446-UP-1R. The downstream homologous arm (500 bp) was also amplified using primers PQ447-DN-2F / PQ448-DN-2R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h. The vector was then dephosphorylated with 3 μl of FastAP and incubated at 37°C for 1 h, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed at 37°C for 30 minutes. Colony PCR was then performed for verification. Correct transformants were inoculated into test tubes, plasmids were extracted, digested, and then sent for assays.

[0170] b) Strain construction

[0171] Plasmid o-QS11 was electroporated and plated on LBHISK15. One recombination was performed on LBK25S and LBK25. The phenotype was correct when the latter grew longer than the former. This was identified using PQ445-UP-1F / P85 and P82 / PQ448-DN-2R. A positive control plasmid and a negative control ATCC 14067 genome were used. One long and one short plasmid were correct. One plasmid from each of the one- and three-digit recombinations were selected and inoculated into antibiotic-free LB tubes overnight. The plasmids were diluted 10, 100, and 1000 times and plated on LBK25, LBK25S, and LBS plates. The plasmid that grew on the LBS plate was plated on LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ449-ID-F / PQ464-ID-R. The correct plasmid was 1280 bp long. Sequencing was then performed using this primer pair. The correct strain was recorded as o-QS13.

[0172] c) Performance Verification

[0173] 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.

[0174] Table 20. Detection of glutamine content in Corynebacterium glutamicum o-QS01, o-QS11, o-QS12, and o-QS13

[0175] Strain o-QS13 is based on strain o-QS01 with its own glnA inserted. Y405F The glutamine yield obtained from the gene and ΔrosR increased from 0.9 g / L to 2.9 g / L, with a 2.2% improvement in conversion rate, and the performance improvement was better than the sum of o-QS11 and o-QS12.

[0176] In combination with embodiments 6, 7, and 8, glnA is introduced in o-QS01. Y405F Strains o-QS11 were obtained, with a 0.5% increase in transformation efficiency. Strains o-QS01 (ΔrosR) were used to obtain strain o-QS12, with a 0.8% increase in transformation efficiency. glnA was also introduced. Y405F The acid production of ΔrosR increased from 0.9 g / L to 2.9 g / L, resulting in a 2.2% increase in conversion rate, which was an unexpected effect (Table 20).

[0177] Examples 2, 3, 4, 6, 7, and 8 demonstrate that combining glnA with different starting bacteria... Y405F The combination of ΔrosR and ΔrosR has yielded unexpected results. Therefore, this combination has shown surprising effectiveness when applied to other glutamine-producing strains.

[0178] By incorporating references

[0179] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0180] Equivalence

[0181] This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

Modified bacteria that produce glutamine, whose genomes contain modifications that increase glutamine synthase (glnA) activity and decrease RosR activity compared to unmodified bacteria. The modified bacteria as described in claim 1, further comprising a modification that reduces glsA activity, preferably a deletion or partial deletion of the glsA gene. The modified bacteria as described in claim 1, wherein the modified bacteria produce glutamine in higher yields than the unmodified bacteria. The modified bacteria as described in claim 1, wherein the bacteria are Corynebacterium bacteria, preferably Corynebacterium glutamicum. The modified bacteria of claim 1, wherein the modification increasing glutamine synthase (glnA) activity is achieved by replacing the 405th amino acid of the glutamine synthase with phenylalanine (glnA). Y405F Preferably, the glnA is derived from Corynebacterium glutamicum or Saccharomyces cerevisiae. The modified bacteria as described in claim 1, wherein the modification that reduces RosR activity is a deletion or partial deletion of the nucleic acid sequence of the RosR gene. The modified bacteria as described in any one of claims 1 to 6, comprising one or more glnA Y40sr Gene copy. The modified bacteria according to any one of claims 1 to 7, wherein the promoter of the glutamine synthase (glnA) gene is the Psod promoter. The modified bacteria of claim 8, wherein the Psod promoter is derived from Corynebacterium glutamicum. Use of the modified bacteria as described in any one of claims 1 to 9 in the production of glutamine. A method for producing glutamine, comprising culturing modified bacteria as described in any one of claims 1 to 9 in a culture medium and isolating glutamine.

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