Method for producing glutamine at high yield

Abstract

The present disclosure provides a modified bacterium that produces glutamine wherein a heterologous polynucleotide of glutamine export protein (rarD) and a modification of reduced rosR activity are included in the genome as compared to a pre-modification bacterium. In addition, methods of increasing glutamine production using the modified bacteria are also provided.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of microbial engineering, and particularly relates to the field of glutamine fermentation production, and a method for producing glutamine by using bacterial fermentation. BACKGROUND

[0002] Glutamine is a non-essential amino acid. The chemical name is 2-amino-4- carbamoyl butyric acid. Glutamine is a coded amino acid in protein synthesis, which can promote the synthesis of protein and inhibit the decomposition of protein, and can be used for treating gastric and duodenal ulcers, and plays an important role in the pharmaceutical industry. At present, the most commonly used method for producing glutamine is fermentation method, mainly using Corynebacterium glutamicum as a production strain to ferment glutamine. Corynebacterium glutamicum is a heterotrophic aerobic gram-positive bacterium, which has the characteristics of fast growth, non-pathogenicity, and weak degradation ability to its own metabolites. The fermentation method has the advantages of wide raw material sources, low production cost, controllable product quality, and single product. The glutamine synthase encoding gene of Corynebacterium glutamicum is inhibited by repressor protein or transcriptional regulator, resulting in a decrease in transcription level and a sharp decrease in enzyme activity, which causes the substrate glutamic acid to be unable to be fully converted. Glutamic acid is the primary precursor of glutamine biosynthesis, and in high-yield glutamine strains, low glutamic acid level is the main factor limiting the synthesis of glutamine.

[0003] Microbial engineering aims to make microorganisms suitable for engineering production by genetic engineering and metabolic engineering of microorganisms. Currently, researchers have found that the production of glutamine in Escherichia coli JGLE1 increased from 122.2 mg / L to 497.4 mg / L after the introduction of the E. coli-derived rarD expression plasmid. In addition to transporting glutamine outside the cell, rarD can also transport serine, threonine, and phenylalanine (US8623619B2); the 405th amino acid of glutamine synthetase from Corynebacterium glutamicum was mutated from tyrosine to phenylalanine, which eliminated the adenylylation modification (CN200610089484); wild-type Corynebacterium glutamicum 14067 containing vgb and glnAY405F can produce high yield of glutamine at low DO level and low cost (CN200310103230). Inactivation of the MarR-type transcriptional regulator rosR gene in Corynebacterium glutamicum G01 increased the production of glutamine from 1.64 g / L to 2.7 g / L (Li X, Bao T, Osire T, et al. MarR-type transcription factor RosR regulates glutamate metabolism network and promotes accumulation of L-glutamate in Corynebacterium glutamicum G01. Biore source Technol. 2021; 342: 125945. doi: 10.1016 / j.biortech.2021.125945). In Lactobacillus brevis ATCC 13869, the expression of PDH was increased by changing the promoter region of the pyruvate dehydrogenase (PDH) E1 subunit gene pdhA; after introducing the promoter mutation that increases the expression of PDH into glutamic acid-producing strain GC 25, the production of glutamic acid was increased (CN1170938C). PDH is composed of E1p(AceE), E2p(AceF), and E3(Lpd), which are encoded by aceE, aceF, and lpd, respectively (Eikmanns BJ, Blombach B. The pyruvate dehydrogenase complex of Corynebacterium glutamicum: an attractive target for metabolic engineering. J Biotechnol. 2014; 192 Pt B: 339-345. doi: 10.1016 / j.jbiotec.2013.12.019).Both pdhA and aceE represent genes encoding pyruvate dehydrogenase E1p. However, there are still some defects in the glutamine-producing strain, such as accumulation of impure acid glutamic acid, insufficient endogenous transport, and insufficient synthesis of precursor glutamic acid, which limit the further synthesis of glutamine by the strain. In order to improve the conversion rate and yield of glutamine, the present disclosure introduces rarD and inactivates rosR on the basis of the glutamine high-yield strain, further strengthens the expression of pyruvate dehydrogenase E1p, improves the transport of glutamine, and improves the synthesis of precursor glutamic acid to improve the conversion rate and yield of glutamine. SUMMARY

[0004] In one aspect, the present disclosure provides a modified bacterium for producing glutamine, wherein the genome of the modified bacterium comprises a heterologous polynucleotide of glutamine efflux protein (rarD) and a modification of reduced rosR activity compared to the bacterium before modification.

[0005] In one embodiment, the modified bacterium produces glutamine at a higher yield than the bacterium before modification.

[0006] In one embodiment, the bacterium is a Corynebacterium bacterium, preferably Corynebacterium glutamicum.

[0007] In one embodiment, the glutamine efflux protein (rarD) is derived from Escherichia coli.

[0008] In one embodiment, the modified bacterium comprises more than one copy of the glutamine efflux protein (rarD) gene, preferably two copies, more preferably three copies.

[0009] In one embodiment, the modified bacterium, wherein the insertion site of the glutamine efflux protein (rarD) gene is selected from within the acetyltransferase-encoding gene CEY17_08220 ORF or within the glutaminase-encoding gene glsA ORF, preferably, the first copy of the glutamine efflux protein (rarD) gene is inserted within the acetyltransferase-encoding gene CEY17_08220 ORF and the second copy is inserted within the glutaminase-encoding gene glsA ORF.

[0010] In one embodiment, the modified bacterium, wherein the modification of reduced rosR activity is a deletion or partial deletion of the nucleic acid sequence of the rosR gene.

[0011] In one embodiment, the modified bacterium further comprises a modification of increased glnA activity, preferably, the modification of increased glnA activity is that glnA comprises a Y405F substitution (glnA Y405F ).

[0012] In one embodiment, the modified bacteria, wherein the glnA is derived from C. glutamicum or S. cerevisiae.

[0013] In one embodiment, the modified bacteria, comprising more than one copy of the glnA gene. Y405F

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

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

[0016] In one embodiment, the modified bacteria, wherein the promoter of the glnA gene is Psod promoter. Y405F

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

[0018] In another aspect, there is provided use of the modified bacteria of the present disclosure in improving glutamine production.

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

[0020] Advantages

[0021] The present disclosure mainly solves the problems of low conversion efficiency and insufficient supply of precursor glutamic acid in the current glutamine production bacteria. The recombinant C. glutamicum glutamine provided by the present disclosure has improved yield and conversion rate. DETAILED DESCRIPTION

[0022] The following description of the present disclosure is merely intended to illustrate various different embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments will be included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0023] ​​​In order to enable a person 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 a part of the embodiments of the present disclosure, not all.

[0024] Table 1. Instruments used in the experiment

[0025] Instrument name Manufacturer Model PCR instrument Bio-Rad S1000 Electrotransformation instrument Bio-Rad Gene Pulser Xcell Constant temperature incubator Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd. SPX-250BSH-II Constant temperature shaker Taicang Experimental Equipment Factory DHZ-DA Pulsating vacuum sterilization cabinet Jiangsu Shennong Sterilization Equipment Co., Ltd. YG-0.36 SBA enzyme electrode analyzer Shandong Academy of Sciences SBA-40C Ultraviolet-visible spectrophotometer Tianmei (China) Scientific Instrument Co., Ltd. UH5300

[0026] Table 2. Reagents used in the experiment

[0027] Reagent name Manufacturer Part number Proteose peptone Oxoid, UK LP0042B NaCl National Pharmaceutical Group Chemical Reagent Co., Ltd. 10019318 Yeast extract Oxoid, UK LP0021B Sorbitol Shenguo Bioengineering Co., Ltd. A610491-0500 Sucrose National Pharmaceutical Group Chemical Reagent Co., Ltd. 10021418 Brain heart infusion Oxoid, UK CM1135B Agar Beijing Aobosan Biotechnology Co., Ltd. 01-023 Glucose National Pharmaceutical Group Chemical Reagent Co., Ltd. 10010518 Urea National Pharmaceutical Group Chemical Reagent Co., Ltd. 10023218 KH2PO4 National Pharmaceutical Group Chemical Reagent Co., Ltd. 10017618 MgSO4.7H2O National Pharmaceutical Group Chemical Reagent Co., Ltd. 10013018 Corn syrup dry powder Baolingbao Biological Co., Ltd. (NH4)2SO4 National Pharmaceutical Group Chemical Reagent Co., Ltd. 10002918 CaCO3 Tianjin Damao Chemical Reagent Factory 1653

[0028] Table 3. Primer sequence table

[0029]

[0030]

[0031]

[0032]

[0033] In this paper, two glutamic acid coryneform bacteria strains (QS12, o-QS04) capable of synthesizing glutamine were used as the starting bacteria, and Psod-rarD and ΔrosR were introduced to improve the conversion rate of glutamine, which proved that the introduction of Psod-rarD and ΔrosR in different strains had unexpected effects.

[0034] 1. Introduction of Psod-rarD and ΔrosR into QS12

[0035] The present disclosure first constructed a glutamine-producing strain QS12, specifically, Corynebacterium glutamicum ATCC14067 was used as the starting bacteria to introduce ino-1S84A, CEY17_06485 A386T , CEY17_05975 V184I , CEY17_04535 T65I , CEY17_04555 R2916C , CEY17_13360 A139T , gyrA A460V , ΔglsA, glnA Y405F , Psod-rarD, Psod-glnA Y405F The yield of glutamine reached 21.5 g / L. In order to further improve the yield of glutamine, two copies of Psod-rarD and ΔrosR were introduced to obtain strain QS15, and the conversion rate was increased by 3.1%, which had unexpected effects.

[0036] 2. Introduction of Psod-rarD and ΔrosR into o-QS04

[0037] To further confirm that the combination of Psod-rarD and ΔrosR is effective in different glutamine synthesis strains, another glutamine production strain o-QS04 was constructed, Psod-rarD and ΔrosR were introduced to obtain strain o-QS09, the conversion rate was increased by 1.5%, and unexpected effect was obtained.

[0038] The genotype of the strain is shown in Table 4.

[0039] Table 4. Genotype of the strain

[0040]

[0041] Table 5. Sequence information

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] Example

[0048] In order 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 a part of the embodiments of the present disclosure, not all.

[0049] Example 1: Construction of QS12 starting strain

[0050] 1.1 Construction of QS01 strain from ATCC14067 and performance verification

[0051] a) Plasmid construction

[0052] The genomic DNA of C. glutamicum ATCC 14067 was used as a template, and the primers PQ28-UP-F / PQ29-UP-R were used to amplify the upstream homology arm UP (594 bp). The genomic DNA of C. glutamicum ATCC 14067 was used as a template, and the primers PQ30-DN-F / PQ31-DN-R were used to amplify the downstream homology arm DN (555 bp). The UP and DN were used as templates, and the primers PQ28-UP-F / PQ31-DN-R were used to amplify the overlap fragment (1120 bp). The overlap fragment and pK18mobsacB were digested with XbaI and PstI at 37°C for 1 hour, the fragment was directly purified, the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 hour, and the gel was recovered. Subsequently, the enzyme was connected, transformed, and verified by colony PCR with primers P82 / P85, with a length of 1.4 kb. The correct transformant was placed in a test tube LBK50, and the plasmid was sent for testing.

[0053] b) Strain construction

[0054] The plasmid was electroporated into C. glutamicum ATCC14067, and LBHISK15 plates were coated. LBK25S and LBK25 plates were spotted once, and the latter was not grown. The correct phenotype was identified by primers PQ28-UP-F / P85, P82 / PQ31-DN-R, positive control plasmid, and negative control ATCC 14067 genome. The correct length was 1.3 kb and 1.2 kb. The primary recombinants were placed in LB test tubes overnight, and were diluted 10, 100, and 1000 times to coat LBK25, LBK25S, and LBS plates. The second time, the LBS plates were grown, and the LBK25 and LB plates were spotted. The former was not grown, and the latter was grown. The appropriate annealing temperature was determined by primers PQ32-id-f / PQ31-DN-R, positive control plasmid, and negative control C. glutamicum ATCC14067 genome. Colony PCR was performed using the annealing temperature, and the correct secondary recombinants were amplified by primers PQ33-ID-F / PQ34-ID-R and sequenced, with a length of 1.4 kb. The correct strain was designated QS01.

[0055] c) Performance verification

[0056] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is as follows:

[0057] The strains stored in glycerol tubes at -80°C were inoculated in BHI slant medium for activation, and after 24 hours of incubation at 33°C, the bacterial lawn was grown. The bacterial lawn from the freshly activated slant was picked and inoculated in the seed medium described below, and incubated at 33°C, 100 rpm for 5 hours until the mid-log phase. The seed liquid was prepared. The seed liquid described above was inoculated in a 500 ml flask containing 20 ml of fermentation medium at a 10% inoculation amount, and incubated at 33°C, 150 rpm for 48 hours. After the glucose was completely consumed, the concentration of glutamine accumulated in the medium was measured by HPLC method.

[0058] The formula of the medium is as follows:

[0059] LB medium: peptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, agar 1.8%, sterilized at 121°C, 0.1 MPa for 20 minutes; LBK25 is LB plus kanamycin 25 μg / mL.

[0060] LBHIS medium: peptone 5 g / L, NaCl 5 g / L, yeast extract 2.5 g / L, brain heart infusion 18.5 g / L, sorbitol 91 g / L, agar 1.8%, sterilized at 121°C, 0.1 MPa for 20 minutes; LBHISK15 is LBHIS plus kanamycin 15 μg / mL.

[0061] 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°C, 0.1 MPa for 20 minutes; LBK25S is LBS plus kanamycin 25 μg / mL.

[0062] BHI slant medium: brain heart infusion 37 g / L, agar 1.8%, sterilized at 121°C, 0.1 MPa for 20 minutes;

[0063] Seed medium: glucose 25 g / L, urea 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.4 g / L, corn syrup dry powder 15 g / L, pH 7.0;

[0064] Fermentation medium: glucose 90.9 g / L, (NH4)2SO4 50 g / L, KH2PO4 2.5 g / L, corn syrup dry powder 2 g / L, CaCO3 40 g / L, pH 7.0.

[0065] Table 6. Glutamine content detection of Corynebacterium glutamicum QS01

[0066] Strain Genotype Growth (OD562nm) gln (g / L) Conversion rate (%) Conversion rate increase (%) ATCC 14067 Wild type 62.3 0.4 0.45 - QS01 Ino-1 S84A ]] 60.7 1.1 1.22 0.77

[0067] Strain QS01 is ino-1 S84A The glutamine yield of the obtained strain QS01 is increased from 0.4 g / L to 1.1 g / L, and the conversion rate is increased by 0.77% as shown in Table 6.

[0068] 1.2 Strain construction and performance verification of QS01→QS02

[0069] a) Plasmid construction

[0070] UP (543 bp) was amplified from the genome of Corynebacterium glutamicum ATCC 14067 using PQ35-UP-F / PQ36-UP-R as primers. DN (548 bp) was amplified from the genome of Corynebacterium glutamicum ATCC 14067 using PQ37-DN-F / PQ38-DN-R as primers. The overlap fragment (1053 bp) was obtained by amplifying UP and DN using PQ35-UP-F / PQ38-DN-R as primers. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour, and the fragment was directly purified. The vector was dephosphorylated by adding 3 μL of FastAP at 37°C for 1 hour, and the gel was recovered. Subsequently, the vector was ligated and transformed, and colony PCR was performed using primers P82 / P85, with a length of 1.3 kb. The correct transformants were inoculated into test tubes of LBK50, and the plasmid was sent for testing.

[0071] b) Strain construction

[0072] The plasmid was electroporated into Corynebacterium glutamicum QS01, and LBHISK15 plates were coated. LBK25S and LBK25 plates were inoculated once, and the latter was not grown. The correct phenotype was identified using PQ35-UP-F / P85, P82 / PQ38-DN-R, positive control plasmid, and negative control ATCC14067 genome. The correct length was 1.1 kb and 1.2 kb. The recombinants were inoculated into test tubes of LB overnight, and LBK25, LBK25S, and LBS plates were coated at dilutions of 10, 100, and 1000 times. The second recombinants were inoculated into LBK25 and LB plates, and the former was not grown. The correct phenotype was identified using PQ39-id-f / PQ38-DN-R, positive control plasmid, and negative control Corynebacterium glutamicum ATCC 14067 genome. The appropriate annealing temperature was determined by colony PCR. The correct second recombinants were identified by amplification and sequencing using primers PQ40-ID-F / PQ41-ID-R, with a length of 1.4 kb. The correct strain was recorded as QS02.

[0073] c) Performance verification

[0074] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0075] Table 7. Glutamine content detection of C. glutamicum QS02

[0076]

[0077]

[0078] Strain QS02 was obtained by introducing CEY17_06485 into strain QS01 A386T As shown in Table 7, the glutamine yield of the obtained strain QS02 was increased from 1.1 g / L to 1.7 g / L, and the conversion rate was increased by 0.65%.

[0079] 1.3 Construction of QS02→QS03 strain and performance verification

[0080] a) Plasmid construction

[0081] The upstream homology arm UP (555 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ42-UP-F / PQ43-UP-R as primers. The downstream homology arm DN (500 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ44-DN-F / PQ45-DN-R as primers. The overlap fragment (1029 bp) was obtained by amplifying UP and DN using PQ42-UP-F / PQ45-DN-R as primers. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour. The fragment was directly purified, the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 hour, and the gel was recovered. Subsequently, the enzyme was connected, transformed, and verified by colony PCR using primers P82 / P85, with a length of 1.3 kb. The correct transformant was placed in a test tube LBK50, and the plasmid was sent for testing.

[0082] b) Strain construction

[0083] Plasmid electroporation of C. glutamicum QS02, spread on LBHISK15 plate, primary counter selection on LBK25S, LBK25 plate, the latter not growing phenotype correct, identified by PQ42-UP-F / P85, P82 / PQ45-DN-R, positive control plasmid, negative control ATCC 14067 genome, correct length 1.1 kb, 1.2 kb. Primary recombinants spread on LB tube overnight, dilution 10, 100, 1000 times spread on LBK25, LBK25S, LBS plate, LBS plate growing secondary counter selection on LBK25, LB plate, the former not growing phenotype correct. Use PQ46-id-f / PQ45-DN-R to find the appropriate annealing temperature, positive control plasmid, negative control C. glutamicum ATCC 14067 genome, use this annealing temperature for colony PCR identification. Correct secondary recombinants amplified by primers PQ47-ID-F / PQ48-ID-R and sequenced, length 1.3 kb, correct strain recorded as QS03.

[0084] c) Performance verification

[0085] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance, and the method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0086] Table 8. Detection of glutamine content of C. glutamicum QS03

[0087]

[0088] Strain QS03 was obtained by introducing CEY17_05975 V184I amino acid mutation into strain QS02, as shown in Table 8, the glutamine yield of the obtained strain QS03 was increased from 1.7 g / L to 2.6 g / L, and the conversion rate was increased by 1.02%.

[0089] 1.4 Construction of QS03→QS04 strain and performance verification

[0090] a) Plasmid construction

[0091] 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, respectively. The fragments were then purified using a Tiangen common DNA product purification kit. The vector was dephosphorylated with 3 μL of FastAP, incubated at 37°C for 1 hour, and then recovered from the gel. 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.

[0092] b) Strain construction

[0093] 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.

[0094] c) Performance Verification

[0095] 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.

[0096] Table 9. Glutamine content detection in Corynebacterium glutamicum QS04

[0097]

[0098] Strain QS04 was introduced CEY17_04535 on the basis of strain QS03 T65I The glutamine yield of the obtained strain QS04 was increased from 2.6 g / L to 3 g / L and the conversion rate was increased by 0.41% as shown in Table 9.

[0099] 1.5 Strain construction and performance verification of QS04→QS05

[0100] a) Plasmid construction

[0101] The upstream homology arm UP (522 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ56-UP-F / PQ57-UP-R as primers. The downstream homology arm DN (523 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ58-DN-F / PQ59-DN-R as primers. The overlap fragment (1020 bp) was obtained by amplifying UP and DN using PQ56-UP-F / PQ59-DN-R as primers. The overlap fragment and pK18mobsacB were digested with XbaI and HindIII at 37°C for 1 hour. The fragment was directly purified, the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 hour, and the gel was recovered. Subsequently, the enzyme was connected, transformed, and verified by colony PCR using primers P82 / P85 with a length of 1.3 kb. The correct transformant was placed in a test tube LBK50, and the plasmid was sent for testing.

[0102] b) Strain construction

[0103] The plasmid was electroporated into Corynebacterium glutamicum QS04, and LBHISK15 plates were coated. LBK25S and LBK25 plates were spotted once, and the latter was not grown. The correct phenotype was identified using PQ56-UP-F / P85, P82 / PQ59-DN-R, positive control plasmid, and negative control Corynebacterium glutamicum ATCC 14067 genome. The correct length was 1.1 kb and 1.2 kb. The recombinant was placed in a test tube overnight, and LBK25, LBK25S, and LBS plates were coated at dilutions of 10, 100, and 1000 times. The second recombinant was spotted on LBK25 and LB plates, and the former was not grown. The correct phenotype was identified using PQ62-id-f / PQ59-DN-R, positive control plasmid, and negative control Corynebacterium glutamicum ATCC 14067 genome. The appropriate annealing temperature was determined by colony PCR identification using the annealing temperature. The correct second recombinant was sequenced after amplification using primers PQ60-ID-F / PQ61-ID-R with a length of 1.2 kb. The correct strain was recorded as QS05.

[0104] c) Performance verification

[0105] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0106] Table 10. Glutamine content detection of C. glutamicum QS05

[0107]

[0108] Strain QS05 was obtained by introducing CEY17_04555 into strain QS04 R2916C As shown in Table 10, the glutamine yield of the obtained strain QS05 was increased from 3 g / L to 3.8 g / L, and the conversion rate was increased by 0.88%.

[0109] 1.6 Construction of QS05→QS06 strain and performance verification

[0110] a) Plasmid construction

[0111] The upstream homology arm UP (539 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ63-UP-F / PQ64-UP-R as primers. The downstream homology arm DN (542 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ65-DN-F / PQ66-DN-R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, 3 μL FastAP was added to dephosphorylate the vector, which was then placed at 37°C for 1 h, and the vector was recovered by gel recovery. The digested vector, UP, and DN were assembled seamlessly at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.3 kb. The correct transformant was placed in a test tube with LBK50, and the plasmid was sent for testing.

[0112] b) Strain construction

[0113] Plasmid electroporation of C. glutamicum QS05, spread on LBHISK15 plate, primary counter selection on LBK25S, LBK25 plate, the latter not growing phenotype correct, identified by PQ63-UP-F / P85, P82 / PQ66-DN-R, positive control plasmid, negative control ATCC 14067 genome, correct length 1.1 kb, 1.2 kb. Primary recombinants spread on LB tube overnight, dilution 10, 100, 1000 times spread on LBK25, LBK25S, LBS plate, LBS plate growing secondary counter selection on LBK25, LB plate, the former not growing the latter growing phenotype correct. Use PQ67-id-f / PQ66-DN-R to find the appropriate annealing temperature, positive control plasmid, negative control C. glutamicum ATCC 14067 genome, use this annealing temperature for colony PCR identification. Correct secondary recombinants amplified by primers PQ68-ID-F / PQ69-ID-R and sequenced, length 1.3 kb, correct strain recorded as QS06.

[0114] c) Performance verification

[0115] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance, and the method for verifying glutamine yield in fermentation was as described in Example 1.1.

[0116] Table 11. Detection of glutamine content of C. glutamicum QS06

[0117]

[0118] Strain QS06 was obtained by introducing the CEY17_13360 A139T amino acid mutation into strain QS05, as shown in Table 11, the glutamine yield of the obtained strain QS06 was increased from 3.8 g / L to 5.2 g / L, and the conversion rate was increased by 1.54%.

[0119] 1.7 Construction of QS06→QS07 strain and performance verification

[0120] a) Plasmid construction

[0121] The upstream homology arm UP (477 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using the primers PQ70-UP-F / PQ71-UP-R. The downstream homology arm DN (550 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using the primers PQ72-DN-F / PQ73-DN-R. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated with 3 μL FastAP for 1 h at 37°C and the vector was recovered from the gel. The digested vector, UP and DN were assembled without gaps for 30 min at 37°C and transformed. Subsequently, the transformants were verified by colony PCR using the primers P82 / P85, which resulted in a fragment of 1.3 kb in length. The correct transformants were inoculated into test tubes with LBK50 and the plasmids were sent for sequencing.

[0122] b) Strain construction

[0123] The plasmid was electroporated into Corynebacterium glutamicum QS06 and plated on LBHISK15 plates. The colonies were patched on LBK25S plates and on LBK25 plates, which were not longer than the former. The correct recombinants were identified using the primers PQ70-UP-F / P85, P82 / PQ73-DN-R. The correct length was 1.1 kb and 1.2 kb. The recombinants were inoculated into test tubes with LB and plated on LBK25, LBK25S and LBS plates at a dilution of 10, 100 and 1000. The colonies grown on LBS plates were patched on LBK25 and LB plates. The former were not longer than the latter. The correct recombinants were identified by colony PCR using the primers PQ74-id-f / PQ73-DN-R. The correct recombinants were amplified with the primers PQ75-ID-F / PQ76-ID-R and sequenced. The correct length was 1.2 kb. The correct strain was designated QS07.

[0124] c) Performance verification

[0125] The recombinant Corynebacterium glutamicum constructed above was fermented and its performance for the production of glutamine was verified. The method for the verification of the glutamine production is described in Example 1.1.

[0126] Table 12. Detection of the glutamine content of Corynebacterium glutamicum QS07

[0127]

[0128] The strain QS07 was obtained by introducing a gyrA A466V amino acid mutation into the strain QS06, as shown in Table 12. The glutamine production of the strain QS07 was increased from 5.2 g / L to 8 g / L and the conversion rate was increased by 3.08%.

[0129] 1.8 QS07→QS08 strain construction and performance verification

[0130] a) Plasmid construction

[0131] The upstream homology arm UP (530 bp) was amplified from Corynebacterium glutamicum ATCC 14067 genome using PQ01-UP-F / PQ02-UP-R as primers. The downstream homology arm DN (550 bp) was amplified from Corynebacterium glutamicum ATCC 14067 genome using PQ03-DN-F / PQ04-DN-R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37℃ for 1h, 3μL FastAP was added to dephosphorylate the vector, which was placed at 37℃ for 1h, and the vector was recovered by gel. The digested vector, UP and DN were assembled at 37℃ for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.4kb. The correct transformant was inoculated into a test tube of LBK50, and the plasmid was sent for sequencing.

[0132] b) Strain construction

[0133] The plasmid was electroporated into Corynebacterium glutamicum QS07, and then plated on LBHISK15 plates. LBK25S and LBK25 plates were inoculated once, and the latter was not grown. The correct phenotype was identified using PQ01-UP-F / P85, P82 / PQ04-DN-R, and the correct length was 1.3kb and 1.2kb. The positive control was a plasmid, and the negative control was the ATCC14067 genome. The recombinant strain was inoculated into a test tube of LB overnight, and then diluted by 10, 100 and 1000 times to inoculate LBK25, LBK25S and LBS plates. The second time, the recombinant strain was inoculated into LBK25 and LB plates, and the former was not grown. The correct phenotype was identified using primers PQ05-ID-F / PQ06-ID-R, and the correct length was 1.3kb. Then, the correct strain was identified by sequencing using the primers for amplification. The correct strain was named QS08.

[0134] c) Performance verification

[0135] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0136] Table 13. Detection of glutamine content of Corynebacterium glutamicum QS08

[0137]

[0138] The strain QS08 was obtained by inactivating glsA based on the strain QS07. As shown in Table 13, the glutamine yield of the obtained strain QS08 was increased from 8g / L to 12.4g / L, and the conversion rate was increased by 4.8%.

[0139] 1.9 QS08→QS09 strain construction and performance verification

[0140] a) Plasmid construction

[0141] The upstream homology arm UP (526 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ07-UP-F / PQ08-UP-R as primers. The downstream homology arm DN (502 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ09-DN-F / PQ10-DN-R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated with 3 μL FastAP at 37°C for 1 h, and the vector was recovered from the gel. The digested vector, UP, and DN were assembled seamlessly at 37°C for half an hour and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the correct transformants were identified by a length of 1.3 kb. The correct transformants were inoculated into LB test tubes, and the plasmids were extracted and sent for sequencing.

[0142] b) Strain construction

[0143] The plasmid was electroporated into Corynebacterium glutamicum QS08, and the bacteria were plated on LBHISK15 plates. The bacteria were then plated on LBK25S and LBK25 plates, and the correct recombinants were identified by a length of 1.1 kb and 1.2 kb using primers PQ07-UP-F / P85, P82 / PQ10-DN-R, and the correct length of 1.1 kb and 1.2 kb. The correct recombinants were inoculated into LB test tubes overnight, and the bacteria were diluted 10, 100, and 1000 times and plated on LBK25, LBK25S, and LBS plates. The bacteria were then plated on LBK25 and LB plates, and the correct recombinants were identified by a length of 551 bp using primers PQ13-id-f / PQ10-DN-R. The correct recombinants were identified by a length of 1.1 kb using primers PQ11-ID-F / PQ12-ID-R, and the correct strain was named QS09.

[0144] c) Performance verification

[0145] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0146] Table 14. Detection of glutamine content in Corynebacterium glutamicum QS09

[0147]

[0148]

[0149] Strain QS09 was constructed by introducing glnA Y405F The glutamine production of strain QS09 was increased from 12.4 g / L to 19.2 g / L, and the conversion rate was increased by 7.5% by amino acid mutation, as shown in Table 14.

[0150] 1.10 Strain construction and performance verification of QS09→QS10

[0151] a) Plasmid construction

[0152] The upstream homology arm (563 bp) was amplified using the Corynebacterium glutamicum ATCC 14067 genome as the template and the primers PQ265-UP-1f / PQ266-UP-1r. The promoter Psod (215 bp) was amplified using the Corynebacterium glutamicum ATCC 13032 (model strain, available from Bomeibowei Biotechnology Co., Ltd.) genome as the template and the primers PQ267-Psod-2f / PQ279-Psod-2r. The rarD (913 bp) was amplified using the Escherichia coli MG1655 (model strain, available from Hubei Aipti Biotechnology Co., Ltd.) genome as the template and the primers PQ280-rarD-3f / PQ281-rarD-3r. The downstream homology arm (536 bp) was amplified using the Corynebacterium glutamicum ATCC 14067 genome as the template and the primers PQ282-DN-4f / PQ272-DN-4r. Subsequently, the 1-2 fragment (751 bp) was obtained by fusion amplification using the upstream homology arm UP and the promoter Psod as the templates and the primers PQ265-UP-1f / PQ279-Psod-2r. The 3-4 fragment (1427 bp) was obtained by fusion amplification using the rarD and the downstream homology arm DN as the templates and the primers PQ280-rarD-3f / PQ272-DN-4r. Finally, the 1-4 fragment (2157 bp) was obtained by amplification using the 1-2 and 3-4 fragments as the templates and the primers PQ265-UP-1f / PQ272-DN-4r. The 1-4 fragment and the pK18mobsacB were subjected to enzyme digestion with EcoRI and NheI at 37°C for 1 hour. The fragment was directly purified, the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 hour, and the product was recovered by electrophoresis. Subsequently, the enzyme ligation and transformation were performed, and the colony PCR was performed using the primers P82 / P85, with a length of 2.3 kb. The correct transformants were inoculated into test tubes containing LBK50, and the plasmid was sent for testing.

[0153] b) Strain construction

[0154] Plasmid electroporation of C. glutamicum QS09, spread on LBHISK15 plate, one recombination pair of LBK25S, LBK25 plate, the latter is not long phenotype correct, identified by PQ265-UP-1f / P85, P82 / PQ272-DN-4r, positive control plasmid, negative control ATCC 14067 genome, correct length 2.3kb, 1.2kb. One recombination subculture in no-antibiotic LB test tube overnight, dilution 10, 100, 1000 times spread on LBK25, LBK25S, LBS plate, LBS plate growth of secondary pair of LBK25, LB plate, the former is not long phenotype correct. Secondary recombinants identified by primers PQ273-ID-F / PQ274-ID-R, correct length 2.3kb, then amplified and sequenced, correct strain is QS10.

[0155] c) Performance verification

[0156] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance, and the method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0157] Table 15. Detection of glutamine content of C. glutamicum QS10

[0158]

[0159] Strain QS10 was obtained by inserting a single copy of RarD gene from E. coli MG1655 into strain QS09, and as shown in Table 15, the glutamine yield of the obtained strain QS10 was increased from 19.2 g / L to 19.8 g / L, and the conversion rate was increased by 0.7%.

[0160] 1.11 Construction of QS10→QS12 strain and performance verification

[0161] a) Plasmid construction

[0162] The genomic DNA of C. glutamicum QS09 was used as template, and the primer of PQ320-UP-1F / PQ321-UP-1R was used to amplify the upstream homologous arm UP (514 bp). The genomic DNA of C. glutamicum ATCC 13032 was used as template, and the primer of PQ322-Psod-2F / PQ323-Psod-2R was used to amplify the promoter Psod (244 bp). The genomic DNA of C. glutamicum QS09 was used as template, and the primer of PQ324-glnA-3F / PQ325-glnA-3R was used to amplify glnAY405F (1447 bp). The genomic DNA of C. glutamicum QS09 was used as template, and the primer of PQ326-DN-4F / PQ327-DN-4R was used to amplify the downstream homologous arm DN (539 bp). Then, the 1-2 fragment (733 bp) was obtained by amplification using UP and Psod as templates and the primers of PQ320-UP-1F / PQ323-Psod-2R. The 3-4 fragment (1956 bp) was obtained by amplification using glnAY405F and DN as templates and the primers of PQ324-glnA-3F / PQ327-DN-4R. Finally, the 1-4 fragment (2832 bp) was obtained by amplification using the 1-2 and 3-4 fragments as templates and the primers of PQ320-UP-1F / PQ327-DN-4R. The 1-4 fragment and pK18mobsacB were digested with XbaI and SalI at 37°C for 1 hour, and the fragment was directly purified. The vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 hour, and the gel was recovered. Then, the vector was ligated and transformed, and the primer P82 / P85 was used for colony PCR verification. The length was 3.1 kb, and the correct transformant was inoculated into a test tube containing LBK50. The plasmid was sent for sequencing.

[0163] b) Strain construction

[0164] The plasmid was electroporated into C. glutamicum QS10, and LBHISK15 plates were coated. LBK25S and LBK25 plates were inoculated once, and the latter was not grown. The correct phenotype was identified by using the primers of PQ320-UP-1F / P85, P82 / PQ327-DN-4R, the positive control plasmid, and the negative control QS09 genome. The correct length was 3 kb and 2.9 kb. The recombinant was inoculated into a test tube containing LB overnight, and LBK25, LBK25S, and LBS plates were coated after dilution by 10, 100, and 1000 times. The second time, the recombinant was inoculated into LBK25 and LB plates, and the former was not grown. The correct phenotype was identified by using the primers of PQ328-ID-F / PQ329-ID-R. The correct length was 3 kb, and then the primer was used for amplification and sequencing. The correct strain was recorded as QS12.

[0165] c) Performance verification

[0166] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0167] Table 16. Glutamine content detection of C. glutamicum QS12

[0168]

[0169] Strain QS12 was obtained by inserting the self glnA gene of strain QS10 at ΔglsA. Y405F The conversion rate of QS12 was increased by 1.9%.

[0170] Example 2: Strain construction and performance verification of QS12→QS13

[0171] 2.1 Plasmid construction

[0172] The upstream homologous arm (500 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ445-UP-1F / PQ446-UP-1R as primers. The downstream homologous arm (500 bp) was amplified from the genome of C. glutamicum ATCC 14067 using PQ447-DN-2F / PQ448-DN-2R as primers. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μl FastAP at 37°C for 1 h, and then recovered by gel. The digested vector, UP, and DN were assembled without a gap at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed for verification, and the correct transformants were inoculated into test tubes, and plasmid digestion was performed for verification, and then sent for testing.

[0173] 2.2 Strain construction

[0174] The plasmid was electroporated into QS12, and then plated on LBHISK15. Once recombination was performed, LBK25S and LBK25 were used for point selection. The correct phenotype was that the latter was longer than the former. The correct ones were identified by PQ445-UP-1F / P85, P82 / PQ448-DN-2R. The positive control was a plasmid, and the negative control was the genome of ATCC 14067. One of each of the one-long and one-short recombinants was selected, inoculated into an antibiotic-free LB test tube overnight, and then diluted by 10, 100, and 1000 times, and plated on LBK25, LBK25S, and LBS plates. The correct ones were identified by PQ449-ID-F / PQ464-ID-R. The correct ones were 1280 bp long, and then sequenced using the primers. The correct strain was recorded as QS13.

[0175] 2.3 Performance verification

[0176] 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.

[0177] Table 17. Glutamine content detection of Corynebacterium glutamicum QS013

[0178]

[0179] Strain QS13 was obtained by ΔrosR based on Corynebacterium glutamicum QS12. The yield of the new strain QS13 increased from 21.5 g / L to 22.6 g / L, and the conversion rate increased by 1.3% (Table 17).

[0180] Example 3: Construction and performance verification of QS12→QS14 strains

[0181] 3.1 Plasmid Construction

[0182] 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 Psod-rarD fragment (1083 bp) was amplified using primers PQ468-Psod-2F / PQ476-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 and incubated at 37°C for 1 h, followed by gel recovery. The digested vector, UP, Psod-rarD, and DN were seamlessly assembled and transformed at 37°C for 30 minutes. Subsequently, colony PCR was performed for verification. Correct transformants were inoculated into test tubes, plasmids were extracted and digested with enzymes for verification, and then sent for testing.

[0183] 3.2 Strain Construction

[0184] 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 2329 bp long. Sequencing was then performed using this primer pair. The correct strain was designated QS14.

[0185] 3.3 Performance verification

[0186] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0187] Table 18. Detection of glutamine content of C. glutamicum QS014

[0188]

[0189] Strain QS14 was obtained by inserting Psod-rarD based on C. glutamicum QS12, and the glutamine yield of the obtained new strain QS14 was increased from 21.5 g / L to 21.8 g / L, and the conversion rate was increased by 0.4% (Table 18).

[0190] Example 4: Construction of QS14→QS15 strain and performance verification

[0191] 4.1 Plasmid construction

[0192] The upstream homologous arm (500 bp) was amplified using C. glutamicum ATCC 14067 genome as a template and using PQ445-UP-1F / PQ446-UP-1R as primers. The downstream homologous arm (500 bp) was amplified using C. glutamicum ATCC 14067 genome as a template and using PQ447-DN-2F / PQ448-DN-2R as primers. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, 3 μl of FastAP was added for dephosphorylation, and the mixture was placed at 37°C for 1 h. The gel was recovered. The digested vector, UP, and DN were assembled at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed for verification, and the correct transformants were inoculated into test tubes, and plasmid digestion was performed for verification. The plasmid was sent for sequencing.

[0193] 4.2 Strain construction

[0194] The plasmid was electroporated into QS14, and LBHISK15 was coated. LBK25S and LBK25 were used for one-time recombination, and the latter was longer than the former. The correct phenotype was identified by using PQ445-UP-1F / P85, P82 / PQ448-DN-2R. The positive control plasmid and the negative control ATCC 14067 genome were used. One of each of the one-time recombination and the three-time recombination was selected, inoculated into an antibiotic-free LB test tube overnight, and then diluted by 10, 100, and 1000 times, and coated on LBK25, LBK25S, and LBS plates. The correct phenotype was identified by using LBK25 and LBK25S for one-time recombination, and LB for three-time recombination. The correct phenotype was identified by using PQ449-ID-F / PQ464-ID-R, and the correct strain was recorded as QS15.

[0195] 4.3 Performance verification

[0196] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0197] Table 19. Glutamine content detection of C. glutamicum QS015

[0198]

[0199] Strain QS15 was obtained based on strain QS14 ΔrosR, i.e., a combination of Psod-rarD and ΔrosR strain. According to the combination of Examples 2, 3, and 4, the conversion rate was increased by 1.3% by introducing ΔrosR, the conversion rate was increased by 0.4% by introducing Psod-rarD, and the conversion rate was increased by 3.1% by combining Psod-rarD and ΔrosR, which had an unexpected effect.

[0200] To confirm that the combination of Psod-rarD and ΔrosR has an effect in different starting strains, strain o-QS04 was constructed to obtain a strain capable of producing glutamine. On this basis, single-point Psod-rarD, ΔrosR verification and Psod-rarD, ΔrosR combination verification were performed, respectively.

[0201] Example 5: Construction of C. glutamicum ATCC 14067→o-QS04 starting strain and performance verification

[0202] 5.1 Construction of C. glutamicum ATCC 14067→o-QS01 strain and performance verification

[0203] a) Plasmid construction

[0204] The upstream homology arm UP (530 bp) was amplified using C. glutamicum ATCC 14067 genome as template and PQ01-UP-F / PQ02-UP-R as primers. The downstream homology arm DN (550 bp) was amplified using C. glutamicum ATCC 14067 genome as template and PQ03-DN-F / PQ04-DN-R as primers. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 h, and the vector was recovered by gel recovery. The digested vector, UP, and DN were assembled seamlessly at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.4 kb. The correct transformant was placed in a test tube containing LBK50, and the plasmid was sent for testing.

[0205] b) Strain construction

[0206] Plasmid electrotransformation of C. glutamicum ATCC 14067, spread on LBHISK15 plates, primary selection on LBK25S, LBK25 plates, the latter not growing phenotype correct, identified with PQ01-UP-F / P85, P82 / PQ04-DN-R, positive control plasmid, negative control ATCC 14067 genome, correct length 1.3kb, 1.2kb. Primary recombinants spread on LB, LBK25S, LBS plates, LBS plates grown, secondary selection on LBK25, LB plates, the former not growing phenotype correct. Secondary recombinants identified with primers PQ05-ID-F / PQ06-ID-R, correct band length 1.3kb, then amplified and sequenced with this primer, correct strain noted o-QS01.

[0207] c) Performance verification

[0208] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0209] Table 20. Detection of glutamine content of C. glutamicum o-QS01

[0210]

[0211] The strain o-QS01 was obtained by inactivating glsA based on C. glutamicum ATCC 14067. The glutamine yield of the obtained new strain o-QS01 increased from 0.4 g / L to 0.9 g / L, and the conversion rate increased by 0.55% (Table 20).

[0212] 5.2 Strain construction and performance verification of o-QS01→o-QS02

[0213] a) Plasmid construction

[0214] The upstream homology arm UP (526 bp) was amplified using the genome of C. glutamicum ATCC 14067 as a template and primers PQ07-UP-F / PQ08-UP-R. The downstream homology arm DN (502 bp) was amplified using the genome of C. glutamicum ATCC 14067 as a template and primers PQ09-DN-F / PQ10-DN-R. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μL FastAP at 37°C for 1 h. The vector was recovered by gel recovery. The digested vector, UP, and DN were assembled seamlessly at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length was 1.3 kb. The correct transformant was inoculated in a test tube with LBK50, and the plasmid was sent for sequencing.

[0215] b) Strain construction

[0216] Plasmid electrotransformation of C. glutamicum o-QS01, spread on LBHISK15 plates, primary counter selection on LBK25S, LBK25 plates, the latter not growing phenotype correct, identified with PQ07-UP-F / P85, P82 / PQ10-DN-R, positive control plasmid, negative control ATCC 14067 genome, correct length 1.1 kb, 1.2 kb. Primary recombinants spread on LB test tubes overnight, diluted 10, 100, 1000 fold and spread on LBK25, LBK25S, LBS plates, secondary counter selection on LBK25, LB plates, the former not growing phenotype correct. Use PQ13-id-f / PQ10-DN-R to find the appropriate annealing temperature, positive control plasmid, negative control C. glutamicum ATCC 14067 genome, use this annealing temperature for colony PCR identification, correct secondary recombinants amplified with primers PQ11-ID-F / PQ12-ID-R and sequenced, length 1.1 kb, correct strain noted as o-QS02.

[0217] c) Performance verification

[0218] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance, the method for verifying glutamine production in fermentation is shown in Example 1.1.

[0219] Table 21. Glutamine content detection of C. glutamicum o-QS02

[0220]

[0221] Strain o-QS02 was obtained by introducing glnA Y405F amino acid mutation based on strain o-QS01, the glutamine yield of the obtained new strain o-QS02 was increased from 0.9 g / L to 1.1 g / L, and the conversion rate was increased by 0.21% (Table 21).

[0222] 5.3 Strain construction and performance verification of o-QS02→o-QS03

[0223] a) Plasmid construction

[0224] The upstream homology arm UP (536 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using primers PQ14-UP-F / PQ15-UP-R. The downstream homology arm DN (536 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using primers PQ16-DN-F / PQ17-DN-R. The pK18mobsacB was digested with Xba I and Hind III at 37 °C for 1 h, the vector was dephosphorylated with 3 μL FastAP for 1 h at 37 °C and the vector was recovered from the gel. The digested vector, UP and DN were assembled without gaps for 30 min at 37 °C and transformed. The correct transformants were identified by colony PCR with primers P82 / P85, which resulted in a fragment of 1.3 kb. The correct transformants were inoculated in LB test tubes and the plasmids were isolated and sent for sequencing.

[0225] b) Strain construction

[0226] The plasmid was electroporated into Corynebacterium glutamicum o-QS02 and plated on LBHISK15 plates. The correct recombinants were selected on LBK25S plates and on LBK25 plates, which do not grow. The correct recombinants were identified by colony PCR with primers PQ14-UP-F / P85, P82 / PQ17-DN-R, which resulted in fragments of 1.1 kb and 1.6 kb. The correct recombinants were inoculated in LB test tubes and plated on LBK25, LBK25S and LBS plates. The correct recombinants were identified by colony PCR with primers PQ18-ID-F / PQ19-ID-R, which resulted in a fragment of 1.2 kb. The correct strain was named o-QS03.

[0227] c) Performance verification

[0228] The recombinant Corynebacterium glutamicum constructed above was fermented and its performance for the production of glutamine was verified. The method for the verification of the glutamine production in the fermentation is described in Example 1.1.

[0229] Table 22. Detection of the glutamine content of Corynebacterium glutamicum o-QS03

[0230]

[0231] The strain o-QS03 was obtained by inactivating glnE in the strain o-QS02. The glutamine production of the new strain o-QS03 was increased from 1.1 g / L to 1.7 g / L and the conversion rate was increased by 0.67% (Table 22).

[0232] 5.4 Strain construction and performance verification of o-QS03→o-QS04

[0233] a) Plasmid construction

[0234] The upstream homology arm UP (520 bp) was amplified using the genome of C. glutamicum ATCC 14067 as template and primers PQ20-UP-F / PQ21-UP-R. The promoter Psod (192 bp) was amplified using the genome of C. glutamicum ATCC 13032 as template and primers PQ22-Psod-F / PQ23-Psod-R. The downstream homology arm DN (526 bp) was amplified using the genome of C. glutamicum ATCC 14067 as template and primers PQ24-DN-F / PQ25-DN-R. The UP-Psod (692 bp) was amplified using the UP and Psod fragments as template and primers PQ20-UP-F / PQ23-Psod-R.

[0235] The vector pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, 3 μL FastAP was added to dephosphorylate the vector, which was incubated at 37°C for 1 h, and the vector was recovered from the gel. The digested vector, UP-Psod, and DN were assembled seamlessly at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed using primers P82 / P85, and the length of the correct transformant was 1.5 kb. The correct transformant was inoculated into a test tube with LBK50, and the plasmid was sent for sequencing.

[0236] b) Strain construction

[0237] The plasmid was electroporated into C. glutamicum o-QS03, which was spread on LBHISK15 plates. The recombinants were then inoculated on LBK25S plates and LBK25 plates, and the recombinants that did not grow on the LBK25 plates were inoculated on LBK25S plates. The recombinants were identified using primers PQ20-UP-F / P85 and P82 / PQ25-DN-R, and the correct length was 1.4 kb and 1.1 kb. The recombinants were inoculated into test tubes with LB and incubated overnight. The recombinants were then inoculated on LBK25, LBK25S, and LBS plates at dilutions of 10, 100, and 1000 times. The recombinants that grew on the LBS plates were inoculated on LBK25 and LB plates, and the recombinants that did not grow on the LBK25 plates were inoculated on the LB plates. The recombinants were identified using primers PQ26-ID-F / PQ27-ID-R, and the correct length was 1.4 kb. The recombinants were then amplified using the primers and sequenced, and the correct strain was designated o-QS04.

[0238] c) Performance verification

[0239] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is described in Example 1.1.

[0240] Table 23. Detection of glutamine content in C. glutamicum o-QS04

[0241]

[0242] Strain o-QS04 was obtained by inserting Psod promoter in front of gdh gene based on strain o-QS03. The glutamine yield of the new strain o-QS04 was increased from 1.7 g / L to 2.4 g / L, and the conversion rate was increased by 0.72% (Table 23).

[0243] Example 6: Construction of o-QS04→o-QS08 strain and performance verification

[0244] 6.1 Plasmid construction

[0245] The upstream homology arm (500 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ445-UP-1F / PQ446-UP-1R as primers. The downstream homology arm (500 bp) was amplified from the Corynebacterium glutamicum ATCC 14067 genome using PQ447-DN-2F / PQ448-DN-2R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μl FastAP and incubating at 37°C for 1 h, and then recovered by gel. The digested vector, UP, and DN were assembled without a gap at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed for verification, and the correct transformants were inoculated into test tubes, and plasmid digestion was performed for verification. The correct transformants were sent for sequencing.

[0246] 6.2 Strain construction

[0247] The plasmid was electroporated into o-QS04, and LBHISK15 was coated. The recombinants were identified by using PQ445-UP-1F / P85 and P82 / PQ448-DN-2R, and the correct recombinants were inoculated into test tubes with LB medium and incubated overnight. The recombinants were diluted by 10, 100, and 1000 times, and then coated on LBK25, LBK25S, and LBS plates. The recombinants that grew on the LBS plate were inoculated into LB plates, and the recombinants that grew on the LBK25 plate did not grow on the LBK25S plate. The correct recombinants were identified by using PQ449-ID-F / PQ464-ID-R, and the correct recombinants had a 1280 bp band. The correct recombinants were sequenced using the primers, and the correct strain was named o-QS08.

[0248] 6.3 Performance verification

[0249] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0250] Table 24. Detection of glutamine content of Corynebacterium glutamicum o-QS08

[0251]

[0252] Strain o-QS08 was obtained by deleting rosR from o-QS04. The glutamine yield of the new strain o-QS08 was increased from 2.4 g / L to 3.0 g / L, and the conversion rate was increased by 0.7% (Table 24).

[0253] Example 7: Construction of o-QS04→o-QS05 strain and performance verification

[0254] 7.1 Plasmid construction

[0255] The upstream homology arm (494 bp) was amplified from the genome of Corynebacterium glutamicum o-QS04 using PQ474-UP-1F / PQ475-UP-1R as primers. The Psod-rarD fragment (1083 bp) was amplified from the genome of Corynebacterium glutamicum o-QS04 using PQ468-Psod-2F / PQ476-rarD-2R as primers. The downstream homology arm (527 bp) was amplified from the genome of Corynebacterium glutamicum o-QS04 using PQ477-DN-3F / PQ478-DN-3R as primers. The pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μl of FastAP and incubating at 37°C for 1 h, followed by gel recovery. The digested vector, UP, Psod-rarD, and DN were assembled at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed to verify the correct transformants, which were then inoculated into test tubes, and the plasmids were digested to verify the correct transformants, which were then sent for sequencing.

[0256] 7.2 Strain construction

[0257] The plasmid was electroporated into o-QS04, and LBHISK15 was coated. The recombinant point LBK25S and LBK25 were selected, and the latter was longer than the former. The correct phenotype was identified by PQ474-UP-1F / P85, P82 / PQ478-DN-3R. The positive control plasmid and the negative control QS12 genome were used. One of each of the one-long and one-short recombinants was selected, inoculated into an antibiotic-free LB test tube overnight, and then diluted by 10, 100, and 1000 times and coated on LBK25, LBK25S, and LBS plates. The recombinant point LBK25 and LBK25S that grew on the LBS plate were selected, and the former did not grow, and the latter grew. The correct phenotype was identified by PQ479-ID-F / PQ480-ID-R. The correct strain was identified by the 2329 bp long band, and then the correct strain was sequenced using the primers. The correct strain was designated as o-QS05.

[0258] 7.3 Performance verification

[0259] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying the glutamine yield in fermentation is shown in Example 1.1.

[0260] Table 25. Glutamine content detection of C. glutamicum o-QS05

[0261]

[0262]

[0263] Strain o-QS05 was obtained by inserting Psod-rarD into C. glutamicum o-QS04, and the glutamine yield of the new strain o-QS05 was increased from 2.4 g / L to 2.9 g / L, and the conversion rate was increased by 0.6% (Table 25).

[0264] Example 8: Construction of o-QS05→o-QS09 strain and performance verification

[0265] 8.1 Plasmid construction

[0266] The upstream homology arm (500 bp) was amplified using C. glutamicum ATCC 14067 genome as template and PQ445-UP-1F / PQ446-UP-1R as primers. The downstream homology arm (500 bp) was amplified using C. glutamicum ATCC 14067 genome as template and PQ447-DN-2F / PQ448-DN-2R as primers. pK18mobsacB was digested with XbaI and HindIII at 37°C for 1 h, and the vector was dephosphorylated by adding 3 μl FastAP and incubating at 37°C for 1 h, and then recovered by gel. The digested vector, UP, and DN were assembled at 37°C for half an hour, and then transformed. Subsequently, colony PCR was performed to verify the correct transformants, which were then inoculated into test tubes, and plasmid digestion was performed to verify the correct transformants, which were then sent for testing.

[0267] 8.2 Strain construction

[0268] The plasmid was electroporated into o-QS05, and LBHISK15 was coated. LBK25S and LBK25 were used for recombination, and the latter was not as long as the former. The correct phenotype was identified by PQ445-UP-1F / P85 and PQ448-DN-2R. The positive control plasmid and the negative control ATCC 14067 genome were used. One of each of the one-long and one-short recombinants was selected, inoculated into an antibiotic-free LB test tube overnight, and then diluted by 10, 100, and 1000 times and coated on LBK25, LBK25S, and LBS plates. The correct phenotype was identified by using PQ449-ID-F / PQ464-ID-R, and the correct strain was identified by using the primers to sequence the 1280 bp fragment. The correct strain was recorded as o-QS09.

[0269] 8.3 Performance verification

[0270] The recombinant C. glutamicum constructed above was fermented to verify its glutamine production performance. The method for verifying glutamine yield in fermentation is shown in Example 1.1.

[0271] Table 26. Glutamine content detection of C. glutamicum o-QS09

[0272]

[0273] Strain o-QS09 was obtained based on strain o-QS05 ΔrosR, i.e. a combination of Psod-rarD and ΔrosR strain. According to the comprehensive Examples 6, 7 and 8, the introduction of ΔrosR increased the conversion rate by 0.7%, the introduction of Psod-rarD increased the conversion rate by 0.6%, and the combination of Psod-rarD and ΔrosR increased the conversion rate by 1.5%, which had an unexpected effect (Table 26).

[0274] INcorporated by reference

[0275] The entire contents of each patent and scientific document referred to herein are incorporated by reference for all purposes.

[0276] Equivalents

[0277] The present disclosure can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments should be regarded as illustrative rather than restrictive, and the scope of the disclosure should be indicated by the appended claims rather than by the description above, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. Modified bacteria producing glutamine, wherein the genome of the modified bacteria comprises a heterologous polynucleotide of glutamine efflux protein (rarD) and a modification of reduced rosR activity as compared to the bacteria before the modification.

2. The modified bacteria of claim 1, wherein the modified bacteria produce glutamine at a higher yield than the bacteria before the modification.

3. The modified bacteria of claim 1, wherein the bacteria are coryneform bacteria, preferably Corynebacterium glutamicum.

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

5. The modified bacteria of any one of claims 1 to 4, comprising more than one copy of the glutamine efflux protein (rarD) gene, preferably two copies, more preferably three copies.

6. The modified bacteria of claim 5, wherein the insertion site of the glutamine efflux protein (rarD) gene is selected from within the acetyltransferase-encoding gene CEY17_08220 ORF or within the glutaminase-encoding gene glsA ORF, preferably the first copy of the glutamine efflux protein (rarD) gene is inserted within the acetyltransferase-encoding gene CEY17_08220 ORF and the second copy is inserted within the glutaminase-encoding gene glsA ORF.

7. The modified bacteria of claim 1, wherein the modification of reduced rosR activity is a deletion or partial deletion of the nucleic acid sequence of the rosR gene.

8. The modified bacteria according to any one of claims 1 to 7, further comprising a modification that increases glnA activity, preferably, said modification that increases glnA activity is glnA comprising Y405F substitution (glnA). Y405F More preferably, the glnA is derived from Corynebacterium glutamicum or Saccharomyces cerevisiae.

9. The modified bacteria of claim 7, comprising one or more copies of the glnA Y405F gene.

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

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

12. The modified bacteria of any one of claims 8 to 10, wherein the glnA Y405F gene has a promoter that is the Psod promoter.

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

14. Use of the modified bacteria of any one of claims 1 to 13 for increasing glutamine production.

15. A method of producing glutamine, comprising culturing the modified bacteria of any one of claims 1 to 13 in a culture medium and isolating glutamine.

Images