A method for producing glutamine

Abstract

Provided are modified bacteria that produce glutamine, wherein the genome has a heterologous polynucleotide for glutamine efflux protein (rarD) and a modification that attenuates expression of CEY17_10535, which can produce under anaerobic or aerobic conditions. Also provided are methods of using the modified bacteria to increase glutamine production.

Description

Technical Field

[0001] This disclosure pertains to the field of bacterial fermentation, and particularly relates to a method for producing glutamine using bacterial fermentation. Background Technology

[0002] Glutamine (2-amino-4-carbamoylbutyric acid) is a non-essential amino acid that encodes proteins. It promotes protein synthesis and inhibits protein breakdown. It can be used to treat gastric and duodenal ulcers and plays an important role in the pharmaceutical industry.

[0003] Currently, glutamine is mainly produced through fermentation using *Corynebacterium glutamicum*. *Corynebacterium glutamicum* is a heterotrophic aerobic bacterium, Gram-positive, characterized by rapid growth, non-pathogenicity, and weak degradation of its own metabolites. However, the fermentation performance of current 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, employing genetic engineering techniques to improve glutamine yield and conversion rates is particularly important.

[0004] Numerous reports have described methods to increase glutamine production. Mutations of serine at position 84 of inositol-3-phosphate synthase to other amino acids enhance glutamine production in *Corynebacterium glutamicum*. Mutations of amino acid position 184 of the CEY17_05975 protein to other amino acids improve the efficiency of microbial glutamine production. Mutations of threonine at position 65 of the CEY17_04535 protein to amino acids other than threonine significantly improve the strain's glutamine production capacity. Substitution of arginine at position 2916 of the CEY17_04555 protein with other amino acids improves the microbial production of L-glutamine. Point mutations in the DNA gyrase of *E. coli*, such as gyrA (Gly821Ser, Asp830Asn), reduce DNA supercoil structure, promoting glutamine accumulation (Tramquon, Susanna. "An Exploratory Study on the Development of an Implicit Measure of Implicit Followership Theory Using the Implicit Association Test." Dissertations & Theses-Gradworks). 79.9(2013):3033-3039.); The mutation of amino acid 405 of glnA from tyrosine (Y) to phenylalanine (F) can undo adenylation of glutamine synthase, effectively increasing glutamine production; Modification of glsA reduces intracellular glutaminase activity, effectively increasing glutamine production; Escherichia coli JM101 simultaneously inactivates glnE and glnB, increasing glutamine content from 0 to 620 mg / L; Modification enhances the activity of glutamine synthase, and further modification enhances the activity of intracellular glutamate dehydrogenase (gdh-encoded) in Corynebacterium bacteria, increasing L-glutamine production; The glutamine synthase encoding gene of Corynebacterium glutamicum itself can be inhibited by repressor proteins or transcriptional regulators, leading to a decrease in transcription level and a sharp decrease in enzyme activity, resulting in the inability to fully utilize the substrate glutamate.

[0005] Currently, these individual modifications have limited effect on increasing glutamine production. How to select these modification methods and integrate them into a single strain of bacteria to significantly increase glutamine production is a pressing technical problem that needs to be solved. Summary of the Invention

[0006] In order to select and integrate existing modification methods into a single chassis bacterium to obtain a glutamine-producing strain with an unexpectedly high yield and thus increase glutamine production, this invention introduces two genetic modifications into Corynebacterium glutamicum: the introduction of rarD and the reduction of CEY17_10535 operon expression. This resulted in a glutamine-producing strain with a significantly increased yield, which was unexpected.

[0007] On the one hand, this disclosure provides a modified bacterium that produces glutamine, wherein the genome contains a heterologous polynucleotide of glutamine efflux protein (rarD) and a modification that attenuates the expression of CEY17_10535.

[0008] In one specific implementation, the rarD gene is derived from Escherichia coli.

[0009] In one specific embodiment, the modified bacteria contains one or more copies of the glutamine efflux protein (rarD) gene, preferably two copies, more preferably three copies.

[0010] In one specific embodiment, the promoter of the glutamine efflux protein (rarD) gene is the Psod promoter.

[0011] In one specific implementation, the modification that weakens CEY17_10535 expression is one or more of the following: partial knockout, complete knockout, base substitution, frameshift mutation, etc. of the CEY17_10535 gene sequence, preferably partial knockout.

[0012] In one specific implementation, the nucleic acid sequence of the CEY17_10535 gene is shown in SEQ ID NO:21 or 23.

[0013] In one specific embodiment, the modified bacteria further comprises a modification that reduces rosR activity, preferably a deletion or partial deletion of the rosR gene nucleic acid sequence.

[0014] In one specific embodiment, the modified bacteria further comprises a modification that reduces glsA activity, preferably a deletion or partial deletion of the glsA gene, and / or a modification that increases glnA activity. Preferably, the modification that increases glnA activity is a glnA gene containing a Y405F substitution (glnA). Y405F More preferably, the glnA is derived from Corynebacterium glutamicum or Saccharomyces cerevisiae.

[0015] On the other hand, this disclosure provides the use of the modified bacteria as described above in increasing glutamine production.

[0016] On the other hand, this disclosure provides a method for producing glutamine, comprising culturing the modified bacteria as described above in a culture medium and isolating glutamine.

[0017] Beneficial effects

[0018] This invention uses two high-yield glutamine-producing bacteria as starting strains. Combining Psod-E.rarD and ΔCEY17_10535 with these strains further improves the glutamine conversion rate, demonstrating that combining Psod-E.rarD and ΔCEY17_10535 with different high-yield strains has unexpected effects.

[0019] 1. Glutamine-producing bacteria superimposed with Psod-E.rarD and ΔCEY17_10535

[0020] This disclosure first constructs a glutamine-producing bacterium, QS15, specifically using ATCC 14067 as the starting strain and introducing ino-1. S84A CEY17_06485 A386T CEY17_05975 V184I CEY17_04535 T65I CEY17_04555 R2916C CEY17_13360 A139T gyrA A466V ,ΔglsA,glnA Y405F , ΔCEY17_08220::Psod-E.rarD, ΔglsA::Psod-glnA Y405F The glutamine yield reached 24.3 g / L by adding ΔglsA::Psod-E.rarD and ΔrosR. To further improve the glutamine yield, three copies of Psod-E.rarD and ΔCEY17_10535 were added to obtain strain QS22, which increased the conversion rate by 1.9%. The combination of the two had an unexpected effect.

[0021] 2. Different glutamine-producing bacteria superimposed with Psod-E.rarD and ΔCEY17_10535

[0022] To further confirm the effectiveness of the Psod-E.rarD and ΔCEY17_10535 combination in different glutamine-producing strains, another glutamine-producing strain, o-QS08, was constructed based on the ATCC 13032 strain. The Psod-E.rarD and ΔCEY17_10535 combination was then added to obtain strain ATCC 13032ΔCEY17_10535Psod-E.rarD and o-QS16, which increased the conversion rate by 1.21% and 1.3%, respectively. The combination of the two strains also showed unexpected positive effects. Detailed Implementation

[0023] 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.

[0024] Table 1. Instruments used in this invention

[0025]

[0026]

[0027] Table 2. Reagents used in this invention

[0028] Reagent Name factory Item number peptone British Oxoid LP0042B NaCl Sinopharm Chemical Reagent Co., Ltd. 10019318 Yeast extract British Oxoid LP0021B Sorbitol Sangon Biotech Co., Ltd. A610491-0500 sucrose Sinopharm Chemical Reagent Co., Ltd. 10021418 Brain and heart immersion fluid British Oxoid CM1135B Agar Beijing Aoboxing Biotechnology Co., Ltd. 01-023 glucose Sinopharm Chemical Reagent Co., Ltd. 10010518 urea Sinopharm Chemical Reagent Co., Ltd. 10023218 <![CDATA[KH2PO4]]> Sinopharm Chemical Reagent Co., Ltd. 10017618 <![CDATA[MgSO4·7H2O]]> Sinopharm Chemical Reagent Co., Ltd. 10013018 Corn liquor dry powder Beijing Hongrun Baoshun Technology Co., Ltd. Y042T <![CDATA[(NH4)2SO4]]> Sinopharm Chemical Reagent Co., Ltd. 10002918 <![CDATA[CaCO3]]> Tianjin Damao Chemical Reagent Factory 1653

[0029] Table 3. Sequences

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041] Table 4. Primer Summary Table

[0042]

[0043]

[0044]

[0045]

[0046] Example

[0047] 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.

[0048] Example 1: Construction and performance verification of Corynebacterium glutamicum ATCC14067→QS01 strain

[0049] 1.1 Plasmid Construction

[0050] 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, T4 ligase was used to ligate and transform DH5α competent cells. Colony PCR was performed using primers P82 / P85 to verify the colony length, which was 1.4 kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0051] 1.2 Strain Construction

[0052] Plasmids were electroporated into Corynebacterium glutamicum ATCC 14067 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 PQ28-UP-F / P85 and P82 / PQ31-DN-R. Positive control plasmids and negative control ATCC 14067 genomes were used, 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 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 PQ32-id-f / PQ31-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 PQ33-ID-F / PQ34-ID-R and sequenced. The length was 1.4kb. The correct strain was recorded as QS01.

[0053] 1.3 Performance Verification

[0054] 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:

[0055] 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.

[0056] The culture medium formula is as follows:

[0057] 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.

[0058] 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.

[0059] 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.

[0060] BHI slant culture medium: brain heart extract 37g / L, agar 1.8%, sterilized at 121℃ and 0.1MPa for 20 minutes;

[0061] 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;

[0062] 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.

[0063] Table 5. Glutamine content detection in Corynebacterium 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 <![CDATA[ino-1 S84A ]]> 60.7 1.1 1.22 0.77

[0065] Strain QS01 is based on Corynebacterium glutamicum ATCC14067 with the introduction of ino-1. S84A As shown in Table 5, 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%.

[0066] Example 2: Construction and performance verification of QS01→QS02 strains

[0067] 2.1 Plasmid Construction

[0068] 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, T4 ligase was used to ligate and transform DH5α competent cells. Colony PCR was performed using primers P82 / P85 to verify the colony length, which was 1.3kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0069] 2.2 Strain Construction

[0070] 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. A positive control plasmid and a negative control ATCC14067 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. 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 PQ40-ID-F / PQ41-ID-R and sequenced. The length was 1.4kb. The correct strain was recorded as QS02.

[0071] 2.3 Performance Verification

[0072] 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 1.3.

[0073] Table 6. Detection of glutamine content in Corynebacterium glutamicum QS02

[0074]

[0075] Strain QS02 is based on strain QS01 with the introduction of CEY17_06485. A386TAs shown in Table 6, 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%.

[0076] Example 3: Construction and performance verification of QS02→QS03 strains

[0077] 3.1 Plasmid Construction

[0078] 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, T4 ligase was used to ligate and transform DH5α competent cells. Colony PCR was performed using primers P82 / P85 to verify the colony length, which was 1.3kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0079] 3.2 Strain Construction

[0080] Plasmids were 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. A positive control plasmid and a negative control ATCC14067 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 optimal annealing temperature was determined using PQ46-id-f / PQ45-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 PQ47-ID-F / PQ48-ID-R and sequenced. The length was 1.3kb. The correct strain was recorded as QS03.

[0081] 3.3 Performance Verification

[0082] 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 1.3.

[0083] Table 7. Detection of glutamine content in Corynebacterium glutamicum QS03

[0084]

[0085] Strain QS03 was developed by introducing CEY17_05975 into strain QS02. V184I As shown in Table 7, 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%.

[0086] Example 4: Construction and performance verification of QS03→QS04 strains

[0087] 4.1 Plasmid Construction

[0088] 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, T4 ligase was used to ligate and transform DH5α competent cells. Colony PCR was performed using primers P82 / P85 to verify the colony length, which was 1.3kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0089] 4.2 Strain Construction

[0090] 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. A positive control plasmid and a negative control ATCC14067 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 comparison of LBS plate growth with LBK25 and LB plates showed that the former did not grow, while the latter showed growth, indicating a correct phenotype. The optimal annealing temperature was determined using PQ55-id-f / PQ52-DN-R. A positive control plasmid and a negative control *Corynebacterium glutamicum* ATCC 14067 genome were used, with 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.

[0091] 4.3 Performance Verification

[0092] 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 1.3.

[0093] Table 8. Detection of glutamine content in Corynebacterium glutamicum QS04

[0094]

[0095] Strain QS04 is based on strain QS03 with the introduction of CEY17_04535. T65I As shown in Table 8, 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%.

[0096] Example 5: Construction and performance verification of QS04→QS05 strain

[0097] 5.1 Plasmid Construction

[0098] 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, T4 ligase was used to ligate and transform DH5α competent cells. Colony PCR was performed using primers P82 / P85 to verify the colony length, which was 1.3kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0099] 5.2 Strain Construction

[0100] 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 ATCC14067 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.

[0101] 5.3 Performance Verification

[0102] 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 1.3.

[0103] Table 9. Glutamine content detection of Corynebacterium glutamicum QS05

[0104]

[0105] Strain QS05 is based on strain QS04 with the introduction of CEY17_04555.R2916C As shown in Table 9, the amino acid mutations obtained by strain QS05 increased the glutamine yield from 3 g / L to 3.8 g / L, and the conversion rate increased by 0.88%.

[0106] Example 6: Construction and performance verification of QS05→QS06 strains

[0107] 6.1 Plasmid Construction

[0108] 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.

[0109] 6.2 Strain Construction

[0110] 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 ATCC14067 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 optimal 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 using primers PQ68-ID-F / PQ69-ID-R and sequenced. The length was 1.3kb, and the correct strain was recorded as QS06.

[0111] 6.3 Performance Verification

[0112] 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 1.3.

[0113] Table 10. Detection of glutamine content in Corynebacterium glutamicum QS06

[0114]

[0115] Strain QS06 was obtained by introducing the CEY17_13360A139T amino acid mutation into strain QS05. As shown in Table 10, the glutamine yield of strain QS6 increased from 3.8 g / L to 5.2 g / L, and the conversion rate increased by 1.54%.

[0116] Example 7: Construction and performance verification of QS06→QS07 strains

[0117] 7.1 Plasmid Construction

[0118] 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.

[0119] 7.2 Strain Construction

[0120] 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. Positive control plasmids and negative control ATCC14067 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 showed 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.

[0121] 7.3 Performance Verification

[0122] 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 1.3.

[0123] Table 11. Detection of glutamine content in Corynebacterium glutamicum QS07

[0124]

[0125] Strain QS07 is based on strain QS06 with the introduction of gyrA. A466V As shown in Table 11, the glutamine yield of strain QS07 obtained by amino acid mutation increased from 5.2 g / L to 8 g / L, and the conversion rate increased by 3.08%.

[0126] Example 8: Construction and performance verification of QS07→QS08 strains

[0127] 8.1 Plasmid Construction

[0128] 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.

[0129] 8.2 Strain Construction

[0130] 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 negative control (ATCC14067 genome). The correct lengths were 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, showing a correct band length of 1.3kb. Amplification with these primers followed by sequencing revealed a correct strain, designated QS08.

[0131] 8.3 Performance Verification

[0132] 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 1.3.

[0133] Table 12. Detection of glutamine content in Corynebacterium glutamicum QS08

[0134]

[0135] Strain QS08 was obtained by inactivating glsA based on strain QS07. As shown in Table 12, 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%.

[0136] Example 9: Construction and performance verification of QS08→QS09 strains

[0137] 9.1 Plasmid Construction

[0138] 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.

[0139] 9.2 Strain Construction

[0140] Plasmids were electroporated into Corynebacterium glutamicum QS08 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, with a positive control plasmid and a negative control ATCC14067 genome, showing 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 LBS plates, comparing growth on LBK25 and LB plates; the former did not grow, while the latter showed a correct phenotype. The appropriate annealing temperature was determined using PQ13-id-f / PQ10-DN-R. The positive control plasmid and the negative control Corynebacterium glutamicum ATCC 14067 genome were used, with a band length of 551 bp. Colony PCR was performed at this annealing temperature for identification. The correct secondary recombinant was amplified with primers PQ11-ID-F / PQ12-ID-R and sequenced, with a length of 1.1 kb. The correct strain was recorded as QS09.

[0141] 9.3 Performance Verification

[0142] 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 1.3.

[0143] Table 13. Detection of glutamine content in Corynebacterium glutamicum QS09

[0144]

[0145] Strain QS09 is based on strain QS08 with the introduction of glnA. Y405F As shown in Table 13, the amino acid mutations resulted in an increase in glutamine production from 12.4 g / L to 19.2 g / L for strain QS09, representing a 7.5% increase in conversion rate.

[0146] Example 10: Construction and performance verification of QS09→QS10 strains

[0147] 10.1 Plasmid Construction

[0148] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (563 bp) was amplified using primers PQ265-UP-1f / PQ266-UP-1r. Using the *Corynebacterium glutamicum* ATCC 13032 genome as a template, the promoter Psod (215 bp) was amplified using primers PQ267-Psod-2f / PQ279-Psod-2r. Using the *E. coli* MG1655 genome as a template, *E. rarD* (913 bp) was amplified using primers PQ280-E.rarD-3f / PQ281-E.rarD-3r. Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the downstream homologous arm DN (536 bp) was amplified using primers PQ282-DN-4f / PQ272-DN-4r. Subsequently, using UP and Psod as templates and PQ265-UP-1f / PQ279-Psod-2r as primers, fragments 1-2 (751 bp) were fused and amplified. Using E. rarD and DN as templates and PQ280-E. rarD-3f / PQ272-DN-4r as primers, fragments 3-4 (1427 bp) were fused and amplified. Finally, using fragments 1-2 and 3-4 as templates and PQ265-UP-1f / PQ272-DN-4r as primers, fragments 1-4 (2157 bp) were fused and amplified. Finally, fragments 1-4 and pK18mobsacB were digested with EcoRI and NheI at 37°C for 1 hour. The fragment products were directly purified, the vector was dephosphorylated with 3 μL of FastAP, incubated at 37°C for 1 hour, and then recovered via gel extraction. Subsequently, T4 ligase was used to ligate and transform DH5α competent cells. Colony PCR was performed using primers P82 / P85 to verify the colony length, which was 2.3 kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0149] 10.2 Strain Construction

[0150] Plasmids were electroporated into Corynebacterium glutamicum QS09 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 PQ265-UP-1f / P85 and P82 / PQ272-DN-4r, serving as a positive control and an ATCC 14067 genome as a negative control, with correct lengths of 2.3kb 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 PQ273-ID-F / PQ274-ID-R, with a correct band length of 2.3kb. The band was then amplified using the same primers and sequenced. The correct strain was designated QS10.

[0151] 10.3 Performance Verification

[0152] 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 1.3.

[0153] Table 14. Detection of glutamine content in Corynebacterium glutamicum QS10

[0154]

[0155] Strain QS10 was obtained by inserting a single copy of the E. rarD gene from E. coli MG1655 into strain QS09. As shown in Table 14, the glutamine yield of strain QS10 increased from 19.2 g / L to 19.8 g / L, and the conversion rate increased by 0.7%.

[0156] Example 11: Construction and performance verification of QS10→QS12 strain

[0157] 11.1 Plasmid Construction

[0158] Using the *Corynebacterium glutamicum* QS09 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* QS09 genome as a template, glnAY405F (1447 bp) was amplified using primers PQ324-glnA-3F / PQ325-glnA-3R. Using the *Corynebacterium glutamicum* QS09 genome as a template, the downstream homologous arm DN (539 bp) was amplified using primers PQ326-DN-4F / PQ327-DN-4R. Subsequently, using UP and Psod as templates and PQ320-UP-1F / PQ323-Psod-2R as primers, fragments 1-2 (733bp) were amplified through fusion. Fragments 3-4 (1956bp) were amplified using glnAY405F and DN as templates and PQ324-glnA-3F / PQ327-DN-4R as primers. 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℃ for 1 hour. The fragment products were directly purified, the vector was dephosphorylated with 3 μL of FastAP, incubated at 37℃ for 1 hour, and then recovered via gel extraction. Subsequently, T4 ligase was used to ligate and transform DH5α competent cells. 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 testing.

[0159] 11.2 Strain Construction

[0160] Plasmids were electroporated into *Corynebacterium glutamicum* QS10 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. The band was then amplified using the same primers and sequenced. The correct strain was designated QS12.

[0161] 11.3 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 1.3.

[0163] Table 15. Detection of glutamine content in Corynebacterium glutamicum QS12

[0164]

[0165] Strain QS12 is based on strain QS10 with the insertion of ΔglsA::Psod-glnA. Y405F Obtained through two copies. As shown in Table 15, the QS12 conversion rate increased by 1.9%.

[0166] Example 12: Construction and performance verification of QS12→QS14 strain

[0167] 12.1 Plasmid Construction

[0168] Using the *Corynebacterium glutamicum* QS12 genome as a template, the upstream homologous arm (494 bp) was amplified using primers PQ474-UP-1F / PQ475-UP-1R. Using the *Corynebacterium glutamicum* QS12 genome as a template, the fragment Psod-E.rarD (1083 bp) was amplified using primers PQ468-Psod-2F / PQ476-E.rarD-2R. Using the *Corynebacterium glutamicum* QS12 genome as a template, the downstream homologous arm (527 bp) was amplified using primers PQ477-DN-3F / PQ478-DN-3R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated with 3 μl of FastAP, incubated at 37°C for 1 h, and then recovered from the gel. The digested vector, UP, Psod-E.rarD, and DN were seamlessly assembled at 37°C for half an hour, transformed into DH5α competent cells, and colony PCR was performed using primers P82 / P85 to verify the colony length, which was 2.4 kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0169] 12.2 Strain Construction

[0170] The plasmid QS12 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 PQ474-UP-1F / P85 and P82 / PQ478-DN-3R. A positive control plasmid and a negative control QS12 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 the plasmids on LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ479-ID-F / PQ480-ID-R. The correct plasmid was 2.3 kb long. Sequencing was then performed using this primer pair. The correct strain was designated QS14.

[0171] 12.3 Performance Verification

[0172] 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 1.3.

[0173] Table 16. Detection of glutamine content in Corynebacterium glutamicum QS14

[0174]

[0175] The strain QS14 was obtained by inserting two copies of ΔglsA::Psod-E.rarD into the base of Corynebacterium glutamicum QS12, as shown in Table 16. The yield of glutamine obtained by QS14 increased from 21.5 g / L to 21.8 g / L, and the conversion rate increased by 0.4%.

[0176] Example 13: Construction and performance verification of QS14→QS15 strains

[0177] 13.1 Plasmid Construction

[0178] 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 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 into DH5α competent cells at 37°C for 30 minutes. Colony PCR was performed using primers P82 / P85, and the transformed cells were 1.3 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0179] 13.2 Strain Construction

[0180] Plasmid QS14 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 1.2 kb long. Sequencing was then performed using the same primer pair. The correct strain was designated QS15.

[0181] 13.3 Performance Verification

[0182] 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 1.3.

[0183] Table 17. Detection of glutamine content in Corynebacterium glutamicum QS15

[0184]

[0185] Strain QS15 was obtained by introducing ΔrosR into Corynebacterium glutamicum QS14. As shown in Table 17, the yield of glutamine obtained by QS17 increased from 21.8 g / L to 24.3 g / L, and the conversion rate increased by 2.7%.

[0186] Example 14: Using QS15 as the starting strain, Psod-rarD(QS15→QS20) was superimposed.

[0187] 14.1 Plasmid Construction

[0188] Using the *Corynebacterium glutamicum* QS15 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ445-UP-1F / PQ527-UP-1R. Using the *Corynebacterium glutamicum* QS15 genome as a template, the fragment Psod-E.rarD (1083 bp) was amplified using primers PQ468-Psod-2F / PQ476-E.rarD-2R. Using the *Corynebacterium glutamicum* QS15 genome as a template, the downstream homologous arm (500 bp) was amplified using primers PQ528-DN-3F / PQ448-DN-2R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated with 3 μl of FastAP, incubated at 37°C for 1 h, and then recovered from the gel. The digested vector, UP, Psod-E.rarD, and DN were seamlessly assembled at 37°C for half an hour, transformed into DH5α competent cells, and colony PCR was performed using primers P82 / P85 to verify the colony length, which was 2.4 kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0189] 14.2 Strain Construction

[0190] The plasmid QS15 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 QS15 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 the plasmid 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 2.3 kb long. Sequencing was then performed using the same primer pair. The correct strain was designated QS20.

[0191] 14.3 Performance Verification

[0192] 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 1.3.

[0193] Table 18. Detection of glutamine content in Corynebacterium glutamicum QS20

[0194]

[0195] The strain QS20 was obtained by inserting three copies of Psod-E.rarD into the base of Corynebacterium glutamicum QS15. As shown in Table 18, the yield of glutamine obtained by QS20 increased from 24.3 g / L to 25 g / L, and the conversion rate increased by 0.8%.

[0196] Example 15: Using QS15 as the starting strain, ΔCEY17_10535 (QS15→QS21) was superimposed.

[0197] 15.1 Plasmid Construction

[0198] Using the *Corynebacterium glutamicum* QS15 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ457-UP-F / PQ458-UP-R. The downstream homologous arm (500 bp) was also amplified using primers PQ459-DN-F / PQ460-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, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed into DH5α competent cells at 37°C for 30 minutes. Colony PCR was performed using primers P82 / P85, and the transformed cells were 1.3 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0199] 15.2 Strain Construction

[0200] The plasmid QS15 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 PQ457-UP-F / P85 and P82 / PQ460-DN-R. A positive control plasmid and a negative control QS15 genome were used. One long and one short genome 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 the plasmid on LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ461-ID-F / PQ462-ID-R. The correct plasmid was 1.3 kb long. Sequencing was then performed using this primer pair. The correct strain was designated QS21.

[0201] 15.3 Performance Verification

[0202] 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 1.3.

[0203] Table 19. Detection of glutamine content in Corynebacterium glutamicum QS21

[0204]

[0205] The strain QS21 was obtained by introducing ΔCEY17_10535 into Corynebacterium glutamicum QS15. As shown in Table 19, the glutamine yield of the new strain QS21 increased from 24.3 g / L to 24.8 g / L, and the conversion rate increased by 0.5%.

[0206] Example 16: Using QS15 as the starting strain, Psod-rarD and ΔCEY17_10535 (QS15→QS22) were superimposed.

[0207] 16.1 Plasmid Construction

[0208] Using the *Corynebacterium glutamicum* QS15 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ457-UP-F / PQ458-UP-R. The downstream homologous arm (500 bp) was also amplified using primers PQ459-DN-F / PQ460-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, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed into DH5α competent cells at 37°C for 30 minutes. Colony PCR was performed using primers P82 / P85, and the transformed cells were 1.3 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0209] 16.2 Strain Construction

[0210] The plasmid QS20 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 PQ457-UP-F / P85 and P82 / PQ460-DN-R. A positive control plasmid and a negative control QS20 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 the plasmids on LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ461-ID-F / PQ462-ID-R. The correct plasmid was 1.3 kb long. Sequencing was then performed using this primer pair. The correct strain was designated QS22.

[0211] 16.3 Performance Verification

[0212] 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 1.3.

[0213] Table 20. Detection of glutamine content in Corynebacterium glutamicum QS22

[0214]

[0215] Based on strain QS20, strain QS22 was obtained by modifying ΔCEY17_10535, which is a combination strain of Psod-E.rarD*3 and ΔCEY17_10535. Combining Examples 14, 15, and 16, the conversion rate of Psod-E.rarD*3 increased by 0.8% when added alone, and the conversion rate of ΔCEY17_10535 increased by 0.5% when added alone. The conversion rate of the combination of ΔrosR::Psod-E.rarD*3 and ΔCEY17_10535 increased by 1.9%. The combination of the two has unexpected effects, as shown in Table 20.

[0216] To confirm that the combination of Psod-E.rarD and ΔCEY17_10535 is effective in different starting strains, strain o-QS08 was constructed, and a strain capable of producing glutamine was obtained. Based on this, single-point verification of Psod-E.rarD and ΔCEY17_10535, as well as verification of the combination of Psod-E.rarD and ΔCEY17_10535, were performed.

[0217] Example 17: Construction and performance verification of Corynebacterium glutamicum ATCC 14067→o-QS01 strain

[0218] 17.1 Plasmid Construction

[0219] 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, then transformed into DH5α competent cells. 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.

[0220] 17.2 Strain Construction

[0221] 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.

[0222] 17.3 Performance Verification

[0223] 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 1.3.

[0224] Table 21. Detection of glutamine content in Corynebacterium glutamicum o-QS01

[0225]

[0226] The strain o-QS01 was obtained by inactivating glsA on the basis of Corynebacterium glutamicum ATCC14067. As shown in Table 21, the glutamine yield of the new strain o-QS01 increased from 0.4 g / L to 0.9 g / L, and the conversion rate increased by 0.55%.

[0227] Example 18: Construction and performance verification of o-QS01→o-QS02 strain

[0228] 18.1 Plasmid Construction

[0229] 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, then transformed into DH5α competent cells. 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.

[0230] 18.2 Strain Construction

[0231] 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.

[0232] 18.3 Performance Verification

[0233] 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 1.3.

[0234] Table 22. Detection of glutamine content in Corynebacterium glutamicum o-QS02

[0235]

[0236] Strain o-QS02 is based on strain o-QS01 with the introduction of glnA. Y405FAs shown in Table 22, the new strain o-QS02 obtained by amino acid mutation increased the glutamine yield from 0.9 g / L to 1.1 g / L, and the conversion rate increased by 0.21%.

[0237] Example 19: Construction and performance verification of o-QS02→o-QS03 strain

[0238] 19.1 Plasmid Construction

[0239] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (536 bp) was amplified using primers PQ14-UP-F / PQ15-UP-R. The downstream homologous arm DN (536 bp) was amplified using primers PQ16-DN-F / PQ17-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, then transformed into DH5α competent cells. Colony PCR was then performed using primers P82 / P85. The transformed cells were 1.3 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0240] 19.2 Strain Construction

[0241] The plasmid was electroporated into *Corynebacterium glutamicum* o-QS02 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 PQ14-UP-F / P85 and P82 / PQ17-DN-R. A positive control plasmid and a negative control ATCC 14067 genome were used, with correct lengths of 1.1kb and 1.6kb respectively. The recombinant was inoculated into LB tubes overnight and diluted 10, 100, and 1000 times, then plated on LBK25, LBK25S, and LBS plates. Growth on the LBS plate was compared to growth on 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 PQ18-ID-F / PQ19-ID-R, with a correct band length of 1.2kb. Amplification with these primers followed by sequencing revealed a correct strain, designated o-QS03.

[0242] 19.3 Performance Verification

[0243] 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 1.3.

[0244] Table 23. Detection of glutamine content in Corynebacterium glutamicum o-QS03

[0245]

[0246]

[0247] The strain o-QS03 was obtained by inactivating glnE on the basis of strain o-QS02, as shown in Table 23. The glutamine yield of the obtained o-QS03 increased from 1.1 g / L to 1.7 g / L, and the conversion rate increased by 0.67%.

[0248] Example 20: Construction and performance verification of o-QS03→o-QS04 strain

[0249] 20.1 Plasmid Construction

[0250] Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the upstream homologous arm UP (520 bp) was amplified using primers PQ20-UP-F / PQ21-UP-R. Using the *Corynebacterium glutamicum* ATCC 13032 genome as a template, the promoter Psod (192 bp) was amplified using primers PQ22-Psod-F / PQ23-Psod-R. Using the *Corynebacterium glutamicum* ATCC 14067 genome as a template, the downstream homologous arm DN (526 bp) was amplified using primers PQ24-DN-F / PQ25-DN-R. Using the UP and Psod fragments as templates, the UP-Psod (692 bp) fragment was fused and amplified using primers PQ20-UP-F / PQ23-Psod-R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 hour. The vector was then dephosphorylated with 3 μL of FastAP and incubated at 37°C for 1 hour. The vector was recovered from the gel. The digested vector, UP-Psod, and DN were seamlessly assembled and reacted at 37°C for half an hour, then transformed into DH5α competent cells. Colony PCR was subsequently performed using primers P82 / P85. The transformed cells were 1.5 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0251] 20.2 Strain Construction

[0252] Plasmids were electroporated into *Corynebacterium glutamicum* o-QS03 and plated on LBHISK15 plates. A second plasmid was used to compare the plasmid lengths on LBK25S and LBK25 plates; the latter showed longer growth than the former, indicating a correct phenotype. The plasmids were identified using PQ20-UP-F / P85 and P82 / PQ25-DN-R, with positive control plasmids and negative control ATCC 14067 genomes showing correct lengths of 1.4 kb and 1.1 kb, 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 plasmids that grew on LBS plates were compared to those on 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 PQ26-ID-F / PQ27-ID-R, showing a correct band length of 1.4 kb. The band was then amplified using these primers and sequenced; the correct strain was designated o-QS04.

[0253] 20.3 Performance Verification

[0254] 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 1.3.

[0255] Table 24. Detection of glutamine content in Corynebacterium glutamicum o-QS04

[0256]

[0257] The o-QS04 strain was obtained by inserting the Psod promoter before the gdh gene on the basis of the o-QS03 strain. As shown in Table 24, the glutamine yield of the obtained o-QS04 increased from 1.7 g / L to 2.4 g / L, and the conversion rate increased by 0.72%.

[0258] Example 21: Construction and performance verification of o-QS04→o-QS08 strain

[0259] 21.1 Plasmid Construction

[0260] 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 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 into DH5α competent cells at 37°C for 30 minutes. Colony PCR was performed using primers P82 / P85, and the transformed cells were 1.3 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0261] 21.2 Strain Construction

[0262] Plasmid o-QS04 was electroporated and plated on LBHISK15. One recombination pair was used on LBK25S and LBK25. The phenotype was correct when the latter was 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 for 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 paired with LBK25 and LB plates. The phenotype was correct when the former grew but the latter did not. This was identified using PQ449-ID-F / PQ464-ID-R. The correct length was 1.2 kb. Sequencing was then performed using this primer pair. The correct strain was recorded as o-QS08.

[0263] 21.3 Performance Verification

[0264] 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 1.3.

[0265] Table 25. Detection of glutamine content in Corynebacterium glutamicum o-QS08

[0266]

[0267] The strain o-QS08 was obtained by inactivating rosR based on the strain o-QS04. As shown in Table 25, the glutamine yield of the obtained o-QS08 increased from 2.4 g / L to 3.0 g / L, and the conversion rate increased by 0.7%.

[0268] Example 22: Using o-QS08 as the starting bacterium, Psod-rarD(o-QS08→o-QS14) was superimposed.

[0269] 22.1 Plasmid Construction

[0270] Using the *Corynebacterium glutamicum* o-QS08 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ445-UP-1F / PQ527-UP-1R. Using the *Corynebacterium glutamicum* o-QS08 genome as a template, the fragment Psod-E.rarD (1083 bp) was amplified using primers PQ468-Psod-2F / PQ476-E.rarD-2R. Using the *Corynebacterium glutamicum* o-QS08 genome as a template, the downstream homologous arm (500 bp) was amplified using primers PQ528-DN-3F / PQ448-DN-2R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated with 3 μl of FastAP, incubated at 37°C for 1 h, and then recovered from the gel. The digested vector, UP, Psod-E.rarD, and DN were seamlessly assembled at 37°C for half an hour, transformed into DH5α competent cells, and colony PCR was performed using primers P82 / P85 to verify the colony length, which was 2.4 kb. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for testing.

[0271] 22.2 Strain Construction

[0272] Plasmid o-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 o-QS08 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 the plasmids 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 2.3 kb long. Sequencing was then performed using the same primer pair. The correct strain was recorded as o-QS14.

[0273] 22.3 Performance Verification

[0274] 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 1.3.

[0275] Table 26. Detection of glutamine content in Corynebacterium glutamicum o-QS14

[0276]

[0277]

[0278] The strain o-QS14 was obtained by inserting Psod-rarD into the base of Corynebacterium glutamicum o-QS08, as shown in Table 26. The glutamine yield of the obtained o-QS14 increased from 3 g / L to 3.6 g / L, and the conversion rate increased by 0.6%.

[0279] Example 23: Starting with o-QS08, ΔCEY17_10535 (o-QS08→o-QS17) was superimposed.

[0280] 23.1 Plasmid Construction

[0281] Using the *Corynebacterium glutamicum* o-QS08 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ457-UP-F / PQ458-UP-R. The downstream homologous arm (500 bp) was also amplified using primers PQ459-DN-F / PQ460-DN-R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h. The vector was dephosphorylated with 3 μl FastAP and incubated at 37°C for 1 h, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed into DH5α competent cells at 37°C for half an hour. Colony PCR was performed using primers P82 / P85, and the transformed cells were 1.3 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0282] 23.2 Strain Construction

[0283] Plasmid o-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 PQ457-UP-F / P85 and P82 / PQ460-DN-R. A positive control plasmid and a negative control o-QS08 genome were used. One long and one short genome 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 the plasmids on LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ461-ID-F / PQ462-ID-R. The correct plasmid was 1.3 kb long. Sequencing was then performed using the same primer pair. The correct strain was recorded as o-QS17.

[0284] 23.3 Performance Verification

[0285] 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 1.3.

[0286] Table 27. Detection of glutamine content in Corynebacterium glutamicum o-QS17

[0287]

[0288] The strain o-QS17 was obtained by introducing ΔCEY17_10535 into Corynebacterium glutamicum o-QS08. As shown in Table 27, the glutamine yield of the obtained o-QS17 increased from 3 g / L to 3.3 g / L, and the conversion rate increased by 0.3%.

[0289] Example 24: Starting with o-QS08, Psod-rarD and ΔCEY17_10535 (o-QS08→o-QS16) were superimposed.

[0290] 24.1 Plasmid Construction

[0291] Using the *Corynebacterium glutamicum* o-QS14 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ457-UP-F / PQ458-UP-R. The downstream homologous arm (500 bp) was also amplified using primers PQ459-DN-F / PQ460-DN-R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h. The vector was dephosphorylated with 3 μl FastAP and incubated at 37°C for 1 h, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed into DH5α competent cells at 37°C for 30 minutes. Colony PCR was performed using primers P82 / P85, and the transformed cells were 1.3 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0292] 24.2 Strain Construction

[0293] Plasmid o-QS14 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 PQ457-UP-F / P85 and P82 / PQ460-DN-R. A positive control plasmid and a negative control o-QS14 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 the plasmids on LBK25 and LB plates. The phenotype was correct when the former grew longer than the latter. This was identified using PQ461-ID-F / PQ462-ID-R. The correct plasmid was 1.3 kb long. Sequencing was then performed using this primer pair. The correct strain was recorded as o-QS016.

[0294] 24.3 Performance Verification

[0295] 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 1.3.

[0296] Table 28. Detection of glutamine content in Corynebacterium glutamicum o-QS16

[0297]

[0298] Based on strain o-QS14, strain o-QS16 was obtained by ΔCEY17_10535, which is a combination strain of ΔrosR::Psod-E.rarD and ΔCEY17_10535. Combining Examples 22, 23, and 24, the conversion rate of Psod-E.rarD at a single point increased by 0.6%, the conversion rate of ΔCEY17_10535 at a single point increased by 0.3%, and the conversion rate of the combination of ΔrosR::Psod-E.rarD and ΔCEY17_10535 increased by 1.3%. The combination of the two has unexpected effects, as shown in Table 28.

[0299] Example 25: Using ATCC 13032 as the starting strain, CEY17_10535 was inactivated (ATCC 13032ΔCEY17_10535).

[0300] To further illustrate that the above combination is unrelated to the originating bacterium, we used Corynebacterium glutamicum ATCC 13032 as the originating bacterium. The Cgl2147 gene (GenBank: BAB99540.1) showed 98.86% homology with the CEY17_10535 base sequence of Corynebacterium glutamicum ATCC 14067. For clarity, this invention will be referred to as CEY17_10535 below. Based on Corynebacterium glutamicum ATCC 13032, CEY17_10535 was inactivated and / or heterologously expressed with rarD derived from Escherichia coli.

[0301] 25.1 Plasmid Construction

[0302] Using the *Corynebacterium glutamicum* ATCC 13032 genome as a template, the upstream homologous arm (500 bp) was amplified using primers PQ457-UP-F / PQ458-UP-R. The downstream homologous arm (500 bp) was also amplified using primers PQ459-DN-F / PQ460-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, followed by gel recovery. The digested vector, UP, and DN were seamlessly assembled and transformed into DH5α competent cells at 37°C for half an hour. Colony PCR was performed using primers P82 / P85, and the transformed cells were 1.3 kb in length. Correct transformants were inoculated into LBK50 tubes, and plasmids were extracted and sent for assays.

[0303] 25.2 Strain Construction

[0304] The plasmid was electroporated with ATCC 13032 and plated on LBHISK15. One recombination was performed on LBK25S and LBK25. The phenotype was correct when the latter grew longer than the former. The plasmids were identified using PQ457-UP-F / P85 and P82 / PQ460-DN-R. A positive control plasmid and a negative control ATCC 13032 genome were used. One plasmid was longer than the former, and both were correct. One plasmid was selected from each of the one- and three-digit recombinations 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 plasmids that grew on LBS plates were compared with those that grew on LBK25 and LB plates. The plasmids that grew longer than the former, and both were correct. The phenotype was correct and identified using PQ461-ID-F / PQ462-ID-R. The correct plasmid was 1.3 kb long. Sequencing was then performed using the same primer pair. The correct strain was named ATCC13032ΔCEY17_10535.

[0305] Example 26: Using ATCC 13032 as the starting strain, Psod-rarD (ATCC 13032Psod-E.rarD) was superimposed.

[0306] 26.1 Plasmid Construction

[0307] Using the *Corynebacterium glutamicum* ATCC 13032 genome as a template, the upstream homologous arm (744 bp) was amplified using primers PQ77-UP-1F / PQ78-UP-1R. Using the *Corynebacterium glutamicum* QS15 genome as a template, the fragment Psod-E.rarD (1083 bp) was amplified using primers PQ468-Psod-2F / PQ476-E.rarD-2R. Using the *Corynebacterium glutamicum* ATCC 13032 genome as a template, the downstream homologous arm (500 bp) was amplified using primers PQ79-DN-3F / PQ80-DN-R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated with 3 μl of FastAP, incubated at 37°C for 1 h, and then recovered from the gel. The digested vector, UP, Psod-E.rarD, and DN were seamlessly assembled at 37°C for half an hour, transformed into DH5α competent cells, and colony PCR was performed using primers P82 / P85 to verify the colony length, which was 2.5 kb. Correct transformants were inoculated into LBK50 test tubes, and plasmids were extracted and sent for testing.

[0308] 26.2 Strain Construction

[0309] The plasmid was electroporated onto ATCC 13032 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 13032 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 the plasmids on LBK25 and LB plates; the phenotype was correct when the former grew longer than the latter. This was identified using PQ81-ID-F / PQ82-ID-R. The correct plasmid was 2.5 kb long. Sequencing was then performed using this primer pair. The correct strain was named ATCC13032Psod-E.rarD.

[0310] Example 27: Using ATCC 13032 as the starting strain, Psod-rarD and ΔCEY17_10535 (ATCC 13032ΔCEY17_10535Psod-E.rarD) were superimposed.

[0311] 27.1 Plasmid Construction

[0312] Using the *Corynebacterium glutamicum* ATCC 13032 genome as a template, the upstream homologous arm (744 bp) was amplified using primers PQ77-UP-1F / PQ78-UP-1R. Using the *Corynebacterium glutamicum* QS15 genome as a template, the fragment Psod-E.rarD (1083 bp) was amplified using primers PQ468-Psod-2F / PQ476-E.rarD-2R. Using the *Corynebacterium glutamicum* ATCC 13032 genome as a template, the downstream homologous arm (500 bp) was amplified using primers PQ79-DN-3F / PQ80-DN-R. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated with 3 μl of FastAP, incubated at 37°C for 1 h, and then recovered from the gel. The digested vector, UP, Psod-E.rarD, and DN were seamlessly assembled at 37°C for half an hour, transformed into DH5α competent cells, and colony PCR was performed using primers P82 / P85 to verify the colony length, which was 2.5 kb. Correct transformants were inoculated into LBK50 test tubes, and plasmids were extracted and sent for testing.

[0313] 27.2 Strain Construction

[0314] The plasmid ATCC 13032ΔCEY17_10535 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 13032 genome were used; one long and one short 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 the plasmids on LBK25 and LB plates; the phenotype was correct when the former grew longer than the latter. This was identified using PQ81-ID-F / PQ82-ID-R. The correct plasmid was 2.5 kb long. Sequencing was then performed using the same primer pair. The correct strain was named ATCC. 13032ΔCEY17_10535Psod-E.rarD.

[0315] 27.3 Performance Verification

[0316] Table 29. Glutamine Content Detection

[0317]

[0318]

[0319] By incorporating via reference

[0320] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0321] Equivalence

[0322] 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

1. Modified bacteria that produce glutamine, wherein the genome contains heterologous polynucleotides of the glutamine efflux protein (rarD) and modifications that reduce the expression of CEY17_10535.

2. The modified bacteria of claim 1, wherein the rarD gene is derived from Escherichia coli.

3. The modified bacteria as described in claim 1 or 2, comprising one or more copies of the glutamine efflux protein (rarD) gene, preferably two copies, more preferably three copies.

4. The modified bacteria according to any one of claims 1 to 3, wherein the promoter of the glutamine efflux protein (rarD) gene is the Psod promoter.

5. The modified bacteria according to any one of claims 1 to 4, wherein the modification that weakens CEY17_10535 expression is one or more of partial knockout, complete knockout, base substitution, and frameshift mutation of the CEY17_10535 gene sequence, preferably partial knockout.

6. The modified bacteria as described in claim 5, wherein the nucleic acid sequence of the CEY17_10535 gene is as shown in SEQ ID NO:21 or 23.

7. The modified bacteria as described in any one of claims 1 to 6, further comprising a modification that reduces rosR activity, preferably a deletion or partial deletion of the nucleic acid sequence of the rosR gene.

8. The modified bacteria as described in any one of claims 1 to 7, further comprising a modification that reduces glsA activity, preferably a deletion or partial deletion of the glsA gene, and / or a modification that increases glnA activity, preferably, the modification that increases glnA activity is a glnA gene containing a Y405F substitution (glnA). Y405F More preferably, the glnA is derived from Corynebacterium glutamicum or Saccharomyces cerevisiae.

9. Use of the modified bacteria as described in any one of claims 1 to 8 in increasing glutamine production.

10. A method for producing glutamine, comprising culturing the modified bacteria as described in any one of claims 1 to 8 in a culture medium and isolating glutamine.

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