Recombinant corynebacterium glutamicum, method for producing glutathione through fermentation of recombinant corynebacterium glutamicum and application of recombinant

Abstract

The invention relates to recombinant corynebacterium glutamicum as well as a method for producing glutathione through fermentation and application thereof. The recombinant corynebacterium glutamicum takes corynebacterium glutamicum as an expression host; a cysteine desulfatase gene and a glutamyltranspeptidase gene are knocked out, and a gene of a glutathione bifunctional enzyme mutant is over-expressed; according to the glutathione bifunctional enzyme mutant, glutathione bifunctional enzyme with the amino acid sequence shown as SEQ ID NO.2 is used as a parent, histidine at the 11th site of the parent glutathione bifunctional enzyme is mutated into proline, and asparagine at the 239th site of the parent glutathione bifunctional enzyme is mutated into glutamic acid. According to the recombinant corynebacterium glutamicum CG05, fermentation parameters such as temperature, pH and supplementary material concentration of a 5L fermentation tank are optimized, the highest yield of glutathione in fermentation liquor of the recombinant corynebacterium glutamicum CG05 is 25.78 + / -0.25 g / L (83.97 + / -0.81 mM), the biomass OD600 is 59.8, the fermentation yield is increased by 2.1 times compared with that of a CG05 strain before optimization, the yield is increased by 5.7 times compared with that of an original strain CG01, and the conversion rate is 83.97%.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a recombinant Corynebacterium glutamicum and a method and application thereof for producing glutathione by fermentation. Background Art

[0002] Glutathione is usually synthesized by γ-glutamylcysteine ​​synthetase (γ-GCS, EC 6.3.2.2, GSH I) and glutathione synthetase (GS, EC 6.3.2.3, GSH II) or bifunctional glutathione synthetase GshF, which requires three precursors, L-glutamate, L-cysteine ​​and L-glycine, and consumes two molecules of ATP. As a non-protein thiol compound, glutathione is widely used in medicine, food, cosmetics and health products, and can be used to stabilize the pigments of chemical and biological reagents. As an environmental pollution indicator; it is used as a food additive to extend the shelf life of meat and fruit, and as a food supplement in capsule form. In recent years, the commercial demand for glutathione has shown an overall trend of growth.

[0003] At present, Yang et al. (Applied Microbiology and Biotechnology (2016), 100(14), 6279-6289. After adding 80mM ATP, the whole-cell catalytic system finally produced 11.2g / L glutathione; Chen et al. (World Journal of Microbiology and Biotechnology (2020), 36(8), 117.) systematically explored factors such as temperature and pH during the fermentation process and studied a method for gradually regulating yeast glutathione synthesis based on oxidative stress and energy metabolism. The glutathione accumulation increased to 5.76g / L, which was 2.84 times that of the control group; Liu et al. (Journal of Industrial Microbiology and Biotechnology (2019), 46 (12), 1685-1695) used GshF in Corynebacterium glutamicum to produce glutathione, transformed the key precursor L-cysteine ​​pathway, and fermented to produce 756 mg / L glutathione; Wei Jianguo et al. needed to add three amino acids, L-glutamate, L-glycine and L-cysteine, during the fermentation process, and used recombinant Corynebacterium glutamicum to synthesize glutathione, with a yield of 24.95 g / L and a conversion rate of 81.2% (Chinese patent, CN112646768A, 2021). However, glutathione synthetase has limitations in ATP energy supply and short half-life in enzymatic synthesis, and chemical synthesis is not specific. The feedback inhibition of GSH in yeast on the key enzyme GSH I and the low tolerance of Escherichia coli to the precursor cysteine ​​lead to the fact that GSH production and productivity are usually quite low; at the same time, fermentation parameters such as temperature, pH, ventilation, and stirring will also affect the stability of the product glutathione; the addition of three substrate amino acids will cause high cost problems.

[0004] Corynebacterium glutamicum is the core strain of the amino acid fermentation industry. It has good substrate tolerance. Due to its biosafety (generally regarded as safe by the US FDA), natural L-glutamate (GSH precursor) secreting strain, and wide substrate spectrum, it is widely used in the fermentation production of glutamate, lysine, and valine. Therefore, the screening of chassis cells, the reconstruction of multi-module metabolic pathways, and the optimization of fermentation parameters are necessary conditions for improving glutathione biosynthesis. The development of a method to increase the production of glutathione by food safety strain Corynebacterium glutamicum can simplify the production process, improve the synthesis efficiency, and reduce the production cost, which has important practical significance for the application of glutathione. In addition, single-cell protein (SCP) obtained from microorganisms has great potential as a successor to plant and insect proteins. SCP based on Corynebacterium glutamicum can be used as animal feed to support the growth of pigs and improve the intestinal flora of animals; it also has an anti-obesity effect. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a recombinant Corynebacterium glutamicum and a method and application thereof for producing glutathione by fermentation. The present invention overexpresses the glutathione bifunctional enzyme GshF from Streptococcus lactis in Corynebacterium glutamicum E01 to produce glutathione by fermentation; through site-directed mutagenesis, the enzyme activity of GshF is increased, and the aceD and ggt genes in the key metabolic pathway are knocked out at the same time, and the fermentation parameters of the temperature, pH and feed concentration of a 5L fermenter are optimized.

[0006] The present invention constructs a recombinant Corynebacterium glutamicum capable of producing high glutathione, and performs amino acid nutrition analysis on the strain to develop its potential value as an alternative protein in animal feed.

[0007] In one embodiment of the present invention, the nucleotide sequence of the glutathione bifunctional enzyme GshF derived from Streptococcus lactis is shown as SEQ ID NO.1.

[0008] In one embodiment of the present invention, the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as a host.

[0009] The present invention is achieved through the following technical solutions:

[0010] The first object of the present invention is to provide a recombinant Corynebacterium glutamicum, wherein the recombinant Corynebacterium glutamicum uses Corynebacterium glutamicum as an expression host; knocks out the cysteine ​​desulfurase gene and the glutamyl transpeptidase gene, and overexpresses the gene of the glutathione bifunctional enzyme mutant;

[0011] The glutathione bifunctional enzyme mutant uses the glutathione bifunctional enzyme with an amino acid sequence as shown in SEQ ID NO.2 as a parent, and the histidine at the 11th position of the parent glutathione bifunctional enzyme is mutated to proline, and the asparagine at the 239th position is mutated to glutamate.

[0012] In one embodiment of the present invention, the nucleotide sequence of the upstream 500bp of the cysteine ​​desulfurase gene is shown as SEQ ID NO 4; the nucleotide sequence of the downstream 500bp of the cysteine ​​desulfurase gene is shown as SEQ ID NO 5; the nucleotide sequence of the upstream 500bp of the glutamyl transpeptidase gene is shown as SEQ ID NO 6; the nucleotide sequence of the downstream 500bp of the glutamyl transpeptidase gene is shown as SEQ ID NO 7.

[0013] In one embodiment of the present invention, the Corynebacterium glutamicum is Corynebacterium glutamicum E01.

[0014] In one embodiment of the present invention, the glutathione bifunctional enzyme is derived from Streptococcus lactis.

[0015] The second object of the present invention is to provide a method for producing glutathione by fermentation, using L-cysteine ​​and L-glycine as substrates, and adding the recombinant Corynebacterium glutamicum described in any one of claims 1 to 4 into the fermentation system to ferment and produce glutathione.

[0016] In one embodiment of the present invention, the concentration of L-cysteine ​​is 30 mM to 100 mM; the concentration of L-glycine is 30 mM to 100 mM.

[0017] In one embodiment of the present invention, the inoculation amount of the recombinant Corynebacterium glutamicum is 0.5 mL-1 mL.

[0018] In one embodiment of the present invention, the fermentation medium in the fermentation system includes: 100g / L-150g / L glucose, 0.5g / L-1g / LK2HPO4, 1g / L-1.5g / LMgSO4, 5g / L-10g / L corn steep liquor, 0.001g / L-0.005g / LFeSO4-7H2O, 0.001g / L-0.005g / LMnSO4-H2O, and 5g / L-10g / L urea.

[0019] In one embodiment of the present invention, the fermentation parameters are: 30°C to 45°C, 300rpm to 800rpm, and a pH value of 7.0 to 8.5 for 24h to 60h.

[0020] The third object of the present invention is to provide the use of the recombinant Corynebacterium glutamicum in preparing animal feed.

[0021] The above technical solution of the present invention has the following advantages compared with the prior art:

[0022] The present invention provides a recombinant Corynebacterium glutamicum and a method and application thereof for producing glutathione by fermentation. The present invention performs combined mutation on a glutathione bifunctional enzyme GshF from Streptococcus lactis, mutates the histidine at position 11 to proline, and mutates the asparagine at position 239 to glutamate. The specific enzyme activity of the mutant enzyme is 3.89 U / mg, which is 3.7 times higher than that before the mutation. H11P-N239E The catalytic activity of the enzyme was significantly improved. Overexpression of GshF in a high-L-glutamic acid-producing Corynebacterium glutamicum E01 strain H11P-N239E , fermentation to produce glutathione; at the same time, the aceD and ggt genes were knocked out, and the fermentation parameters of temperature, pH, and feed concentration of the 5L fermenter were optimized. The maximum glutathione production of the recombinant Corynebacterium glutamicum CG05 was 25.78±0.25g / L (83.97±0.81mM), and the biomass OD 600 The yield was 59.8, which was 2.1 times higher than that of the fermentation yield of the CG05 strain before optimization and 5.7 times higher than that of the starting strain CG01. The conversion rate was 83.97%.

[0023] Compared with previous studies on glutathione production, the present invention provides a method for constructing an engineered bacterium for producing glutathione by Corynebacterium glutamicum, and no substrate L-glutamate needs to be added during the fermentation process, thereby reducing production costs and increasing conversion rates. After optimizing fermentation parameters, glutathione production is significantly improved. These strategies provide effective strategies for metabolic modification of glutathione production by other strains and optimization of fermentation parameters, and are of great significance to the fermentation production of glutathione.

[0024] Recombinant Corynebacterium glutamicum CG05 was prepared into bacterial protein, wherein the crude protein content was 65.6% and the crude fat content was 3.1%, which was higher than some common additives in animal feed. Rich branched-chain amino acids (19.51%) are beneficial to improving athletic ability and delaying fatigue, which is beneficial to the growth of animals; sulfur-containing amino acids (16.92%) are beneficial to the balance and stability of free radicals in animals; at the same time, the biomass of recombinant Corynebacterium glutamicum CG05 bacterial protein has 15.04% glutamic acid, which is a flavoring substance that can increase the feed intake of animals, thereby improving the growth performance of animals. In addition, there is still 1.12% glutathione in the bacterial protein of recombinant Corynebacterium glutamicum CG05, which can improve the antioxidant effect in animals and maintain important physiological functions related to animal nutrition and immunity. Therefore, the fermentation of recombinant Corynebacterium glutamicum CG05 to produce glutathione, and its bacterial protein can be used as a nutritional additive for animal feed, which has potential application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0026] Figure 1 is the biomass and yield of glutathione produced by fermentation of the engineered strains CG01, CG02, CG04 and CG05 in Example 6 of the present invention;

[0027] Figure 2 It is the biomass and yield of glutathione produced by the CG05 engineered strain after the fermentation parameters are optimized in Example 7 of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0030] The Corynebacterium glutamicum E01 involved in the following examples is recorded in the patent application with publication number CN103215198A.

[0031] The culture medium involved in the following embodiments is:

[0032] Corynebacterium glutamicum competent medium: 1% NaCl, 1% tryptone, 0.5% yeast extract, 0.3% glycine, 0.7% glucose, 0.1% tween-80.

[0033] LB liquid medium: 1% NaCl, 1% tryptone, 0.5% yeast extract. Solid medium: add 2% agar powder.

[0034] Corynebacterium glutamicum BHI liquid medium: 3.85% brain heart infusion broth powder.

[0035] Corynebacterium glutamicum fermentation medium: 15% glucose, 0.1% K2HPO4, 0.06% MgSO4, 0.5% corn steep liquor, 0.005% FeSO4-7H2O, 0.005% MnSO4-H2O, 0.7% urea.

[0036] The detection methods involved in the following embodiments are as follows:

[0037] The content of glucose in the fermentation broth was determined using a biosensor analyzer (SBA-40ES, Institute of Biology, Shandong Academy of Sciences); the biomass OD was detected using a spectrophotometer. 600 ; The content of glutathione was determined by HPLC (Thermo Fisher), using a C18 column, the mobile phase was a 95% phosphate aqueous solution by volume, the flow rate was set to 1 mL / min, the column temperature was maintained at 30°C, and the quantitative detection was performed at an ultraviolet wavelength of 210 nm. The amino acid composition of biomass was detected by high performance liquid chromatography, using a C18 column, the mobile phase was a 92% sodium acetate aqueous solution and an 8% methanol acetonitrile solution by volume, the flow rate was set to 1 mL / min, the column temperature was maintained at 40°C, and the quantitative detection was performed at an ultraviolet wavelength of 338 nm. GB / T6432 determines crude protein in biomass, and GB5009.6-2016 determines crude fat in biomass.

[0038] Example 1: Combination mutation of glutathione bifunctional enzyme GshF

[0039] (1) Preparation of competent E. coli

[0040] Streak E. coli BL21 on an antibiotic-free LB plate, place in a 37°C incubator, pick a colony and inoculate it into a 10mL LB vial for 12 hours, transfer 1% of the inoculation volume to a 50mL LB medium bottle, and when the bacterial concentration reaches 0.4-0.6, prepare the preparation of the competent state of the large intestine. Pre-cool the relevant reagents and instruments in advance, prepare the calcium chloride solution, the mixed solution of calcium chloride and glycerol, the 1.5mL EP tube and the 50mL centrifuge tube and place them on ice, and control the temperature of the centrifuge to 4°C. Divide 50mL of the bacterial solution on the sterile operating table and centrifuge (8000r·min -1 , 5min), remove the supernatant. Pipette 5mL of calcium chloride solution for suspension by suction, and centrifuge (8000r·min -1 , 5min), remove the supernatant. Pipette 5mL of calcium chloride and glycerol mixed solution to suspend by pipetting, and dispense into pre-cooled EP tubes, with 100μL in each tube.

[0041] (2) Construction of recombinant E. coli / pET28a-GshF

[0042] According to the gene sequence of the glutathione bifunctional enzyme GshF from Streptococcus agalactiae published on the NCBI website (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2), it was sent to Suzhou Genewise Co., Ltd. for synthesis. The PCR program was: 95°C, 10

[0043] min; 95℃, 30s; 58℃, 30s; 72℃, 1min; 72℃, 10min, 30 cycles. The obtained gshF gene fragments were purified and connected to the linearized plasmid pET-28a (P1 / P2) through the homologous recombinase In-FusionSnapAssemblyMasterMi (Takara), and transformed into E.coliBL21 (DE3) competent cells to obtain transformants. The transformants were spread on LB solid culture medium containing 50μg / mL kanamycin, cultured at 37℃ for 12h, and positive colonies were picked. P3 / P4 was used as primers to verify the single colony by colony PCR with Taq DNA polymerase; the positive single colony with the target band size was inoculated in a vial containing LB liquid culture medium and cultured for 12h, and the plasmid was extracted and sent to Jinweizhi Company for sequencing. If it is correct, the recombinant E. coli E.coli / pET28a-GshF was successfully constructed. The primer sequences involved are as follows:

[0044] p1:5'-caaatgggtcgcggatccgaattcgtgattattgatagactgctgcag-3'

[0045] p2:5'-ctggccaaactgtttcctgaactgtaaaagcttgcggccgcactcgag-3'

[0046] P3: 5'-agaggatcgagatctcgatcccgc-3'

[0047] P4: 5'-atccggatatagttcctcctttca-3'

[0048] (3) Recombinant E. coli / pET28a-GshF H11P-N239E Construction

[0049] Using pET28a-GshF as a template, 11 mutation sites were amplified by PCR using P5 / P6 as primers, respectively, and transformed into E. coli BL21 (DE3) competent cells to obtain transformants. The transformants were spread on LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 hours. Positive colonies were picked and verified by colony PCR using Taq DNA polymerase using P3 / P4 as primers for single colonies. Positive single colonies with the target band size were inoculated in a vial containing LB liquid medium and cultured for 12 hours. The plasmid was extracted and sent to Genewise for sequencing to verify that the 11 mutation sites of GshF were successfully mutated. The single mutant strain E. coli / pET28a-GshF H11P The construction was successful. H11P As templates, PCR amplification of the 239 mutation site was performed with P7 / P8 as primers, and the site was transformed into E. coli BL21 (DE3) competent cells to obtain transformants. The transformants were spread on LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. Positive colonies were picked and verified by colony PCR using Taq DNA polymerase with P3 / P4 as primers. The positive single colony with the target band size was inoculated in a vial containing LB liquid medium and cultured for 12 h. The plasmid was extracted and sent to GeneWeichi for sequencing to verify that the 239 mutation site of GshF was successfully mutated. The mutant strain E. coli / pET28a-GshF H11P-N239E The construction was successful (the amino acid sequence after combined mutations is shown in SEQ ID NO.3).

[0050] P5:5'-attgatagactgctgcagagaagcccaagccatctg-3'

[0051] P6: 5'-gcagagaagcccaagccatctgccaattctgcaagcca-3'

[0052] P7: 5'-ctatactagcctgaaagattatgtggaagatctggaaa-3'

[0053] P8:5'-aagattatgtggaagatctggaaaatgcagtgaaaagt-3'

[0054] Example 2: Inducible expression and enzyme activity determination of glutathione bifunctional enzyme and mutant enzyme

[0055] Take the E.coli / pET28a-GshF and E.coli / pET28a-GshF successfully constructed in Example 1 H11P-N239E A single colony was inoculated into 10 mL of LB liquid medium and cultured at 37 °C for 12 h. A 1% inoculum was inoculated into 50 mL of LB liquid medium and cultured until OD 600 The pH value is about 0.8. IPTG is added and cultured at 20℃ for 16h. Wash the cells three times with PBS, resuspend the collected bacteria with PBS, and break the cells with an ultrasonic disruptor. Break E. coli for 1s and stop for 3s, and break for a total of 15min. Centrifuge at 10000rpm for 20min, take the supernatant and run on protein gel. Purify the crude enzyme solution through a protein purification nickel column, run the pure enzyme on protein gel for verification, and set aside.

[0056] The original enzyme GshF and the mutated pure enzyme GshF were respectively H11P-N239E Add 100mM Tris-HCl buffer, which contains 50mM L-glutamic acid, 50mM L-cysteine, 50mM L-glycine, and 100mM ATP. React at 37°C for 20min, add 10% trichloroacetic acid to terminate the reaction, centrifuge at 10000rpm for 10min, and measure the absorbance of the supernatant at 305nm. One unit of GshF enzyme activity (1U) is defined as the amount of enzyme required to produce 1μmol of glutathione per minute.

[0057] The experimental results are shown in Table 1. The specific activity of the original enzyme was 1.05 U / mg. The specific activity of the mutant enzyme after the histidine at position 11 was mutated to proline and the asparagine at position 239 was mutated to glutamate was 3.89 U / mg, which was 3.7 times higher than that before the mutation. H11P-N239E Significantly improved the catalytic activity of the enzyme.

[0058] Table 1 Specific enzyme activity of original enzyme and mutant enzyme

[0059]

[0060] Example 3: Recombinant Corynebacterium glutamicum Cg / pXMJ-19-GshF and Cg / pXMJ-19-GshF H11P-N239E Construction

[0061] (1) Preparation of competent cells of Corynebacterium glutamicum

[0062] Corynebacterium glutamicum E01 was selected and inoculated into 10 mL of BHI liquid medium, and cultured at 30 °C in a shaking incubator for 24 h. The cultured bacterial liquid was transferred into a BHI competent medium so that the initial cell OD 600 The cell OD reached 0.3. Culture at 30°C and 200 rpm until the cell OD 600 After the cell culture is completed, precool the bacterial solution for 30 minutes, then collect the cells by centrifugation. Wash the cells three times with precooled 10% glycerol, and finally resuspend the cells with 1mL 10% glycerol, and dispense them into 1.5mL tubes, with 100μL in each tube directly used for electroporation.

[0063] (2) Overexpression plasmids pXMJ-19-GshF and pXMJ-19-GshF H11P-N239E Construction

[0064] gshF and gshF H11P-N239E After purification, the gene fragments were connected with the linearized plasmid pXMJ-19 (P9 / P10) through the homologous recombination enzyme In-Fusion SnapAssembly MasterMi (Takara), transformed into E. coli BL21 (DE3) competent cells to obtain transformants, which were spread on LB solid culture medium containing chloramphenicol at a concentration of 20 μg / mL and cultured at 37°C for 12 hours. Positive colonies were picked and verified by colony PCR using Taq DNA polymerase with P11 / P12 as primers for single colonies; positive single colonies with the target band size were inoculated in a vial containing LB liquid culture medium and cultured for 12 hours, and the plasmids were extracted and sent to GeneWeichi for sequencing. If the sequence is correct, pXMJ-19-GshF and pXMJ-19-GshF H11P-N239E The plasmid was constructed successfully. The primer sequences involved are as follows:

[0065] P9:5'-acacaggaaacagaattaattaagctt-3'

[0066] P10: 5'-gcatgcctgcaggtcgactctagag-3'

[0067] P11: 5'-ctggcaaatattctgaaatgagctg-3'

[0068] P12: 5'-gcagttccctactctcgcat-3'

[0069] (3) Recombinant Corynebacterium glutamicum Cg / pXMJ-19-GshF (CG01) and Cg / pXMJ-19-GshF H11P-N239E Construction of (CG02)

[0070] The pXMJ-19-GshF and pXMJ-19-GshF obtained in step (2) were H11P-N239E The plasmids were electroporated into competent cells of Corynebacterium glutamicum E01 to obtain transformants, which were then spread on a BHI solid medium containing 20 μg / mL chloramphenicol resistance and cultured at 30°C for 2-4 days. Positive colonies were picked and verified by colony PCR using Taq DNA polymerase using P3 / P4 as primers. Positive single colonies with the target band size indicated that the recombinant strains Cg / pXMJ-19-GshF (CG01) and Cg / pXMJ-19-GshF H11P-N239E (CG02) Build successful.

[0071] Example 4: Recombinant Corynebacterium glutamicum Cg ΔaceD Construction of (CG03)

[0072] (1) Knockout plasmid PK18-aceD 上500bp -aceD 下500bp Construction

[0073] To reduce the degradation of substrate L-cysteine, the cysteine ​​desulfurase aceD gene in the genome of Corynebacterium glutamicum was knocked out. According to the upstream and downstream 500bp of aceD in the whole genome nucleic acid sequence of Corynebacterium glutamicum in NCBI, primers P13 / P14 and P15 / P16 were designed to obtain aceD 上500bp and aceD 下500bp(The nucleotide sequences of 500bp upstream and downstream of aceD are shown in SEQ ID NO.4 and SEQ ID NO.5), the obtained gene fragment was fused with the linearized plasmid pK18mobsacB (P17 / P18), connected by homologous recombinase In-Fusion SnapAssembly MasterMi (Takara), and transformed into E. coli BL21 (DE3) competent cells to obtain transformants, which were spread on LB solid culture medium containing 50 μg / mL kanamycin, cultured at 37°C for 12 hours, and positive colonies were picked. P19 / P20 was used as primers to verify the single colony by colony PCR with Taq DNA polymerase; the positive single colony with the target band size was inoculated in a vial containing LB liquid culture medium and cultured for 12 hours, and the plasmid was extracted and sent to Jinweizhi Company for sequencing. If it is correct, then PK18-aceD 上500bp -aceD 下500bp The plasmid was constructed successfully. The primer sequences involved are as follows:

[0074] P13: 5'-Gtgggtggatcgatggcgtcagct-3'

[0075] P14: 5'-Acctgcccgccgttgaaggaagca-3'

[0076] P15:5'-Ataatgagtaaaaagtctgtcctg-3'

[0077] P16:5'-gtcggtagccaagttggatggcgc-3'

[0078] P17:5'-acagctatgacatgattacgaattcgtgggtggatcgatggcgtcagc-3'

[0079] P18:5'-gtcggtagccaagttggatggcgcaagcttggcactggccgtcgt-3'

[0080] P19: 5'-ccgactggaaagcgggcagtgagc-3'

[0081] P20: 5'-attaagttgggtaacgccagggtt-3'

[0082] (2) Recombinant Corynebacterium glutamicum Cg ΔaceD Construction of (CG02)

[0083] The PK18-aceD obtained in step (1)上500bp -aceD 下500bp The plasmid was electroporated into competent cells of Corynebacterium glutamicum E01 to obtain transformants, which were then spread on a solid medium containing 50 μg / mL kanamycin-resistant BHI and cultured at 30°C for 2-4 days. Positive colonies were picked and verified by colony PCR using Taq DNA polymerase using P11 / P12 as primers. A positive single colony with the target band size indicated that the recombinant strain Cg / PK18-aceD 上500bp -aceD 下500bp The correct transformants were then inoculated into seed liquid without kanamycin for screening, and diluted and coated into seed culture medium containing 10% sucrose to induce a second round of homologous recombination. Finally, sequencing verification confirmed that the aceD gene was successfully knocked out, and the knockout strain Cg ΔaceD (CG03) Build successful.

[0084] Example 5: Recombinant Corynebacterium glutamicum Cg ΔaceDΔggt / pXMJ-19-GshF(CG04) and Cg ΔaceDΔggt / pXMJ-19-GshF H11P-N239E Construction of (CG05)

[0085] (1) Knockout plasmid PK18-ggt 上500bp -ggt 下500bp Construction

[0086] In order to reduce the degradation of product glutathione, the glutamyl transpeptidase ggt gene in the genome of Corynebacterium glutamicum was knocked out. According to the upstream and downstream 500bp of ggt in the whole genome nucleic acid sequence of Corynebacterium glutamicum in NCBI, primers P21 / P22 and P23 / P24 were designed to obtain ggt 上500bp and ggt 下500bp (The nucleotide sequences of 500bp upstream and downstream of ggt are shown in SEQ ID NO.6 and SEQ ID NO.7). The obtained gene fragments were fused with the linearized plasmid pK18mobsacB (P25 / P26), connected with the homologous recombinase In-Fusion Snap Assembly MasterMi (Takara), and transformed into E. coli BL21 (DE3) competent cells to obtain transformants. The transformants were spread on LB solid culture medium containing 50μg / mL kanamycin, cultured at 37°C for 12h, and positive colonies were picked. P19 / P20 was used as primers to verify the single colony by colony PCR with Taq DNA polymerase; the positive single colony with the target band size was inoculated in a vial containing LB liquid culture medium and cultured for 12h, and the plasmid was extracted and sent to Jinweizhi Company for sequencing. If it is correct, then PK18-ggt上500bp -ggt 下500bp The plasmid was constructed successfully. The primer sequences involved are as follows:

[0087] P21:5'-ttatgcagccaagaggtggctcgg-3'

[0088] P22: 5'-aaagttccttgataggctcgagag-3'

[0089] P23: 5'-ttagcgcgcagaccacgctgacca-3'

[0090] P24:5'-tgaccatcgcgatcttctggcgca-3'

[0091] P25:5'-tgaccatcgcgatcttctggcgcaaagcttggcactggccgtcgtttt-3'

[0092] P26:5'-cagctatgacatgattacgaattctttatgcagccaagaggtggctcgg-3'

[0093] (2) Recombinant Corynebacterium glutamicum Cg ΔaceDΔggt Construction

[0094] The PK18-aceD obtained in step (1) 上500bp -aceD 下500bp glutamicum Cg ΔaceD The competent cells were transformed and the transformants were spread on a BHI solid medium containing 50 μg / mL kanamycin resistance and cultured at 30°C for 2-4 days. Positive colonies were picked and verified by colony PCR using Taq DNA polymerase with P11 / P12 as primers. A positive single colony with the target band size indicated that the recombinant strain Cg ΔaceD / PK18-ggt 上500bp -ggt 下500bp The construction was successful. The correct transformants were then inoculated into seed liquid without kanamycin for screening, and diluted and coated into seed culture medium containing 10% sucrose to induce a second round of homologous recombination. Finally, sequencing verification proved that the ggt gene was successfully knocked out, and the knockout strain Cg ΔaceDΔggt Build successful.

[0095] (3) Recombinant Corynebacterium glutamicum Cg ΔaceDΔggt / pXMJ-19-GshF(CG04) and Cg ΔaceDΔggt / pXMJ-19-GshF H11P-N239E Construction of (CG05)

[0096] The pXMJ-19-GshF and pXMJ-19-GshF H11P-N239E glutamicum Cg ΔaceDΔggt The competent cells were transformed and the transformants were spread on a BHI solid medium containing 20 μg / mL chloramphenicol resistance and cultured at 30°C for 2-4 days. Positive colonies were picked and verified by colony PCR using Taq DNA polymerase with P11 / P12 as primers. A positive single colony with the target band size indicated that the recombinant strain Cg ΔaceDΔggt / pXMJ-19-GshF(CG04) and Cg ΔaceDΔggt / pXMJ-19-GshF H11P-N239E (CG05) Build successful.

[0097] Example 6: Fermentation of glutathione by recombinant Corynebacterium glutamicum

[0098] 1. Bacterial strains: Recombinant bacteria Corynebacterium glutamicum CG01, CG02, CG04 and CG05 were used for fermentation production of glutathione.

[0099] 2. Fermentation medium: 100g / L-150g / L glucose, 0.5g / L-1g / L K2HPO4, 1g / L-1.5g / LMgSO4, 5g / L-10g / L corn steep liquor, 0.001g / L-0.005g / LFeSO4-7H2O, 0.001g / L-0.005g / LMnSO4-H2O, 5g / L-10g / L urea.

[0100] 3. Fermentation culture of Corynebacterium glutamicum: Use an inoculation loop to streak CG01, CG02, CG04 and CG05 strains on seed culture medium solid plates, place the plates in a constant temperature incubator at 30°C to culture single colonies, inoculate into primary seed BHI liquid culture medium, culture at 30°C, 120rpm to mid-logarithmic growth period, inoculate 0.5mL-1mL into 100mL of secondary seed fermentation medium, and ferment at 30°C, 120rpm.

[0101] 4. Fermentation and production of glutathione in a 5L fermenter: Transfer the secondary seed liquid of CG01, CG02, CG04 and CG05 to 5L fermentation medium at a transfer volume of 200mL-400mL, respectively, and ferment for 72h at 30℃, 300rpm-800rpm, ventilation volume 2L / min-4L / min, and pH7.0. Add IPTG with a final concentration of 0.5mM for induction after 12h of culture, and add 30mM-100mM L-cysteine ​​and L-glycine for 24h of fermentation (Corynebacterium glutamicum E01 is the dominant strain for producing L-glutamic acid, so L-glutamic acid does not need to be added during fermentation). Take samples every 12h, and measure the biomass (OD 600 ). The concentration of glutathione in the fermentation broth was determined by HPLC. The experimental results are shown in Figure 1 As shown in Table 2:

[0102] Table 2 Biomass and yield of glutathione produced by fermentation of CG01, CG02, CG04 and CG05 engineered strains

[0103]

[0104] Depend on Figure 1 As shown in Table 2, the CG01 strain without knockout of aceD and ggt genes overexpressed the glutathione bifunctional enzyme GshF, and the maximum glutathione production by fermentation was 4.56±0.13 g / L at 48 h, and the biomass OD 600 was 45.4; while overexpression of mutant enzyme GshF H11P-N239E After that, the maximum yield of glutathione produced by fermentation was 6.56±0.26g / L at 48h, and the biomass OD 600 After knocking out the aceD and ggt genes and overexpressing the glutathione bifunctional enzyme GshF, the maximum glutathione production of CG04 strain was 8.13±0.20 g / L at 48 h, and the biomass OD 600 Knockout of aceD and ggt genes and overexpression of mutant enzyme GshF H11P-N239E After fermentation, the glutathione production of CG05 strain increased to 12.56±0.32g / L, which was 1.5 times higher than that of CG04. 600 In general, after knocking out the aceD and ggt genes and overexpressing the mutant enzyme GshF H11P-N239E After that, the production of glutathione by fermentation of engineered strain CG05 was significantly increased, 2.8 times higher than that of the original strain CG01. H11P-N239E The catalytic efficiency is enhanced.

[0105] Example 7: Preparation of recombinant Corynebacterium glutamicum CG05 bacterial protein and analysis of its nutritional properties

[0106] 1. Bacterial strain: The recombinant bacterium Corynebacterium glutamicum CG05 was used for the fermentation production of glutathione, and the fermentation parameters were optimized. The culture medium and fermentation culture were the same as those in Example 6.

[0107] 2. Fermentation parameters of the fermenter: Transfer the secondary seed liquid of CG05 to 5L fermentation medium in a transfer volume of 200mL-400mL, and ferment for 72h at 300rpm-800rpm and aeration volume of 2L / min-4L / min. Add IPTG with a final concentration of 0.5mM for induction after 12h of cultivation. The pH, temperature and time of adding fermentation substrates during fermentation were optimized. The specific content of the fermentation parameter optimization is as follows: During the fermentation process, the pH was controlled between 7.0 and 8.5, the temperature was controlled between 30℃ and 45℃, and the substrate concentration added within 24h to 60h of fermentation was 30mM to 100mM of L-cysteine ​​and L-glycine (the control and addition of pH, temperature and fermentation substrates are dynamic processes). Samples were taken every 12h, and the biomass (OD 600 ).

[0108] 3. Preparation of single cell raw materials: The concentration of glutathione in the fermentation supernatant was determined by HPLC. The cells were collected by centrifugation and washed with sterile water. The collected cells were further freeze-dried and ground to obtain powdered biomass as the single cell protein raw material. The experimental results are shown in Figure 2 and Table 3:

[0109] Table 3 Biomass and glutathione production capacity of CG05 strain after optimization of fermentation parameters

[0110]

[0111] Depend on Figure 2 As can be seen from Table 3, in order to further increase the glutathione production, the fermentation parameters were optimized. At 30℃~45℃, pH value was 7.0~8.5, 30mM~100mM L-cysteine ​​and L-glycine were added within 24h~60h, and after 48h of fermentation in a 5L fermenter, the maximum glutathione production of recombinant Corynebacterium glutamicum CG05 in the supernatant fermentation liquid was 25.78±0.25g / L (83.97±0.81mM), and the biomass OD 600 The conversion rate of the CG05 engineered strain was 83.97%, which was calculated based on the substrate L-cysteine.

[0112] The nutritional components of the recombinant Corynebacterium glutamicum CG05 bacterial protein of the present invention are shown in Table 4, wherein the crude protein content is 65.6% and the crude fat content is 3.1%. The crude protein content in the Corynebacterium glutamicum CG05 bacterial body is higher than the common additives in animal feed (whole cottonseed: 23%; soybean hulls: 13%; corn stalks: 5% to 9%) (data source: China Feed Composition and Nutritional Value Table (2022, 33rd Edition)); the crude fat content is higher than that of feed raw materials such as wheat (1.7%) and soybean meal (1.9%) (data source: China Feed Composition and Nutritional Value Table (2022, 33rd Edition)). Therefore, the bacterial protein of recombinant Corynebacterium glutamicum CG05 can not only be used as a high-quality alternative protein raw material, but also a source of high-quality fat.

[0113] Table 4 Analysis of nutritional components of protein in CG05 strain

[0114]

[0115] The quality of recombinant Corynebacterium glutamicum CG05 is determined by the protein content and the amino acid composition of the protein, especially the content of essential amino acids. Table 5 shows the amino acid analysis results of the recombinant Corynebacterium glutamicum CG05 bacterial protein. There are 9 essential amino acids, namely lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine and histidine. Recombinant Corynebacterium glutamicum CG05 contains all essential amino acids, accounting for 42.34% of the total amino acid content, which is higher than the 40% specified in the FAO / WHO standard. Soybean is a common additive in feed. Table 5 lists the amino acid content of soybean (data source: U.S. Department of Agriculture Food Database, NDB Number: 16119). Compared with soybean, the content of branched-chain amino acids (leucine, valine, isoleucine) in CG05 is 19.51%, which is higher than soybean (17%). Supplementing branched-chain amino acids can improve exercise capacity and delay fatigue, which is beneficial to the growth of animals. The content of sulfur-containing amino acids (methionine, cysteine) in CG05 is 16.92%, which is significantly higher than 2.80% of soybeans. Sulfur-containing amino acids are beneficial to the balance and stability of free radicals in animals; at the same time, the recombinant Corynebacterium glutamicum CG05 bacterial protein biomass has a high content of flavor substances, such as glutamic acid (15.04%), which can increase the feed intake of animals, thereby improving the growth performance of animals. In addition, there is still 1.12% glutathione in the recombinant Corynebacterium glutamicum CG05 bacterial protein, which can improve the antioxidant effect in animals and maintain important physiological functions related to animal nutrition and immunity. Therefore, the fermentation of recombinant Corynebacterium glutamicum CG05 to produce glutathione, and its bacterial protein can be used as a nutritional additive for animal feed, which has potential application prospects.

[0116] Table 5 Analysis of amino acid content of CG05 bacterial protein

[0117]

[0118] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A recombinant Corynebacterium glutamicum, characterized in that The recombinant Corynebacterium glutamicum uses Corynebacterium glutamicum as an expression host; knocks out the cysteine ​​desulfurase gene and the glutamyl transpeptidase gene, and overexpresses the gene of the glutathione bifunctional enzyme mutant; The glutathione bifunctional enzyme mutant uses the glutathione bifunctional enzyme with an amino acid sequence as shown in SEQ ID NO.2 as a parent, and the histidine at the 11th position of the parent glutathione bifunctional enzyme is mutated to proline, and the asparagine at the 239th position is mutated to glutamate.

2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that The nucleotide sequence of the upstream 500bp of the cysteine ​​desulfurase gene is shown as SEQ ID NO 4; the nucleotide sequence of the downstream 500bp of the cysteine ​​desulfurase gene is shown as SEQ ID NO 5; the nucleotide sequence of the upstream 500bp of the glutamyl transpeptidase gene is shown as SEQ ID NO 6; and the nucleotide sequence of the downstream 500bp of the glutamyl transpeptidase gene is shown as SEQ ID NO 7.

3. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that The Corynebacterium glutamicum is Corynebacterium glutamicum E01.

4. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that The glutathione bifunctional enzyme is derived from Streptococcus lactis.

5. A method for producing glutathione by fermentation, characterized in that: L-cysteine ​​and L-glycine are used as substrates, and the recombinant Corynebacterium glutamicum described in any one of claims 1 to 4 is added to the fermentation system to ferment and produce glutathione.

6. The method according to claim 5, characterized in that The concentration of the L-cysteine ​​is 30mM to 100mM; the concentration of the L-glycine is 30mM to 100mM.

7. The method according to claim 5, characterized in that The inoculation amount of the recombinant Corynebacterium glutamicum is 0.5 mL-1 mL.

8. The method according to claim 5, characterized in that The fermentation medium in the fermentation system includes: 100g / L-150g / L glucose, 0.5g / L-1g / L K2HPO4, 1g / L-1.5g / L MgSO4, 5g / L-10g / L corn steep liquor, 0.001g / L-0.005g / L FeSO4-7H2O, 0.001g / L-0.005g / L MnSO4-H2O, and 5g / L-10g / L urea.

9. The method according to claim 5, characterized in that The fermentation parameters are: 30°C to 45°C, 300rpm to 800rpm, and a pH value of 7.0 to 8.5 for 24h to 60h.

10. Use of the recombinant Corynebacterium glutamicum according to any one of claims 1 to 4 in preparing animal feed.

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