Recombinant Corynebacterium glutamicum and method and application for fermenting and producing glutathione by using the same

Abstract

The present invention relates to a recombinant Corynebacterium glutamicum and a method and application for fermenting and producing glutathione by using the same. The recombinant Corynebacterium glutamicum in the present invention uses Corynebacterium glutamicum as an expression host; the cysteine desulfurase gene and the glutamyl transpeptidase gene are knocked out, and the gene of a glutathione bifunctional enzyme mutant is overexpressed; the glutathione bifunctional enzyme mutant uses the glutathione bifunctional enzyme with the amino acid sequence shown in SEQ ID NO.2 as a parent, and the histidine at the 11th site of the parent glutathione bifunctional enzyme is mutated into proline, and the asparagine at the 239th site is mutated into glutamic acid. The present invention optimizes the fermentation parameters of temperature, pH and feeding concentration in a 5L fermenter. The highest yield of glutathione in the fermentation broth of the recombinant Corynebacterium glutamicum CG05 is 25.78±0.25 g / L (83.97±0.81 mM), and the biomass OD 600 is 59.8, which is 2.1 times higher than the fermentation yield of the CG05 strain before optimization and 5.7 times higher than the yield of the starting strain CG01, and the conversion rate is 83.97%.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant Corynebacterium glutamicum and a method and application for fermenting and producing glutathione therefrom. 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, and requires three precursors, L-glutamic acid, 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 fruits, and is used as a food supplement in capsule form. In recent years, the commercial demand for glutathione has shown an overall growth trend.

[0003] Currently, Yang et al. (Applied Microbiology and Biotechnology (2016), 100(14), 6279 - 6289.) produced 11.2 g / L glutathione in the whole - cell catalysis system after adding 80 mM ATP; Chen et al. (World Journal of Microbiology and Biotechnology (2020), 36(8), 117.) studied a method for gradually regulating glutathione synthesis in yeast based on oxidative stress and energy metabolism by systematically exploring factors such as temperature and pH during fermentation, and the glutathione accumulation increased to 5.76 g / 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) produced glutathione using GshF in Corynebacterium glutamicum, modified the key precursor L - cysteine pathway, and fermented to produce 756 mg / L glutathione; Wei Jianguo et al. needed to supplement 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 synthase has limitations in ATP energy supply and short half - life in enzymatic synthesis, and chemical synthesis lacks specificity. Problems such as feedback inhibition of GSH on the key enzyme GSH I in yeast and low tolerance of Escherichia coli to the precursor cysteine result in generally quite low GSH yield and productivity; at the same time, fermentation parameters such as temperature, pH, aeration, and stirring also affect the stability of the product glutathione; the addition of three substrate amino acids causes high - cost problems.

[0004] Corynebacterium glutamicum is the core strain in the amino acid fermentation industry, with good substrate tolerance. Due to its advantages such as biosafety (recognized as GRAS by the US FDA, Generally regarded as safe), being a natural L-glutamic acid (precursor of GSH) secreting strain, and having a wide substrate spectrum, it is widely used in the fermentation production of glutamic acid, lysine, valine, etc. 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; developing a method to improve the glutathione production of the food safety strain Corynebacterium glutamicum can simplify the production process, improve synthesis efficiency, and reduce production costs, which has important practical significance for the application of glutathione. In addition, single cell protein (SCP) obtained from microorganisms has great potential after plant and insect proteins. SCP based on Corynebacterium glutamicum can be used as animal feed to support the growth of pigs, improve the animal intestinal flora; and has an anti-obesity effect. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a recombinant Corynebacterium glutamicum and its method and application for fermentative production of glutathione. In the present invention, the glutathione bifunctional enzyme GshF derived from Streptococcus lactis is overexpressed in Corynebacterium glutamicum E01 for fermentative production of glutathione; through site-directed mutagenesis, the enzyme activity of GshF is increased, and at the same time, the aceD and ggt genes in the key metabolic pathway are knocked out, and the fermentation parameters such as temperature, pH, and feeding concentration in a 5L fermenter are optimized.

[0006] The present invention constructs a recombinant Corynebacterium glutamicum capable of high-yielding glutathione, and conducts amino acid nutrition analysis on this 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 as shown in SEQ ID NO.1.

[0008] In one embodiment of the present invention, the recombinant Escherichia coli uses Escherichia coli BL21(DE3) as the 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, which 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 the amino acid sequence shown in SEQ ID NO.2 as the parent, and mutates the histidine at the 11th position of the parent glutathione bifunctional enzyme to proline and the asparagine at the 239th position to glutamate.

[0012] In one embodiment of the present invention, the nucleotide sequence of the 500 bp upstream of the cysteine desulfurase gene is as shown in SEQ ID NO 4; the nucleotide sequence of the 500 bp downstream of the cysteine desulfurase gene is as shown in SEQ ID NO 5; the nucleotide sequence of the 500 bp upstream of the glutamyl transpeptidase gene is as shown in SEQ ID NO 6; the nucleotide sequence of the 500 bp downstream of the glutamyl transpeptidase gene is as shown in 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 fermentatively producing glutathione, using L-cysteine and L-glycine as substrates, and adding the recombinant Corynebacterium glutamicum described in any one of claims 1-4 to the fermentation system to fermentatively 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 inoculum 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: 100 g / L - 150 g / L glucose, 0.5 g / L - 1 g / L K2HPO4, 1 g / L - 1.5 g / L MgSO4, 5 g / L - 10 g / L corn steep liquor, 0.001 g / L - 0.005 g / L FeSO4·7H2O, 0.001 g / L - 0.005 g / L MnSO4·H2O, 5 g / L - 10 g / L urea.

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

[0020] The third object of the present invention is to provide the use of the recombinant Corynebacterium glutamicum in the preparation of 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 for fermenting and producing glutathione. The present invention performs combinatorial mutagenesis on a glutathione bifunctional enzyme GshF derived from Streptococcus lactis. After mutating histidine at position 11 to proline and asparagine at position 239 to glutamic acid, the specific enzyme activity of the mutant enzyme is 3.89 U / mg, which is 3.7 times higher than the enzyme activity before mutation. The mutated GshF H11P-N239E significantly improves the catalytic activity of the enzyme. Overexpress GshF in a high-yield L-glutamic acid-producing Corynebacterium glutamicum E01 strain H11P-N239E , and ferment to produce glutathione; at the same time, knockout the aceD and ggt genes, and optimize the fermentation parameters of temperature, pH, and feeding concentration in a 5 L fermenter. The highest yield of glutathione of the recombinant Corynebacterium glutamicum CG05 is 25.78 ± 0.25 g / L (83.97 ± 0.81 mM), and the biomass OD 600 is 59.8, which is 2.1 times higher than the fermentation yield of the CG05 strain before optimization and 5.7 times higher than the yield of the starting strain CG01, and the conversion rate is 83.97%.

[0023] Compared with previous studies on the production of glutathione, the present invention provides a method for constructing an engineered strain for producing glutathione by Corynebacterium glutamicum, and there is no need to add the substrate L-glutamic acid during the fermentation process, reducing the production cost, having a high conversion rate, and significantly increasing the glutathione yield after optimizing the fermentation parameters. These strategies provide effective strategies for the metabolic modification and fermentation parameter optimization of other strains for fermenting and producing glutathione, and are of great significance for the fermentation production of glutathione.

[0024] The recombinant Corynebacterium glutamicum CG05 was prepared as microbial protein, with a crude protein content of 65.6% and a crude fat content of 3.1%, which is higher than the contents of some common additives in animal feed. The rich branched-chain amino acids (19.51%) are beneficial to improving exercise ability and delaying fatigue, and are beneficial to the growth of animals; the sulfur-containing amino acids (16.92%) are beneficial to the free radical balance and stability in animals; meanwhile, the microbial protein biomass of the recombinant Corynebacterium glutamicum CG05 has 15.04% of glutamic acid, and this flavoring substance can increase the feed intake of animals, thereby improving the growth performance of animals. In addition, 1.12% of glutathione still exists in the microbial protein of the recombinant Corynebacterium glutamicum CG05, which can enhance the antioxidant effect in animals and maintain important physiological functions related to animal nutrition and immunity. Therefore, when the recombinant Corynebacterium glutamicum CG05 is fermented to produce glutathione, its microbial protein can be used as a nutritional additive for animal feed and has potential application prospects. Description of the Drawings

[0025] To make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, wherein,

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

[0027] Figure 2 are the biomass and yield of glutathione produced by the fermentation of the engineering strain CG05 after optimizing the fermentation parameters in Example 7 of the present invention. Detailed Embodiments

[0028] The following further describes the present invention in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not used as a limitation to the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

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

[0031] The culture media involved in the following embodiments:

[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. 2% agar powder is added to the solid medium.

[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 examples are as follows:

[0037] The content of glucose in the fermentation broth was determined using a biosensing 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 chromatographic column, the mobile phase was an aqueous phosphate solution with a volume fraction of 95%, the flow rate was set at 1 mL / min, the column temperature was maintained at 30 °C, and quantitative detection was performed at a UV wavelength of 210 nm. The amino acid composition of the biomass was detected by high performance liquid chromatography, using a C18 chromatographic column, the mobile phase was an aqueous sodium acetate solution with a volume fraction of 92% and a methanol acetonitrile solution with a volume fraction of 8%, the flow rate was set at 1 mL / min, the column temperature was maintained at 40 °C, and quantitative detection was performed at a UV wavelength of 338 nm. The crude protein in the biomass was determined by GB / T 6432, and the crude fat in the biomass was determined by GB 5009.6—2016.

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

[0039] (1) Preparation of Escherichia coli competent cells

[0040] Streak Escherichia coli BL21 on an antibiotic-free LB plate, incubate it in an incubator at 37°C, pick colonies after incubation, inoculate them into a 10 mL LB vial and culture for 12 h, transfer with an inoculation amount of 1%, transfer to a 50 mL LB medium bottle, and when the cell concentration reaches 0.4 - 0.6, prepare for the preparation of E. coli competent cells. Pre-cool relevant reagents and instruments in advance, prepare calcium chloride solution, calcium chloride and glycerol mixed solution, 1.5 mL EP tubes and 50 mL centrifuge tubes, place them on ice, and control the temperature of the centrifuge to 4°C. Aliquot 50 mL of the bacterial solution in a sterile workbench, centrifuge (8000 r·min -1 , 5 min), and discard the supernatant. Pipette 5 mL of calcium chloride solution and blow and suck to suspend, centrifuge (8000 r·min -1 , 5 min), and discard the supernatant. Pipette 5 mL of calcium chloride and glycerol mixed solution and blow and suck to suspend, aliquot into pre-cooled and pre-prepared EP tubes, with 100 μL in each tube.

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

[0042] According to the gene sequence of 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 GenScript Corporation in Suzhou for synthesis. The PCR program is as follows: 95°C, 10

[0043] min; 95°C, 30 s; 58°C, 30 s; 72°C, 1 min; 72°C, 10 min, with 30 cycles. The obtained gshF gene fragment was purified and ligated to the linearized plasmid pET-28a (P1 / P2) through the homologous recombinase In-Fusion Snap Assembly Master Mix (Takara), transformed into E. coli BL21(DE3) competent cells to obtain transformants. The transformants were spread on an LB solid medium containing kanamycin at a concentration of 50 μg / mL and cultured at 37°C for 12 h. Pick positive colonies, and perform colony PCR verification on single colonies with Taq DNA polymerase using P3 / P4 as primers; after inoculating positive single colonies with the size of the target band into a vial containing LB liquid medium and culturing for 12 h, extract the plasmid and send it to GenScript Corporation for sequencing. If the sequencing is correct, the recombinant Escherichia coli E.coli / pET28a-GshF is 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) Construction of recombinant Escherichia coli E.coli / pET28a-GshF H11P-N239E Construction

[0049] Using pET28a-GshF as a template, PCR amplification of the 11 mutation sites was performed with P5 / P6 as primers respectively, and then 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 colony PCR verification of the single colonies was carried out with P3 / P4 as primers through Taq DNA polymerase; the positive single colonies with the target band size were inoculated into small bottles containing LB liquid medium and cultured for 12 h, then the plasmids were extracted and sent to Genewiz for sequencing. If the 11 mutation sites of GshF were successfully mutated, then the single mutant strain E.coli / pET28a-GshF H11P was successfully constructed. Using pET28a-GshF H11P as a template, PCR amplification of the 239 mutation sites was performed with P7 / P8 as primers respectively, and then 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 colony PCR verification of the single colonies was carried out with P3 / P4 as primers through Taq DNA polymerase; the positive single colonies with the target band size were inoculated into small bottles containing LB liquid medium and cultured for 12 h, then the plasmids were extracted and sent to Genewiz for sequencing. If the 239 mutation sites of GshF were successfully mutated, then the mutant strain E.coli / pET28a-GshF H11P-N239E was successfully constructed (the amino acid sequence after combined mutation 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: Construction of Recombinant Corynebacterium glutamicum Cg / pXMJ-19-GshF and Cg / pXMJ-19-GshF H11P-N239E Construction

[0061] (1) Preparation of Corynebacterium glutamicum Competent Cells

[0062] Pick Corynebacterium glutamicum E01 and inoculate it into 10 mL of BHI liquid medium. Culture it on a shaker at 30 °C for 24 h. Transfer the cultured bacterial liquid into BHI competent medium so that the initial cell OD 600 reaches 0.3. Culture it at 30 °C and 200 rpm until the cell OD 600 reaches 1.0. After the cell culture is completed, pre-cool the bacterial liquid for 30 min, and then centrifuge to collect the bacterial cells. Wash the bacterial cells 3 times with pre-cooled 10% glycerol, and finally resuspend the cells with 1 mL of 10% glycerol. Aliquot with 1.5 mL tubes, 100 μL per tube, and directly use for electroporation.

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

[0064] gshF and gshF H11P-N239E After the gene fragments of gshF and gshF are purified, they are respectively ligated with the linearized plasmid pXMJ-19 (P9 / P10) by the homologous recombinase In-Fusion SnapAssembly MasterMi (Takara), and transformed into E. coli BL21(DE3) competent cells to obtain transformants. Spread the transformants on LB solid medium containing 20 μg / mL chloramphenicol and culture at 37 °C for 12 h. Pick positive colonies and verify the single colonies by colony PCR with P11 / P12 as primers using Taq DNA polymerase; After the positive single colonies with the target band size are inoculated into small bottles containing LB liquid medium and cultured for 12 h, the plasmids are extracted and sent to Genewiz for correct sequencing, then the plasmids pXMJ-19-GshF and pXMJ-19-GshF H11P-N239E are successfully constructed. 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) Construction of recombinant Corynebacterium glutamicum Cg / pXMJ-19-GshF (CG01) and Cg / pXMJ-19-GshF H11P-N239E (CG02)

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

[0071] Example 4: Construction of recombinant Corynebacterium glutamicum C.g ΔaceD (CG03)

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

[0073] To reduce the degradation of the substrate L-cysteine, the cysteine desulfurase aceD gene in the genome of Corynebacterium glutamicum was knocked out. According to the 500 bp upstream and downstream 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 500 bp upstream and downstream of aceD are shown in SEQ ID NO.4 and SEQ ID NO.5). After the obtained gene fragments were fused and ligated with the linearized plasmid pK18mobsacB (P17 / P18) by the homologous recombinase In-Fusion Snap Assembly Master Mix (Takara), they were transformed into competent E. coli BL21(DE3) 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 of single colonies using P19 / P20 as primers with Taq DNA polymerase. After positive single colonies with the target band size were inoculated into vials containing LB liquid medium and cultured for 12 h, plasmids were extracted. If the sequencing by Genewiz was correct, then PK18-aceD 上500bp -aceD 下500bp The plasmid construction was successful. 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) Construction of recombinant Corynebacterium glutamicum C.g ΔaceD (CG02)

[0083] The PK18-aceD obtained in step (1)上500bp -aceD 下500bp The plasmid was electrotransformed into competent cells of Corynebacterium glutamicum E01 to obtain transformants, and the transformants were spread on BHI solid medium containing 50 μg / mL kanamycin resistance. The cells were cultured at 30 °C for 2 - 4 days, and positive colonies were picked. Colony PCR verification of single colonies was performed using P11 / P12 as primers and Taq DNA polymerase; positive single colonies with the target band size indicated the recombinant strain C.g / PK18-aceD 上500bp -aceD 下500bp The construction was successful. Then the correct transformants were inoculated into seed liquid without kanamycin for screening, and diluted and spread on seed medium containing 10% sucrose to induce the occurrence of the second round of homologous recombination. Finally, sequencing verification proved that the aceD gene knockout was successful, and the knockout strain C.g ΔaceD (CG03) was successfully constructed.

[0084] Example 5: Construction of recombinant Corynebacterium glutamicum C.g ΔaceDΔggt / pXMJ-19-GshF(CG04) and C.g ΔaceDΔggt / pXMJ-19-GshF H11P-N239E (CG05)

[0085] (1) Construction of the knockout plasmid PK18-ggt 上500bp -ggt 下500bp construction

[0086] To reduce the degradation of the product glutathione, the ggt gene in the genome of Corynebacterium glutamicum was knocked out. According to the 500 bp upstream and downstream 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 500 bp upstream and downstream of ggt are shown in SEQ ID NO.6 and SEQ ID NO.7). After the obtained gene fragments were fused, they were ligated with the linearized plasmid pK18mobsacB (P25 / P26) through the homologous recombinase In-Fusion Snap Assembly Master Mix (Takara), and transformed into competent cells of E.coli BL21(DE3) 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 colony PCR verification of single colonies was performed using P19 / P20 as primers and Taq DNA polymerase. After positive single colonies with the target band size were inoculated into a vial containing LB liquid medium and cultured for 12 h, the plasmid was extracted and sent to Genewiz for correct sequencing, then PK18-ggt上500bp -ggt 下500bp The plasmid was successfully constructed. 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’-cagctatgacatgattacgaattcttatgcagccaagaggtggctcgg-3’

[0093] (2) Construction of recombinant Corynebacterium glutamicum C.g ΔaceDΔggt of

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

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

[0096] The obtained pXMJ-19-GshF and pXMJ-19-GshF H11P-N239E plasmids were respectively electrotransformed into the competent cells of Corynebacterium glutamicum C.g ΔaceDΔggt to obtain transformants, and the transformants were spread on BHI solid medium containing 20 μg / mL chloramphenicol resistance and cultured at 30 °C for 2 - 4 days. Positive colonies were picked, and colony PCR verification of the single colonies was carried out by Taq DNA polymerase using P11 / P12 as primers; the positive single colonies with the size of the target band indicated the recombinant strains C.g ΔaceDΔggt / pXMJ-19-GshF (CG04) and C.g ΔaceDΔggt / pXMJ-19-GshF H11P-N239E (CG05) was successfully constructed.

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

[0098] 1. Strains: The recombinant strains Corynebacterium glutamicum CG01, CG02, CG04 and CG05 were used for the fermentation production of glutathione.

[0099] 2. Fermentation medium: 100 g / L - 150 g / L glucose, 0.5 g / L - 1 g / L K2HPO4, 1 g / L - 1.5 g / L MgSO4, 5 g / L - 10 g / L corn steep liquor, 0.001 g / L - 0.005 g / L FeSO4·7H2O, 0.001 g / L - 0.005 g / L MnSO4·H2O, 5 g / L - 10 g / L urea.

[0100] 3. Fermentation culture of Corynebacterium glutamicum: Using an inoculation loop, the strains CG01, CG02, CG04 and CG05 were streaked on the solid plate of the seed medium, and the plate was placed in a constant temperature incubator at 30 °C until single colonies grew. Then they were inoculated into the first-stage seed BHI liquid medium and cultured at 30 °C and 120 rpm until the mid-logarithmic growth phase, and then inoculated into 100 mL of the fermentation medium of the second-stage seed according to an inoculation amount of 0.5 mL - 1 mL and fermented at 30 °C and 120 rpm.

[0101] 4. Fermentation production of glutathione in a 5L fermenter: The secondary seed solutions of CG01, CG02, CG04, and CG05 were respectively transferred to a 5L fermentation medium with a transfer volume of 200 mL - 400 mL, and fermented for 72 h under the conditions of 30 °C, 300 rpm - 800 rpm, an aeration rate of 2 L / min - 4 L / min, and pH 7.0. IPTG with a final concentration of 0.5 mM was added for induction at 12 h of cultivation, and 30 mM - 100 mM of L-cysteine and L-glycine were supplemented at 24 h of fermentation (Corynebacterium glutamicum E01 is a dominant strain for the production of L-glutamate, so L-glutamate does not need to be supplemented during fermentation). Samples were taken every 12 h, and the biomass (OD 600 ) was measured using a spectrophotometer. The concentration of glutathione in the fermentation broth was measured by HPLC. The experimental results are shown in Figure 1 and Table 2:

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

[0103]

[0104] As can be seen from Figure 1 and Table 2, for the CG01 strain without the aceD and ggt genes knocked out, after overexpressing the glutathione bifunctional enzyme GshF, the highest yield of glutathione production by fermentation was 4.56 ± 0.13 g / L at 48 h, and the biomass OD 600 was 45.4; after overexpressing the mutant enzyme GshF H11P-N239E , the highest yield of glutathione production by fermentation was 6.56 ± 0.26 g / L at 48 h, and the biomass OD 600 slightly increased to 51.8, and the yield was 1.4 times higher than that of CG01. After knocking out the aceD and ggt genes and overexpressing the glutathione bifunctional enzyme GshF, the highest yield of glutathione production by the CG04 strain was 8.13 ± 0.20 g / L at 48 h, and the biomass OD 600 decreased to 40.7; after knocking out the aceD and ggt genes and overexpressing the mutant enzyme GshF H11P-N239E , the yield of glutathione production by the CG05 strain increased to 12.56 ± 0.32 g / L, the yield was 1.5 times higher than that of CG04, and the biomass OD 600 increased to 48.1. Generally speaking, after knocking out the aceD and ggt genes and overexpressing the mutant enzyme GshF H11P-N239E , the yield of glutathione production by the engineering strain CG05 by fermentation increased significantly, which was 2.8 times higher than that of the starting strain CG01 strain, and the catalytic efficiency of the mutant enzyme GshF H11P-N239E was enhanced.

[0105] Example 7: Preparation of Recombinant Corynebacterium glutamicum CG05 Cell Protein and Analysis of Its Nutritional Characteristics

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

[0107] 2. Fermentation parameters of the fermenter: The secondary seed liquid of CG05 was transferred to a 5 L fermentation medium with a transfer volume of 200 mL - 400 mL, and fermented for 72 h under the conditions of 300 rpm - 800 rpm and an aeration rate of 2 L / min - 4 L / min. IPTG with a final concentration of 0.5 mM was added for induction at 12 h of cultivation. The pH, temperature, and addition time of the fermentation substrate were optimized. The specific content of the fermentation parameter optimization was as follows: during the fermentation process, the pH was controlled between 7.0 and 8.5, the temperature was controlled between 30 °C and 45 °C, and the substrate concentration of L-cysteine and L-glycine added within 24 h - 60 h of fermentation was 30 mM - 100 mM (the control and addition of pH, temperature, and fermentation substrate were all dynamic processes). Samples were taken every 12 h, and the biomass (OD 600 ) was measured using a spectrophotometer.

[0108] 3. Preparation of single-cell raw material: The concentration of glutathione in the fermentation supernatant was determined by HPLC. At the same time, the cells were collected by centrifugation, washed with sterile water, and the collected cells were further freeze-dried and ground to obtain a powdery biomass, which was used as the single-cell 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 Fermentation Parameter Optimization

[0110]

[0111] From Figure 2 and Table 3, it can be seen that in order to further improve the glutathione yield, the fermentation parameters were optimized. At 30 °C - 45 °C, pH value of 7.0 - 8.5, and 30 mM - 100 mM of L-cysteine and L-glycine added within 24 h - 60 h, after 48 h of fermentation in a 5 L fermenter, the highest yield of glutathione in the supernatant fermentation broth of recombinant Corynebacterium glutamicum CG05 was 25.78 ± 0.25 g / L (83.97 ± 0.81 mM), and the biomass OD 600 was 59.8, which was 2.1 times higher than the fermentation yield of the CG05 strain before optimization and 5.7 times higher than the yield of the starting strain CG01. The conversion rate was calculated based on the substrate L-cysteine, and the conversion rate of the CG05 engineering strain was 83.97%.

[0112] The nutritional components of the recombinant Corynebacterium glutamicum CG05 cell protein are shown in Table 4. Among them, the crude protein content is 65.6%, and the crude fat content is 3.1%. The crude protein content in the cells of Corynebacterium glutamicum CG05 is higher than the content of common additives in animal feed (whole cottonseed: 23%; soybean hull: 13%; corn straw: 5% - 9%) (data source: China Feed Composition and Nutritional Value Table (33rd Edition, 2022)); 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 (33rd Edition, 2022)). Therefore, the cell protein of recombinant Corynebacterium glutamicum CG05 can not only be used as a high-quality alternative protein raw material but also be a source of high-quality fat.

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

[0114]

[0115] The quality of recombinant Corynebacterium glutamicum CG05 is determined by the protein content and the amino acid composition that makes up the protein, especially the content of essential amino acids. Table 5 shows the amino acid analysis results of the cell protein of recombinant Corynebacterium glutamicum CG05. 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% stipulated by the FAO / WHO standard. Soybean is a common additive in feed. Table 5 lists the amino acid content of soybean (data source: USDA 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 that of soybean (17%). Supplementing branched-chain amino acids can improve exercise ability 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 soybean. Sulfur-containing amino acids are beneficial to the free radical balance and stability in animals; at the same time, the cell protein biomass of recombinant Corynebacterium glutamicum CG05 has a relatively high content of flavor substances, such as glutamic acid (15.04%), which can improve the feed intake of animals, thereby improving the growth performance of animals. In addition, there is still 1.12% of glutathione in the cell 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, when recombinant Corynebacterium glutamicum CG05 is fermented to produce glutathione, its cell protein can be used as a nutritional additive for animal feed and has potential application prospects.

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

[0117]

[0118] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill 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 enumerate all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A recombinant Corynebacterium glutamicum, characterized in that The recombinant Corynebacterium glutamicum is Corynebacterium glutamicum ( Corynebacterium glutamicum ) as the expression host; knocking out the cysteine ​​desulfurase gene and the glutamyl transpeptidase gene, and overexpressing 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 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 30 mM to 100 mM; the concentration of the L-glycine is 30 mM to 100 mM.

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: 100 g / L-150 g / L glucose, 0.5 g / L-1 g / L K2HPO4, 1 g / L-1.5 g / L MgSO4, 5 g / L-10 g / L corn steep liquor, 0.001 g / L-0.005 g / L FeSO4-7H2O, 0.001 g / L-0.005 g / L MnSO4-H2O, and 5 g / L-10 g / L urea.

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

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

Images