CN112646768A - Preparation method of recombinant corynebacterium glutamicum and glutathione - Google Patents
Preparation method of recombinant corynebacterium glutamicum and glutathione Download PDFInfo
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- CN112646768A CN112646768A CN202011635514.6A CN202011635514A CN112646768A CN 112646768 A CN112646768 A CN 112646768A CN 202011635514 A CN202011635514 A CN 202011635514A CN 112646768 A CN112646768 A CN 112646768A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
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Abstract
The invention relates to a preparation method of recombinant corynebacterium glutamicum and glutathione, wherein genes of bifunctional glutathione synthetase are overexpressed in corynebacterium glutamicum genetic engineering bacteria. The corynebacterium glutamicum genetically engineered bacterium is used as a starting strain, glutathione is produced by a fermentation method, the yield of glutathione is high, the operation is simple and convenient, and meanwhile, the food-grade corynebacterium glutamicum is safe and non-toxic, accords with the production concept of resource saving and environmental protection, and provides reference and reference for industrial production of glutathione by recombinant corynebacterium glutamicum.
Description
Technical Field
The invention relates to the field of bioengineering, and in particular relates to a method for preparing glutathione by using recombinant corynebacterium glutamicum.
Background
Glutathione (GSH) is an active tripeptide compound formed by condensing L-glutamic acid, L-cysteine and glycine, and is widely present in various organisms. Glutathione of reduced type has a plurality of important functions in living tissues, can improve human immunity and resist aging, has greater efficacy on the delayed cells of the old than that of the young, and has very obvious effect on treating symptoms such as leucopenia caused by radioactive rays and radiopharmaceuticals.
At present, glutathione is prepared by a plurality of methods, which commonly comprise a solvent extraction method, a chemical synthesis method, a biological fermentation method and an enzyme method. At present, glutathione is produced at home and abroad mainly by adopting a fermentation method or an enzyme method, wherein the fermentation method is mainly characterized in that genes for coding and synthesizing a glutathione enzyme system or bifunctional enzymes are cloned into escherichia coli or yeast, glutathione is obtained by fermentation culture, and the yeast fermentation method in the fermentation method is mature in process. Patents CN201810844388 and CN201680013630 exogenously express glutathione synthesis bifunctional enzyme genes in yeast and escherichia coli respectively to achieve the purpose of producing glutathione by fermentation, but the yield is very low. Escherichia coli is also a conditional pathogen, and relevant antigens of the Escherichia coli are remained in products such as generated recombinant protein, organic acid and the like so as to cause immune reaction; coli is also a good host for bacteriophages, and is highly susceptible to bacteriophage infection. Therefore, the existing fermentation method is not suitable for producing food-grade glutathione and does not conform to the production concept of saving resources and being environment-friendly.
Disclosure of Invention
The invention aims to provide a recombinant corynebacterium glutamicum genetically engineered bacterium, which is used for producing glutathione and has the advantages of high yield, high safety, no toxicity and the like.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a corynebacterium glutamicum genetically engineered bacterium, wherein genes of bifunctional glutathione synthetase are overexpressed in the corynebacterium glutamicum genetically engineered bacterium.
Preferably, the nucleotide sequence of the bifunctional glutathione synthetase gene is shown as SEQ ID NO. 1.
In the invention, the bifunctional glutathione synthetase gene is derived from Streptococcus thermophilus (gshFst).
The second aspect of the present invention provides a preparation method of corynebacterium glutamicum genetically engineered bacteria, comprising the following steps:
(1) cloning the gene of the bifunctional glutathione synthetase to pEC-XK99E plasmid to obtain recombinant plasmid;
(2) transforming the recombinant plasmid into corynebacterium glutamicum to obtain the corynebacterium glutamicum genetic engineering strain.
In the invention, the bifunctional glutathione synthetase gene can be obtained by a conventional genetic engineering means. For example, it can be obtained by PCR amplification and isolation using Streptococcus thermophilus DNA as a template, or by artificial synthesis based on its sequence.
Preferably, the specific method of step (1) is: and (2) amplifying DNA by taking the plasmid pET28a-gshFst as a template, carrying out double enzyme digestion on the amplification product or the plasmid by using BamHI and PstI, and then connecting the amplification product or the plasmid with the pEC-XK99E plasmid subjected to enzyme digestion treatment by using BamHI and PstI to obtain the recombinant plasmid.
In the present invention, the recombinant plasmid is transferred into Corynebacterium glutamicum by electrotransfer.
The third aspect of the invention provides an application of the corynebacterium glutamicum genetically engineered bacterium in glutathione production.
The fourth aspect of the invention provides a preparation method of glutathione, which adopts the corynebacterium glutamicum genetic engineering bacteria to produce the glutathione.
Preferably, the glutathione is produced by a fed-batch fermentation process.
Preferably, glutamic acid, glycine and cysteine are added during production of the glutathione, and initial concentrations of the added glutamic acid, glycine and cysteine in a reaction system are respectively 80-120 mM, more preferably 90-110 mM, and even more preferably 95-105 mM.
The invention synthesizes the glutathione from the exogenously added glutamic acid, glycine and cysteine by high-density culture and over-expression of the bifunctional glutathione synthetase gene, thereby achieving high yield and high yield.
Preferably, after the corynebacterium glutamicum genetic engineering bacteria are subjected to amplification culture, inoculating the corynebacterium glutamicum genetic engineering bacteria to a fermentation culture medium for fermentation culture, adding a feed liquid in batches during fermentation culture, controlling the temperature of the fermentation culture to be 25-35 ℃, controlling the pH to be 6.5-7.5, and controlling the fermentation culture time to be 40-60 h; and adding an inducer for continuous culture for 5-15 h, adding glutamic acid, glycine and cysteine, and raising the temperature to 35-40 ℃ for culture until the yield of the glutathione is not increased any more.
Further preferably, the temperature of the fermentation culture is 28-32 ℃, the pH value is 6.5-7.2, and the fermentation culture time is 45-50 h; and adding an inducer for continuous culture for 9-12 h, adding glutamic acid, glycine and cysteine, and raising the temperature to 36-38 ℃ for culture until the yield of the glutathione is not increased any more.
In the invention, the method for the expanded culture of the corynebacterium glutamicum genetically engineered bacteria is as follows:
A. plate culture: inoculating the corynebacterium glutamicum genetic engineering bacteria stored in the glycerin tube into an LB culture medium, and culturing at 30 ℃ for 8-12h to obtain first-class seeds.
B. Liquid seed culture: inoculating the genetically engineered bacteria (primary seeds) of the corynebacterium glutamicum cultured by a plate into a secondary seed culture medium, and culturing at 30 ℃ for 8-12h to obtain a secondary seed culture solution. In the present invention, the seed medium is LB liquid medium.
Further preferably, the inoculation amount during the fermentation culture is controlled to be 7-10%.
In the invention, the secondary seed culture solution is inoculated into the fermentation medium in a fermentation tank according to the inoculation amount of 7-10%.
In the present invention, IPTG is used as the inducer, and the concentration of IPTG is 1 mmol/L.
Further preferably, the fermentation medium for the fermentation culture comprises the following components: 5-30 g/L carbon source, 1-4 g/L nitrogen source, 0.5-15 g/L inorganic salt and trace elements, and 0.001-1 g/L growth factor which can be optionally added.
Further preferably, the carbon source is one or more of starch hydrolysis sugar, corn steep liquor, molasses, cane sugar, glucose and glycerol; the nitrogen source is one or more of yeast extract, peptone, tryptone, yeast extract and urea; the inorganic salt and the trace elements are one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, ammonium chloride, potassium chloride, sodium chloride, calcium carbonate, ammonium sulfate and magnesium sulfate, and the growth factors comprise one or more of biotin, vitamin B1, vitamin B6 and glutamic acid.
Further preferably, the feed liquid is one or more of glucose solution, yeast extract, sucrose, starch hydrolysis sugar, tryptone, corn steep liquor, magnesium sulfate and glycerol, and the concentration of the feed liquid is 400-500 g/L.
The corynebacterium glutamicum is a food-grade microorganism, is safe and nontoxic, and therefore, the corynebacterium glutamicum is more favorable for producing glutathione by taking the corynebacterium glutamicum as a starting strain.
The recombinant corynebacterium glutamicum disclosed by the invention is used for producing glutathione by a fermentation method, so that the operation is simple and convenient, the yield is high, the production concept of resource saving and environmental friendliness is met, and reference is provided for industrial production of glutathione by the recombinant corynebacterium glutamicum.
Compared with the prior art, the invention has the following advantages:
the recombinant corynebacterium glutamicum genetically engineered bacterium obtained by the invention has the advantages of high yield, high safety, no toxicity and the like when used for producing glutathione.
Drawings
FIG. 1: production profile of GSH in the fermentation broth of example 2 of the present invention;
FIG. 2 is a drawing: production profile of GSH in the fermentation broth of example 3 of the present invention;
FIG. 3: production profile of GSH in the fermentation broth of example 4 of the present invention;
FIG. 4 is a drawing: production profile of GSH in the fermentation broth of example 5 of the present invention;
FIG. 5: production profile of GSH in the fermentation broth of example 6 of the invention;
FIG. 6: production profile of GSH in the fermentation broth of example 7 of the present invention;
FIG. 7: production profile of GSH in the fermentation broth of example 8 of the present invention;
FIG. 8: production profile of GSH in the fermentation broth of example 9 of the present invention;
FIG. 9: production profile of GSH in the fermentation broth of example 10 of the present invention;
FIG. 10: production profile of GSH in the fermentation broth of example 11 of the present invention;
FIG. 11: production profile of GSH in the fermentation broth of example 12 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to specific related embodiments. The described embodiments are only some, but not all embodiments of the invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any inventive work belong to the scope of the present invention. The experimental procedure used in the examples below. Unless otherwise specified, all the methods are conventional, and materials, reagents and the like used therein may be commercially available. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
Construction of recombinant Corynebacterium glutamicum engineering bacteria for GSH production
The method comprises the steps of selecting a bifunctional glutathione synthetase gene (SEQ ID NO: 1) derived from Streptococcus thermophilus (gshFst), amplifying a gene fragment of the bifunctional glutathione synthetase by using a recombinant plasmid pET28a-gshFst as a template, carrying out double enzyme digestion by BamHI and SmaI to obtain a linearized plasmid, and carrying out ligation under the action of T4 ligase to obtain a recombinant plasmid pHT-ST.
Preparation of DNA fragments: DNA amplification was carried out using a recombinant plasmid pET28a-gshFst available in the laboratory as a template. The reaction conditions are as follows: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 deg.C for 10s, annealing at 60 deg.C for 30s, and extension at 72 deg.C for 5min for 30 cycles; after completion, final extension was carried out at 72 ℃ for 10 min. The amplified product was verified by running gel by nucleic acid electrophoresis, purified by PCR Purification Kit, and then digested with DpnI. Finally carrying out double digestion by using BamHI and SmaI.
The enzyme cutting system is as follows: PCR product or 50ul of pEC-XK99E plasmid, 1ul each of BamHI and SmaI, 10ul of buffer, ddH2O38 ul, total volume 100 ul.
The PCR product after enzyme digestion is connected with pEC-XK99E plasmid after the same enzyme treatment, and the connecting system is: 4ul of PCR product after enzyme digestion, 4ul of pEC-XK99E plasmid after enzyme digestion, 1ul of T4 ligase and 1ul of buffer solution are connected to obtain the recombinant plasmid pEC-XK 99E-ST. And (3) electrically transferring the recombinant plasmid into corynebacterium glutamicum to obtain a recombinant strain PST.
Example 2:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 9h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 11h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 7% for fed-batch fermentation, wherein the fermentation culture medium adopts 10g/L of starch hydrolysis sugar, 2g/L of yeast extract, 0.5g/L of urea, 0.2g/L of potassium dihydrogen phosphate, 0.2g/L of dipotassium hydrogen phosphate, 0.1g/L of sodium chloride and 0.5g/L of magnesium sulfate, the feed liquid is 400g/L of glucose solution, the culture temperature is 30 ℃, the culture pH is 7.0, the fermentation is carried out for 50h, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 10h, finally 100mM of glutamic acid, glycine and cysteine are added to carry out GSH synthesis, the fermentation temperature is increased to 37 ℃, and the fermentation is stopped when the yield of GSH is not increased any more.
The content of GSH in the fermentation broth is measured, and a GSH production curve is drawn, wherein the GSH production curve of the embodiment is shown in figure 1. As shown in FIG. 1, the final GSH yield was 25.0g/L, calculated as 81.3% based on the amount of amino acid added.
Example 3:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 8h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 11h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 7.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 8g/L of starch hydrolysis sugar, 1g/L of molasses, 1g/L of yeast extract, 1g/L of urea, 0.5g/L of potassium dihydrogen phosphate, 0.5g/L of dipotassium hydrogen phosphate, 0.2g/L of sodium chloride and 1.5g/L of magnesium sulfate, the feed solution is 500g/L of glucose solution, the culture temperature is 30 ℃, the culture pH is 6.5, the fermentation is carried out for 45 hours, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 10 hours, finally 100mM of each of glutamic acid, glycine and cysteine are added to carry out GSH synthesis, the fermentation temperature is increased to 37 ℃, and the fermentation is stopped when the yield of the GSH is not increased any more.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of the embodiment is shown in figure 2, as shown in figure 2, the final yield of GSH is 24.05g/L, and the GSH yield is 78.3% based on the addition amount of amino acid.
Example 4:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 9h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 12h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 9% for fed-batch fermentation, wherein the fermentation culture medium adopts 10g/L of sucrose, 1g/L of peptone, 2g/L of yeast extract, 2g/L of ammonium chloride, 0.3g/L of potassium dihydrogen phosphate, 0.3g/L of dipotassium hydrogen phosphate, 0.4g/L of sodium chloride and 1g/L of magnesium sulfate, the feed liquid is 450g/L of glucose solution, the culture temperature is 30 ℃, the culture pH is 6.7, the fermentation is carried out for 49h, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 10h, finally 100mM of glutamic acid, glycine and cysteine are added to carry out GSH synthesis, the fermentation temperature is increased to 37 ℃, and the fermentation is stopped when the yield of the GSH is not increased any more.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of the example is shown in figure 3, as shown in figure 3, the final yield of GSH is 23.95g/L, and the GSH yield is 77.9% based on the addition amount of amino acid.
Example 5:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 10h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 10h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 8.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 10mL/L of corn steep liquor, 1g/L of molasses, 3g/L of yeast extract, 2g/L of ammonium sulfate, 0.3g/L of potassium dihydrogen phosphate, 0.3g/L of dipotassium hydrogen phosphate, 0.5g/L of sodium chloride and 1.2g/L of magnesium sulfate, the feed solution is 400g/L of glucose and 1g/L of yeast extract, the culture temperature is 30 ℃, the culture pH is 6.8, the fermentation is carried out for 47h, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 10h, 100mM of glutamic acid, glycine and cysteine are finally added to carry out GSH synthesis, the fermentation temperature is increased to 37 ℃, and the fermentation is stopped when the GSH yield is not increased any more.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of the embodiment is shown in figure 4, as shown in figure 4, the final yield of GSH is 24.95g/L, and the calculated GSH yield is 81.2% based on the addition amount of amino acid.
Example 6:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 8h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 11h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 10% for fed-batch fermentation, wherein the fermentation culture medium adopts 11g/L of molasses, 2g/L of glycerol, 3g/L of yeast extract, 2g/L of ammonium sulfate, 0.2g/L of potassium dihydrogen phosphate and 1g/L of disodium hydrogen phosphate, 0.5g/L potassium chloride and 0.6g/L magnesium sulfate, 400g/L glucose, 50g/L glycerol and 2g/L yeast extract, the culture temperature is 30 ℃, the culture pH is 6.6, the fermentation is carried out for 48h, 1mM inducer IPTG is added into the culture medium, the culture is continued for 10h, finally 100mM of glutamic acid, glycine and cysteine are added to synthesize GSH, the fermentation temperature is increased to 37 ℃, and the fermentation is stopped when the GSH yield is not increased any more.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of this example is shown in figure 5, as shown in figure 5, the final GSH yield is 24.90g/L, and the GSH yield is 81.0% based on the addition amount of amino acid.
Example 7:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 9h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 12h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 7.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 12g/L of glycerol, 3g/L of sucrose, 5g/L of calcium carbonate, 1g/L of glutamic acid, 1.5g/L of tryptone, 1g/L of ammonium chloride, 0.3g/L of potassium dihydrogen phosphate, 1.5g/L of disodium hydrogen phosphate, 0.5g/L of potassium chloride and 1.2g/L of magnesium sulfate, the feed liquid is 400g/L of glucose, 50g/L of sucrose, 1.5g/L of yeast extract, the culture temperature is 30 ℃, the culture pH is 7.0, the fermentation is 46h, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 10h, 100mM of glutamic acid, glycine and cysteine are finally added to synthesize GSH, the fermentation temperature is increased to 37 ℃, the fermentation was stopped when the production of GSH no longer increased.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of the example is shown in figure 6, as shown in figure 6, the final GSH yield is 24.50g/L, and the GSH yield is 79.7% based on the addition amount of amino acid.
Example 8:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 8h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 9h to obtain a secondary seed culture solution.
(3) Inoculating the second-stage seed culture solution into a 2L fermentation tank according to the inoculation amount of 7.9% for fed-batch fermentation, wherein the fermentation culture medium adopts sucrose 12g/L, molasses 3g/L, biotin 20ug/L, vitamin B11 mg/L, tryptone 2g/L, ammonium sulfate 3g/L and potassium dihydrogen phosphate 0.4g/L, 0.4g/L of dipotassium phosphate, 0.6g/L of sodium chloride and 0.5g/L of magnesium sulfate, the feed liquid is 400g/L of glucose and 1g/L of tryptone, the culture temperature is 30 ℃, the culture pH is 7.2, the fermentation is carried out for 49h, 1mM inducer IPTG is added into the culture medium, the culture is continued for 10h, finally 100mM of each of glutamic acid, glycine and cysteine are used for GSH synthesis, the fermentation temperature is increased to 37 ℃, and the fermentation is stopped when the GSH yield is not increased any more.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of this example is shown in figure 7, as shown in figure 7, the final GSH yield is 23.70g/L, and the GSH yield is 77.1% based on the addition amount of amino acid.
Example 9:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 11h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 11h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 9.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 9g/L of glucose, 1g/L of glycerol, 20mL/L of corn steep liquor, 40ug/L of biotin, 12 mg/L of vitamin B, 3g/L of yeast extract, 4g/L of ammonium sulfate, 0.3g/L of potassium dihydrogen phosphate, 0.3g/L of dipotassium hydrogen phosphate, 1.5g/L of sodium chloride, 0.8g/L of magnesium sulfate and 2g/L of calcium carbonate, the supplement liquid is 450g/L of glucose, 50mL/L of corn steep liquor and 5g/L of magnesium sulfate, the culture temperature is 30 ℃, the culture pH is 7.2, the fermentation is carried out for 45h, 1mM of inducer G is added into the culture medium, the culture is continued for 10h, finally, 100mM of glutamic acid, glycine and cysteine are added to carry out GSH synthesis, and the fermentation temperature is increased to 37 ℃, the fermentation was stopped when the production of GSH no longer increased.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of this example is shown in figure 8, as shown in figure 8, the final GSH yield is 23.20g/L, and the GSH yield is 75.5% based on the addition amount of amino acid.
Example 10:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 12h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 8h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 7% for fed-batch fermentation, wherein the fermentation culture medium adopts 15ml/L of corn steep liquor, 1g/L of sucrose, 2g/L of starch hydrolysis sugar, 8g/L of glucose, 10ug/L of biotin, 62 mg/L of vitamin B, 1g/L of vitamin B12 mg/L of yeast extract, 1g/L of ammonium sulfate, 1g/L of urea, 0.4g/L of potassium dihydrogen phosphate, 0.4g/L of dipotassium hydrogen phosphate, 0.6g/L of sodium chloride, 0.8g/L of magnesium sulfate and 1g/L of calcium carbonate, the feed solution is 200g/L of glucose, 200g/L of starch hydrolysis sugar, 20g/L of glycerol and 4g/L of magnesium sulfate, the culture temperature is 30 ℃, the culture pH is 7.0, and the fermentation is 46 hours, adding 1mM inducer IPTG into the culture medium, continuing culturing for 10h, finally adding 100mM of each of glutamic acid, glycine and cysteine to synthesize GSH, raising the fermentation temperature to 37 ℃, and stopping fermentation until the yield of GSH is not increased any more.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of this example is shown in figure 9, as shown in figure 9, the final GSH yield is 24.83g/L, and the GSH yield is 80.8% based on the addition amount of amino acid.
Example 11:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 11h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 9h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 5L fermentation tank according to the inoculation amount of 9% for fed-batch fermentation, wherein the formulas of a fermentation culture medium and the feed solution are the same as those in example 2, the culture temperature is 30 ℃, the culture pH is 7.0, the fermentation is carried out for 48h, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 10h, finally 100mM of each of glutamic acid, glycine and cysteine are added for GSH synthesis, the fermentation temperature is increased to 37 ℃, and the fermentation is stopped when the yield of the GSH is not increased any more.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of this example is shown in figure 10, as shown in figure 10, the final GSH yield is 23.90g/L, and the GSH yield is 77.8% based on the addition amount of amino acid.
Example 12:
and (3) producing GSH by using recombinant bacteria PST:
(1) inoculating the recombinant strain PST engineering strain stored in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 8h to serve as a first-level seed.
(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 10h to obtain a secondary seed culture solution.
(3) Inoculating the secondary seed culture solution into a 50L fermentation tank according to the inoculation amount of 10% for fed-batch fermentation, wherein the formulas of a fermentation culture medium and the feed solution are the same as those in example 2, the culture temperature is 30 ℃, the culture pH is 7.0, the fermentation is 49h, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 10h, finally 100mM of each of glutamic acid, glycine and cysteine are added for GSH synthesis, the fermentation temperature is increased to 37 ℃, and the fermentation is stopped when the yield of the GSH is not increased any more.
Measuring the content of GSH in the fermentation broth, and drawing a GSH production curve, wherein the GSH growth curve of this example is shown in figure 11, as shown in figure 11, the final maximum yield of GSH is 24.7g/L, and the GSH yield is 80.4% based on the addition amount of amino acid.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Sequence listing
<110> Shanghai Qingping pharmaceutical Co., Ltd
<120> preparation method of recombinant corynebacterium glutamicum and glutathione
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2265
<212> DNA
<213> Streptococcus thermophilus bifunctional glutathione synthetase gene (Streptococcus thermophilus gshFst)
<400> 1
atgacattaa accaacttct tcaaaaactg gaagctacca gccctattct ccaagctaat 60
tttggaatcg agcgcgagag tctacgtgtc gataggcaag gacaactggt gcatacacct 120
cacccatcct gtctaggagc tcgtagtttc cacccctata ttcagactga tttttgcgag 180
tttcagatgg aactcatcac accagttgcc aaatctacta ctgaggctcg ccgatttctg 240
ggagctatta ctgatgtagc tggccgctct attgctacag acgaggttct ctggccttta 300
tccatgccac ctcgtctaaa ggcagaggag attcaagttg ctcaactgga aaatgacttc 360
gaacgccatt atcgtaacta tttggctgaa aaatacggaa ctaaactaca agctatctca 420
ggtatccact ataatatgga actgggtaaa gatttagttg aggccttgtt ccaagaaagt 480
ggtcagaccg atatgattgc cttcaaaaac gccctctatc ttaagctggc tcagaactac 540
ttgcgctacc gttgggtgat tacctatctc tttggggcct cacccatcgc cgaacaaggt 600
ttctttgacc aggaagttcc agaacctgtg cgttccttcc gtaacagtga ccacggctat 660
gtcaataagg aagagattca agtatccttt gtaagtctag aagattatgt ctcagccatt 720
gaaacctata tcgaacaagg agatttgaat gcagagaaag aattttactc agctgttcgt 780
ttccgtggac aaaaggttaa tcgttccttc cttgacaaag gaatcaccta cctagagttc 840
cgtaatttcg accttaaccc ttttgagcgt atcggtatta gtcagactac tatggacact 900
gtgcacttac tcattttagc cttcctttgg cttgatagcc ctgaaaatgt cgaccaagct 960
cttgcacaag gccacgcgtt aaatgagaaa attgccctct ctcatcctct agaacctcta 1020
ccttcggagg ctaaaactca ggacattgta actgccctag accaactggt gcaacacttt 1080
ggacttggtg actatcatca agatctggtt aagcaagtta aggcagcctt tgcggatcca 1140
aatcaaacgc tctctgccca gctcttaccc tatatcaaag acaaatctct agccgaattt 1200
gctttaaaca aggctcttgc ctatcatgat tacgactgga ctgcccacta tgctctcaag 1260
ggctatgaag agatggaact ctccacccag atgttgctct ttgatgccat ccaaaagggg 1320
attcactttg aaatattgga tgagcaagat caattcctaa aactttggca ccaagaccat 1380
gttgaatacg tcaaaaacgg taacatgacc tcaaaagaca actacgtggt tccccttgct 1440
atggctaata agaccgtaac caagaagatt ctagcagatg ctggctttcc agttccttca 1500
ggagacgaat ttaccagtct tgaggaagga cttgcctact accctcttat caaggataag 1560
caaattgttg tcaaacccaa gtcaactaac tttggtctgg gaatttccat tttccaagaa 1620
cctgccagtc ttgacaacta tcaaaaagcc cttgaaattg ctttcgcaga agatacctct 1680
gtccttgttg aagaatttat tccaggaacc gaataccgtt tcttcatctt ggatgggcgt 1740
tgtgaggctg ttcttctgcg tgtcgctgcc aatgttattg gtgatggcaa acacaccatt 1800
cgtgaactag tcgctcagaa aaatgctaat ccattgcgtg gccgtgatca ccggtcacct 1860
ctggaaatca ttgagctagg agacatcgaa caactaatgt tagctcaaca gggttacaca 1920
cctgatgata ttctcccaga aggaaaaaag gtcaatctgc gtcgtaattc caacatctct 1980
acaggtggtg actctattga tatcactgag accatggatt cctcttacca agaattagcc 2040
gcagccatgg caactagcat gggcgcctgg gcttgcgggg ttgatctgat aattccagat 2100
gaaactcaaa ttgccaccaa ggaaaatcct cattgcacct gcattgagct caactttaac 2160
ccttcgatgt atatgcacac ctactgtgct gagggtcctg gccaagctat cactactaaa 2220
atcctagata aactttttcc agaaatagtg gctggtcaaa cttaa 2265
Claims (10)
1. A corynebacterium glutamicum genetically engineered bacterium is characterized in that: the gene of the bifunctional glutathione synthetase is overexpressed in the corynebacterium glutamicum genetic engineering bacteria.
2. The genetically engineered bacterium of Corynebacterium glutamicum of claim 1, wherein the strain of Corynebacterium glutamicum comprises: the nucleotide sequence of the bifunctional glutathione synthetase gene is shown as SEQ ID NO. 1.
3. A method for producing a genetically engineered bacterium of corynebacterium glutamicum according to claim 1 or 2, comprising: the method comprises the following steps:
(1) cloning the gene of the bifunctional glutathione synthetase to pEC-XK99E plasmid to obtain recombinant plasmid;
(2) transforming the recombinant plasmid into corynebacterium glutamicum to obtain the corynebacterium glutamicum genetic engineering strain.
4. The production method according to claim 3, characterized in that: the specific method of the step (1) is as follows: and (2) amplifying DNA by taking the plasmid pET28a-gshFst as a template, carrying out double enzyme digestion on the amplification product or the plasmid by using BamHI and PstI, and then connecting the amplification product or the plasmid with the pEC-XK99E plasmid subjected to enzyme digestion treatment by using BamHI and PstI to obtain the recombinant plasmid.
5. A preparation method of glutathione is characterized by comprising the following steps: the glutathione is produced by using the corynebacterium glutamicum genetically engineered strain of claim 1 or 2.
6. The method for producing glutathione according to claim 5, wherein: producing the glutathione by adopting a fed-batch fermentation method; glutamic acid, glycine and cysteine are added during production of the glutathione, and the initial concentration ranges of the added glutamic acid, the added glycine and the added cysteine in a reaction system are respectively 80-120 mM.
7. The method for producing glutathione according to claim 6, wherein: carrying out amplification culture on the corynebacterium glutamicum genetically engineered bacteria, inoculating the corynebacterium glutamicum genetically engineered bacteria to a fermentation culture medium for fermentation culture, adding a feed liquid in batches during fermentation culture, controlling the temperature of the fermentation culture to be 25-35 ℃, controlling the pH to be 6.5-7.5, and carrying out the fermentation culture for 40-60 hours; and adding an inducer for continuous culture for 5-15 h, adding glutamic acid, glycine and cysteine, and raising the temperature to 35-40 ℃ for culture until the yield of the glutathione is not increased any more.
8. The method for producing glutathione according to claim 7, wherein: the fermentation medium for fermentation culture comprises the following components: 5-30 g/L carbon source, 1-4 g/L nitrogen source, 0.5-15 g/L inorganic salt and trace elements, and 0.001-1 g/L growth factor which can be selectively added.
9. The method for producing glutathione according to claim 8, wherein: the carbon source is one or more of starch hydrolysis sugar, corn steep liquor, molasses, cane sugar, glucose and glycerol; the nitrogen source is one or more of yeast extract, peptone, tryptone, yeast extract and urea; the inorganic salt and the trace elements are one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, ammonium chloride, potassium chloride, sodium chloride, calcium carbonate, ammonium sulfate and magnesium sulfate, and the growth factors are one or more of biotin, vitamin B1, vitamin B6 and glutamic acid.
10. The method for producing glutathione according to claim 7, wherein: the feed liquid is one or more of glucose solution, yeast extract, sucrose, starch hydrolysis sugar, tryptone, corn steep liquor, magnesium sulfate and glycerol, and the concentration of the feed liquid is 400-500 g/L.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101715490A (en) * | 2007-04-06 | 2010-05-26 | 协和发酵生化株式会社 | Method for production of glutathione or gamma-glutamylcysteine |
| JP2012085637A (en) * | 2010-09-22 | 2012-05-10 | Ajinomoto Co Inc | METHOD FOR MANUFACTURING γ-Glu-X-Y |
| CN102586369A (en) * | 2011-01-10 | 2012-07-18 | 华东理工大学 | Method for producing glutathione by fermentation of recombinant Escherichia coli |
-
2020
- 2020-12-31 CN CN202011635514.6A patent/CN112646768B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101715490A (en) * | 2007-04-06 | 2010-05-26 | 协和发酵生化株式会社 | Method for production of glutathione or gamma-glutamylcysteine |
| US20100203592A1 (en) * | 2007-04-06 | 2010-08-12 | Kyowa Hakko Bio Co., Ltd. | Method for production of glutathione or gamma-glutamylcysteine |
| JP2012085637A (en) * | 2010-09-22 | 2012-05-10 | Ajinomoto Co Inc | METHOD FOR MANUFACTURING γ-Glu-X-Y |
| CN102586369A (en) * | 2011-01-10 | 2012-07-18 | 华东理工大学 | Method for producing glutathione by fermentation of recombinant Escherichia coli |
Non-Patent Citations (2)
| Title |
|---|
| WEI LIU等: "Efficient production of glutathione with multi-pathway engineering in Corynebacterium glutamicum", 《JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY》 * |
| 万俊仙等: "大肠杆菌半胱氨酸合成途径强化对谷胱甘肽生物合成的影响", 《华东理工大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116621920A (en) * | 2023-07-24 | 2023-08-22 | 山东睿鹰制药集团有限公司 | Preparation method of reduced glutathione |
| CN116621920B (en) * | 2023-07-24 | 2023-09-29 | 山东睿鹰制药集团有限公司 | Preparation method of reduced glutathione |
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Denomination of invention: A Preparation Method for Recombinant Corynebacterium Glutamate and Glutathione Granted publication date: 20230523 Pledgee: Agricultural Bank of China Limited Shanghai Yangtze River Delta Integrated Demonstration Zone Sub branch Pledgor: SHANGHAI QINGPING PHARMACEUTICAL Co.,Ltd. Registration number: Y2024310000191 |
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