CN112646768B - Recombinant corynebacterium glutamicum and preparation method of glutathione - Google Patents

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 gene engineering bacterium is adopted as an original strain, and the glutathione is produced by a fermentation method, so that the production of the glutathione is high, the yield is high, the operation is simple and convenient, the food-grade corynebacterium glutamicum is safe and nontoxic, the production concept of saving resources and being environment-friendly is met, and the method provides reference and reference for the industrial production of the glutathione for the recombinant corynebacterium glutamicum.

Description

A kind of preparation method of recombinant Corynebacterium glutamicum and glutathione

technical field

The invention relates to the field of bioengineering, in particular to a method for preparing glutathione by using recombinant Corynebacterium glutamicum.

Background technique

Glutathione (γ-L-glutamyl-cysteinyl-glycine, GSH) is an active tripeptide compound formed by the condensation of three amino acids, L-glutamic acid, L-cysteine and glycine. in various organisms. Reduced glutathione has many important functions in living tissues. Glutathione can improve human immunity and anti-aging. Its effect on the retarded cells of the elderly is greater than that of young people. It is also very effective in treating symptoms such as leukopenia caused by radiation and radiopharmaceuticals.

At present, there are many preparation methods of glutathione, common ones include solvent extraction, chemical synthesis, biological fermentation and enzymatic method. At present, the production of glutathione at home and abroad mainly adopts fermentation method or enzymatic method. The fermentation method is mainly to clone the gene coding for the synthesis of glutathione enzyme system or bifunctional enzyme into E. coli or yeast, and obtain glutathione through fermentation culture. Glycerin, the yeast fermentation process is relatively mature in the fermentation method. Patents CN201810844388 and CN201680013630 exogenously express glutathione synthesis bifunctional enzyme genes in yeast and Escherichia coli, respectively, to achieve the purpose of fermentation and production of glutathione, but the yield is very low. Escherichia coli is also an opportunistic pathogenic bacterium, and the recombinant protein, organic acid and other products produced contain Escherichia coli-related antigens to cause an immune response; Escherichia coli is also a good host of phages, and is extremely susceptible to phage infection. Therefore, the current fermentation method is not suitable for the production of food-grade glutathione, and does not meet the production concept of saving resources and being environmentally friendly.

Contents of the invention

The object of the present invention is to provide a genetic engineering bacterium of recombinant Corynebacterium glutamicum, which is used to produce glutathione, which has the advantages of high yield, high yield, safety and non-toxicity.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

The first aspect of the present invention provides a genetically engineered bacterium of Corynebacterium glutamicum, wherein the gene of bifunctional glutathione synthase is overexpressed in the genetically engineered bacterium of Corynebacterium glutamicum.

Preferably, the nucleotide sequence of the bifunctional glutathione synthetase gene is shown in SEQ ID NO.1.

In the present invention, the bifunctional glutathione synthetase gene is derived from Streptococcusthermophilus (gshFst).

The second aspect of the present invention provides a kind of preparation method of described Corynebacterium glutamicum genetic engineering bacterium, comprises the steps:

(1) Cloning the gene of the bifunctional glutathione synthase into the pEC-XK99E plasmid to obtain a recombinant plasmid;

(2) Transforming the recombinant plasmid into Corynebacterium glutamicum to obtain the genetic engineering bacteria of Corynebacterium glutamicum.

In the present invention, the bifunctional glutathione synthetase gene can be obtained by conventional genetic engineering means. For example, Streptococcus thermophilus DNA can be used as a template to obtain it through PCR amplification and isolation, and it can also be obtained through artificial synthesis according to its sequence.

Preferably, the specific method of the step (1) is: use the plasmid pET28a-gshFst as a template to amplify the DNA, then use BamHI and PstI to carry out double digestion of the amplified product or plasmid, and then combine with BamHI and PstI The pEC-XK99E plasmid after restriction treatment is ligated to obtain the recombinant plasmid.

In the present invention, the recombinant plasmid is transferred into Corynebacterium glutamicum by electroporation.

The third aspect of the present invention provides an application of the Corynebacterium glutamicum genetically engineered bacteria in the production of glutathione.

The fourth aspect of the present invention provides a method for preparing glutathione, using the Corynebacterium glutamicum genetically engineered bacteria to produce the glutathione.

Preferably, the glutathione is produced by a fed-batch fermentation method.

Preferably, glutamic acid, glycine, and cysteine are added when producing the glutathione, and the added glutamic acid, glycine, and cysteine are in the reaction system The initial concentration of each is 80-120 mM, more preferably 90-110 mM, more preferably 95-105 mM.

The present invention synthesizes glutathione from exogenously added glutamic acid, glycine and cysteine through high-density culture and overexpression of a bifunctional glutathione synthetase gene, thereby achieving high yield and high yield.

Preferably, after expanding the culture of the Corynebacterium glutamicum genetically engineered bacteria, inoculate it into the fermentation medium to carry out fermentation culture, add feed liquid in batches while fermentation culture, and control the temperature of fermentation culture to be 25 ～35℃, pH 6.5～7.5, fermentation and cultivation time 40～60h; then add inducer and continue to cultivate for 5～15h, then add glutamic acid, glycine, cysteine, and raise the temperature to 35～40℃ for cultivation Until the production of glutathione is no longer increased.

Further preferably, the temperature of the fermentation culture is 28-32°C, the pH is 6.5-7.2, and the fermentation culture time is 45-50 hours; then add the inducer and continue the culture for 9-12 hours, then add glutamic acid, glycine, half cystine, and raise the temperature to 36-38° C. to cultivate until the glutathione production no longer increases.

In the present invention, Corynebacterium glutamicum genetically engineered bacterium expands culture method as follows:

A. Plate culture: Inoculate the genetically engineered bacteria of Corynebacterium glutamicum preserved in glycerol tubes into LB medium, and culture at 30°C for 8-12 hours to obtain first-grade seeds.

B. Liquid seed culture: inoculate the Corynebacterium glutamicum genetically engineered bacteria (first-class seed) cultured on the plate into the second-level seed medium, and cultivate at 30° C. for 8-12 hours to obtain the second-level 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 present invention, the secondary seed culture liquid is inoculated into the fermentation medium in the fermenter according to the inoculum amount of 7%-10%.

In the present invention, the inducer used is IPTG, and the concentration of IPTG is 1 mmol/L.

Further preferably, the fermentation medium of the fermentation culture includes the following components: carbon source 5-30g/L, nitrogen source 1-4g/L, inorganic salts and trace elements 0.5-15g/L, optionally added The growth factor of 0.001～1g/L.

Further preferably, the carbon source is one or more of starch hydrolysis sugar, corn steep liquor, molasses, sucrose, glucose, glycerin; the nitrogen source is yeast extract, peptone, tryptone, yeast extract , one or more of urea; the inorganic salts and trace elements are potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, ammonium chloride, potassium chloride, sodium chloride, calcium carbonate, sulfuric acid One or more of ammonium and magnesium sulfate, and the growth factor includes 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 hydrolyzed sugar, tryptone, corn steep liquor, magnesium sulfate, glycerol, and the feed liquid The concentration is 400-500g/L.

Corynebacterium glutamicum in the present invention is a food-grade microorganism, safe and non-toxic, so it is more advantageous to use Corynebacterium glutamicum as a starting strain to produce glutathione.

Using the recombinant Corynebacterium glutamicum of the present invention to produce glutathione by fermentation is not only simple and convenient to operate, but also has high yield and high yield, which conforms to the production concept of resource saving and environmental friendliness. It is a recombinant glutamic acid rod Bacillus industrial production of glutathione provides a reference and reference.

Compared with the prior art, the present invention has the following advantages:

The invention obtains a recombinant Corynebacterium glutamicum genetically engineered bacterium, utilizes it to produce glutathione, and has the advantages of high yield, high yield, safety and non-toxicity, and the like.

Description of drawings

Accompanying drawing 1: the production curve of GSH in the fermented liquid of embodiment 2 of the present invention;

Accompanying drawing 2: the production curve of GSH in the fermented liquid of the embodiment of the present invention 3;

Accompanying drawing 3: the production curve of GSH in the fermented liquid of embodiment 4 of the present invention;

Accompanying drawing 4: the production curve of GSH in the fermented liquid of the embodiment of the present invention 5;

Accompanying drawing 5: the production curve of GSH in the fermented liquid of embodiment 6 of the present invention;

Accompanying drawing 6: the production curve of GSH in the fermented liquid of the embodiment of the present invention 7;

Accompanying drawing 7: the production curve of GSH in the fermented liquid of the embodiment of the present invention 8;

Accompanying drawing 8: the production curve of GSH in the fermented liquid of the embodiment of the present invention 9;

Accompanying drawing 9: the production curve of GSH in the fermented liquid of embodiment 10 of the present invention;

Accompanying drawing 10: the production curve of GSH in the fermented liquid of the embodiment of the present invention 11;

Accompanying drawing 11: The production curve of GSH in the fermented liquid of embodiment 12 of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with specific related embodiments. However, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention. Experimental method used in the following examples. Unless otherwise specified, all are conventional methods, and the used materials, reagents, etc., unless otherwise specified, can be obtained from commercial sources. It should be understood that these examples are only used to illustrate the present invention 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 bifunctional glutathione synthase gene (SEQ ID NO: 1) derived from Streptococcus thermophilus (gshFst) was selected, and the gene fragment of the bifunctional glutathione synthase was amplified using the recombinant plasmid pET28a-gshFst as a template, The linearized plasmid was obtained by double digestion with BamHI and SmaI, and the recombinant plasmid pHT-ST was obtained by ligation under the action of T4 ligase.

Preparation of DNA fragments: The recombinant plasmid pET28a-gshFst already in the laboratory was used as a template for DNA amplification. The reaction conditions were: pre-denaturation at 98°C for 5 minutes; denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 5 min, a total of 30 cycles; after the end, final extension at 72°C for 10 min. The amplified product was verified by nucleic acid electrophoresis and purified by PCR Purification Kit kit, and then the template plasmid was digested by DpnI. Finally, it was double digested with BamHI and SmaI.

The enzyme digestion system is: PCR product or pEC-XK99E plasmid 50ul, BamHI and SmaI each 1ul, buffer 10ul, ddH ₂ O 38ul, total volume 100ul.

The digested PCR product was ligated with the pEC-XK99E plasmid treated with the same enzyme. The ligation system was: 4ul of the digested PCR product, 4ul of the digested pEC-XK99E plasmid, 1ul of T4 ligase, buffer 1ul, the recombinant plasmid pEC-XK99E-ST was obtained by ligation. The recombinant plasmid was electrotransfected into Corynebacterium glutamicum to obtain the recombinant strain PST.

Example 2:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the engineered strains of recombinant PST strains stored in glycerol tubes into LB medium, and culture them at 30°C for 9 hours as primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 11 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture liquid into the fermenter of 2L by 7% inoculum size and carry out fed-batch fermentation. L, Potassium dihydrogen phosphate 0.2g/L, Dipotassium hydrogen phosphate 0.2g/L, Sodium chloride 0.1g/L, Magnesium sulfate 0.5g/L, feed liquid is 400g/L glucose solution, culture temperature 30°C, Cultivate at pH 7.0, ferment for 50 h, add 1 mM inducer IPTG to the culture medium, continue culturing for 10 h, finally add 100 mM each of glutamic acid, glycine, and cysteine to synthesize GSH and raise the fermentation temperature to 37°C. Stop fermentation when the output of GSH no longer increases.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH production curve of this embodiment is shown in Figure 1. As shown in Figure 1, the final GSH yield is 25.0 g/L, and the GSH yield is 81.3% based on the amount of amino acid added.

Example 3:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the recombinant PST engineered strains stored in the glycerol tube into the LB medium, and cultivate them at 30° C. for 8 hours as the primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 11 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture solution into a 2L fermenter with an inoculum size of 7.5% and carry out fed-batch fermentation. The fermentation medium adopts starch hydrolysis sugar 8g/L, molasses 1g/L, and yeast extract 1g/L , urea 1g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, sodium chloride 0.2g/L, magnesium sulfate 1.5g/L, feed liquid is 500g/L glucose solution, culture Temperature 30°C, culture pH 6.5, ferment for 45h, add 1mM inducer IPTG to the medium, continue to culture for 10h, finally add 100mM each of glutamic acid, glycine, and cysteine to synthesize GSH and raise the fermentation temperature to 37°C, stop the fermentation when the output of GSH no longer increases.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 2, as shown in accompanying drawing 2, the output of final GSH is 24.05g/L, after calculation, based on the addition of amino acid The yield of GSH was 78.3%.

Example 4:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the engineered strains of recombinant PST strains stored in glycerol tubes into LB medium, and culture them at 30°C for 9 hours as primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 12 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture solution into a 2L fermenter with an inoculum size of 9% and carry out fed-batch fermentation. The fermentation medium adopts sucrose 10g/L, peptone 1g/L, and yeast extract 2g/L , ammonium chloride 2g/L, potassium dihydrogen phosphate 0.3g/L, dipotassium hydrogen phosphate 0.3g/L, sodium chloride 0.4g/L, magnesium sulfate 1g/L, feeding liquid is 450g/L glucose solution, Culture temperature 30°C, culture pH 6.7, ferment for 49h, add 1mM inducer IPTG to the medium, continue to culture for 10h, finally add 100mM each of glutamic acid, glycine, and cysteine to synthesize GSH and increase the fermentation temperature To 37 ℃, stop the fermentation when the output of GSH no longer increases.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 3, as shown in accompanying drawing 3, the output of final GSH is 23.95g/L, after calculation, based on the addition of amino acid The yield of GSH was 77.9%.

Example 5:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the engineered strains of the recombinant PST bacteria stored in the glycerol tube into LB medium, and cultivate them at 30° C. for 10 h as primary seeds.

(2) The primary seeds were transferred into LB liquid medium, and cultured at 30° C. for 10 h to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture solution into a 2L fermenter with an inoculation amount of 8.5% and carry out fed-batch fermentation. The fermentation medium adopts corn steep liquor 10mL/L, molasses 1g/L, and yeast extract 3g/L. L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 0.3g/L, dipotassium hydrogen phosphate 0.3g/L, sodium chloride 0.5g/L, magnesium sulfate 1.2g/L, feeding liquid is glucose 400g/L, Yeast extract 1g/L, culture temperature 30°C, culture pH 6.8, ferment for 47h, add 1mM inducer IPTG to the medium, continue to culture for 10h, finally add 100mM each of glutamic acid, glycine, and cysteine Synthesize GSH and increase the fermentation temperature to 37°C, and stop the fermentation when the output of GSH no longer increases.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 4, as shown in accompanying drawing 4, the output of final GSH is 24.95g/L, after calculation, based on the addition of amino acid The yield of GSH was 81.2%.

Embodiment 6:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the recombinant PST engineered strains stored in the glycerol tube into the LB medium, and cultivate them at 30° C. for 8 hours as the primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 11 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture solution into a 2L fermenter with 10% inoculum size and carry out fed-batch fermentation. The fermentation medium adopts 11g/L of molasses, 2g/L of glycerol, and 3g/L of yeast extract. , Ammonium Sulfate 2g/L, Potassium Dihydrogen Phosphate 0.2g/L, Disodium Hydrogen Phosphate 1g/L, Potassium Chloride 0.5g/L, Magnesium Sulfate 0.6g/L, Feeding Liquid is Glucose 400g/L, Glycerin 50g /L, yeast extract 2g/L, culture temperature 30°C, culture pH 6.6, ferment for 48h, add 1mM inducer IPTG to the medium, continue to culture for 10h, finally add glutamic acid, glycine, cysteine Each 100 mM was used to synthesize GSH and the fermentation temperature was increased to 37° C., and the fermentation was stopped when the GSH production no longer increased.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 5, as shown in accompanying drawing 5, the output of final GSH is 24.90g/L, after calculation, based on the addition of amino acid The yield of GSH was 81.0%.

Embodiment 7:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the engineered strains of recombinant PST strains stored in glycerol tubes into LB medium, and culture them at 30°C for 9 hours as primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 12 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture liquid into the fermenter of 2L by the inoculation amount of 7.5% and carry out fed-batch fermentation, and the fermentation medium adopts glycerol 12g/L, sucrose 3g/L, calcium carbonate 5g/L, grain Ammonium chloride 1g/L, tryptone 1.5g/L, ammonium chloride 1g/L, potassium dihydrogen phosphate 0.3g/L, disodium hydrogen phosphate 1.5g/L, potassium chloride 0.5g/L, magnesium sulfate 1.2g /L, the feed solution is glucose 400g/L, sucrose 50g/L, yeast extract 1.5g/L, culture temperature 30℃, culture pH 7.0, fermentation 46h, add 1mM inducer IPTG to the medium, continue Cultivate for 10 h, finally add 100 mM each of glutamic acid, glycine, and cysteine to synthesize GSH and raise the fermentation temperature to 37° C., and stop the fermentation when the output of GSH no longer increases.

Measure the content of GSH in the fermented liquid, and draw GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 6, as shown in accompanying drawing 6, the output of final GSH is 24.50g/L, after calculation, based on the addition of amino acid The yield of GSH was 79.7%.

Embodiment 8:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the recombinant PST engineered strains stored in the glycerol tube into the LB medium, and cultivate them at 30° C. for 8 hours as the primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 9 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture liquid into the fermenter of 2L by 7.9% inoculum size and carry out fed-batch fermentation, and the fermentation medium adopts sucrose 12g/L, molasses 3g/L, biotin 20ug/L, vitamin B1 1mg/L, tryptone 2g/L, ammonium sulfate 3g/L, potassium dihydrogen phosphate 0.4g/L, dipotassium hydrogen phosphate 0.4g/L, sodium chloride 0.6g/L, magnesium sulfate 0.5g/L, The feed solution was glucose 400g/L, tryptone 1g/L, culture temperature 30°C, culture pH 7.2, fermentation for 49h, 1mM inducer IPTG was added to the culture medium, and culture was continued for 10h, finally glutamic acid, glycine, semi 100 mM of photoacid to synthesize GSH and raise the fermentation temperature to 37° C., and stop the fermentation when the output of GSH no longer increases.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 7, as shown in accompanying drawing 7, the output of final GSH is 23.70g/L, after calculation, based on the addition of amino acid The yield of GSH was 77.1%.

Embodiment 9:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the recombinant bacteria PST engineered strains stored in the glycerol tube into LB medium, and cultivate them at 30° C. for 11 hours as the primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 11 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture solution into a 2L fermenter with an inoculum size of 9.5% and carry out fed-batch fermentation. The fermentation medium adopts glucose 9g/L, glycerol 1g/L, corn steep liquor 20mL/L, biological Vegetarian 40ug/L, vitamin B1 2mg/L, yeast extract 3g/L, ammonium sulfate 4g/L, potassium dihydrogen phosphate 0.3g/L, dipotassium hydrogen phosphate 0.3g/L, sodium chloride 1.5g/L , magnesium sulfate 0.8g/L, calcium carbonate 2g/L, feed liquid 450g/L glucose, corn steep liquor 50ml/L, magnesium sulfate 5g/L, culture temperature 30°C, culture pH 7.2, fermentation 45h, to the culture medium Add 1mM inducer IPTG to the medium, continue culturing for 10h, finally add 100mM each of glutamic acid, glycine, and cysteine to synthesize GSH and raise the fermentation temperature to 37°C, and stop the fermentation when the output of GSH no longer increases.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 8, as shown in accompanying drawing 8, the output of final GSH is 23.20g/L, after calculation, based on the addition of amino acid amount, the GSH yield was 75.5%.

Example 10:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the recombinant bacteria PST engineered strains stored in the glycerol tube into LB medium, and cultivate them at 30° C. for 12 hours as the primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 8 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture solution into a 2L fermenter with a 7% inoculum size and carry out fed-batch fermentation. The fermentation medium adopts corn steep liquor 15ml/L, sucrose 1g/L, starch hydrolyzed sugar 2g/L , glucose 8g/L, biotin 10ug/L, vitamin B6 2mg/L, vitamin B1 2mg/L yeast extract 1g/L, ammonium sulfate 1g/L, urea 1g/L, potassium dihydrogen phosphate 0.4g/L , dipotassium hydrogen phosphate 0.4g/L, sodium chloride 0.6g/L, magnesium sulfate 0.8g/L, calcium carbonate 1g/L, feeding liquid is glucose 200g/L, starch hydrolyzed sugar 200g/L, glycerin 20g/L L. Magnesium sulfate 4g/L, culture temperature 30°C, culture pH 7.0, ferment for 46h, add 1mM inducer IPTG to the medium, continue to culture for 10h, finally add 100mM each of glutamic acid, glycine, and cysteine Synthesize GSH and increase the fermentation temperature to 37°C, and stop the fermentation when the output of GSH no longer increases.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 9, as shown in accompanying drawing 9, the output of final GSH is 24.83g/L, after calculation, based on the addition of amino acid The yield of GSH was 80.8%.

Example 11:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the recombinant bacteria PST engineered strains stored in the glycerol tube into LB medium, and cultivate them at 30° C. for 11 hours as the primary seeds.

(2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 9 hours to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture solution into a 5L fermenter with an inoculum size of 9% and carry out fed-batch fermentation. The formulation of the fermentation medium and the feeding solution is the same as in Example 2, with a culture temperature of 30° C. and a culture pH of 7.0 , fermented for 48h, added 1mM inducer IPTG to the medium, continued to cultivate for 10h, finally added 100mM each of glutamic acid, glycine, and cysteine to synthesize GSH and raised the fermentation temperature to 37°C. Stop fermentation when no more increase.

Measure the content of GSH in the fermentation broth, and draw the GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 10, as shown in accompanying drawing 10, the output of final GSH is 23.90g/L, after calculation, based on the addition of amino acid The yield of GSH was 77.8%.

Example 12:

Production of GSH using recombinant bacteria PST:

(1) Inoculate the recombinant PST engineered strains stored in the glycerol tube into the LB medium, and cultivate them at 30° C. for 8 hours as the primary seeds.

(2) The primary seeds were transferred into LB liquid medium, and cultured at 30° C. for 10 h to obtain the secondary seed culture solution.

(3) Inoculate the secondary seed culture solution into a 50L fermenter with a 10% inoculum size and carry out fed-batch fermentation. The formulation of the fermentation medium and the feeding solution is the same as in Example 2, with a culture temperature of 30° C. and a culture pH of 7.0 , fermented for 49 hours, added 1 mM inducer IPTG to the medium, continued to cultivate for 10 hours, and finally added 100 mM of glutamic acid, glycine, and cysteine to synthesize GSH and raised the fermentation temperature to 37 ° C. Stop fermentation when no more increase.

Measure the content of GSH in the fermented liquid, and draw GSH production curve, the GSH growth curve of the present embodiment is shown in accompanying drawing 11, as shown in accompanying drawing 11, the highest output of final GSH is 24.7g/L, after calculation, based on amino acid Added amount, GSH yield is 80.4%.

The above-mentioned embodiments are only to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.

sequence listing

<110> Shanghai Qingping Pharmaceutical Co., Ltd.

<120> A preparation method of recombinant Corynebacterium glutamicum and glutathione

<160> 1

<170> SIP Sequence Listing 1.0

<210> 1

<211> 2265

<212>DNA

<213> Streptococcus thermophilus gshFst bifunctional glutathione synthase gene

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gtccttgttg aagaatttt 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 ttctccccaga 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 (4)

1. A method for preparing glutathione, which is characterized in that: the glutathione is produced by adopting corynebacterium glutamicum genetic engineering bacteria,

the gene of the difunctional glutathione synthetase is overexpressed in the corynebacterium glutamicum genetic engineering bacteria, the nucleotide sequence of the difunctional glutathione synthetase gene is shown as SEQ ID NO.1,

after carrying out enlarged culture on the corynebacterium glutamicum genetically engineered bacterium, inoculating the corynebacterium glutamicum genetically engineered bacterium into a fermentation culture medium for fermentation culture, adding feed liquid in batches while carrying out fermentation culture, controlling the temperature of the fermentation culture to be 28-32 ℃, controlling the pH to be 6.5-7.2, and controlling the fermentation culture time to be 45-50 h; then adding an inducer to continue culturing for 9-12 hours, adding glutamic acid, glycine and cysteine, and raising the temperature to 36-38 ℃ to culture until the yield of the glutathione is not increased any more,

the initial concentration ranges of the glutamic acid, the glycine and the cysteine in the reaction system are respectively 80-120 mM,

the fermentation medium for fermentation culture comprises the following components: 5-30 g/L of carbon source, 1-4 g/L of nitrogen source, 0.5-15 g/L of inorganic salt and 0.001-1 g/L of trace element which can be selectively added,

the feed supplement liquid is one or more of glucose solution, yeast extract, sucrose, tryptone, corn steep liquor and glycerin, and the concentration of the feed supplement liquid is 400-500 g/L.

2. The method for producing glutathione according to claim 1, wherein: the preparation method of the corynebacterium glutamicum genetically engineered bacterium comprises the following steps:

(1) Cloning the gene of the difunctional glutathione synthetase to pEC-XK99E plasmid to obtain recombinant plasmid;

(2) And transforming the recombinant plasmid into corynebacterium glutamicum to obtain the corynebacterium glutamicum genetic engineering bacterium.

3. The method for producing glutathione according to claim 2, characterized in that: the specific method of the step (1) is as follows: the plasmid pET28a-gshFst is used as a template for DNA amplification, then the amplified product is subjected to double digestion by BamH І and SmaI, and then is connected with the pEC-XK99E plasmid which is subjected to enzyme digestion by BamH І and SmaI, so as to obtain the recombinant plasmid.

4. The method for producing glutathione according to claim 1, wherein: the carbon source is one or more of corn steep liquor, molasses, sucrose, glucose and glycerol; the nitrogen source is one or more of peptone, yeast extract and urea; the inorganic salt and microelements 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 and vitamin B6.

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