CN112779173A - High-yield glutathione pichia pastoris strain G3-SF and application thereof - Google Patents

Abstract

The invention discloses a high-yield glutathione Pichia pastoris strain G3-SF and application thereof, Pichia pastoris GS115 is taken as a host, Scgsh1 and Scgsh2 genes from saccharomyces cerevisiae are heterologously expressed, the excessive production of GSH is obtained, and an engineering bacterium is named as G3; on the basis, heterologous expression is derived from a saccharomyces cerevisiae adenosine kinase Scadk1 gene and a codon-optimized StgshF gene derived from streptococcus sanguis, and aims to enhance energy supply in the fermentation process of engineering bacteria and reconstruct a GSH synthesis path. The highest horizontal yield of glutathione in a shake flask can reach (527.14 +/-17.92) mg/L, and the yield of intracellular glutathione is (19.96 +/-0.05) mg/L/OD. The constructed engineering bacteria G3-SA are fermented in a 5L fermentation tank, the highest yield of the glutathione in 56h is 5950mg/L, the yield of the intracellular glutathione is 25.54mg/L/OD, and a new idea is provided for the industrial production of the GSH.

Description

High-yield glutathione pichia pastoris strain G3-SF and application thereof

Technical Field

The invention belongs to the technical field of biosynthesis, and particularly relates to a high-yield glutathione pichia pastoris strain G3-SF and application thereof.

Background

Glutathione is a tripeptide active substance widely present in animals, plants and microorganisms, and has the effects of protecting and regulating intracellular redox balance. The molecular weight of the compound is 307.33, the compound is composed of glutamic acid, cysteine and glycine, the content of the compound in various organisms is different, and the content of the compound in yeast and animal viscera is higher. GSH also plays a wide role in the clinical field, and has significant efficacy in liver diseases, kidney diseases and cardiovascular diseases.

GSH is primarily characterized by having one gamma amide bond and one sulfhydryl group. In most procaryotic and eucaryotic organisms, glutamylcysteine is generated by glutamic acid and cysteine under the action of GSH1 in the first step, and then the product is reacted with glycine under the action of GSH2 to generate GSH, wherein the enzyme in the first step is a key enzyme for generating GSH and is inhibited by the feedback of the product, the two steps of enzyme are ATP dependent, and 1 molecule of ATP is consumed in the reaction.

Methods for synthesizing GSH have been reported to be chemical synthesis, enzymatic conversion, and fermentation. The chemical synthesis method adopts three raw materials and utilizes a chemical process to synthesize the GSH, so the cost is high and the GSH has pollution to the environment. The enzymatic method adopts three raw materials, ATP and enzyme reaction to generate GSH, and the core of the method is to screen out synthetase with high activity, but the cost is high, and most of the synthetases are researched at present. The fermentation method is that microorganisms use cheap raw materials to grow and accumulate high-concentration GSH, and then the GSH is separated and purified in the next step. Low cost, high purity and no pollution, and is the main method for producing GSH at present.

For this reason, attempts have been made to achieve the production of GSH using various microorganisms. In this regard, previous studies have often employed mutagenesis or genetic engineering to increase enzyme activity, or to increase precursor utilization by optimizing fermentation conditions. Since the yeast has a high content of GSH and a clear genetic background, it is often used as an excellent host for the production of GSH. At present, the main industrial production is mainly fermentation method, and the production host is yeast, large intestine and the like. The pichia pastoris is in a state of food-safe microorganisms, exogenous expression is mainly integrated, and an expensive inducer is not needed, so that the pichia pastoris becomes an excellent host for producing exogenous proteins and other biological products by high-density fermentation.

Three factors limiting the fermentation yield of GSH are the activity of GSH synthase, the supply of cysteine and ATP. Cysteine can be supplemented by exogenous addition, but excess cysteine can result in insufficient supply of ATP during synthesis.

Alfafara et al found cysteine (Cys) to be a key amino acid for increasing GSH. However, when high-density fermentation is carried out, Cys synthesized by cells through metabolism is far from meeting the requirement of GSH synthesis. Exogenous addition of Cys is therefore an effective strategy and indeed also leads to large scale production of GSH. However, when Cys is added in excess, ATP, which acts as an energy carrier, is made the limiting factor for GSH production. To solve this problem, Liang et al increase GSH yield by 11% by directly adding ATP, which is an expensive raw material, and cannot be scaled up. As another example, Wang et al studied that citrate was used as a co-energy substrate to promote the production of NADH, and further strengthened the production of ATP by an electron transport chain, the SAM and GSH yields of the strain and the co-production yield thereof were improved by 27.5%, however, this method could not improve enough ATP to meet the production requirement of high GSH.

Increasing the supply of ATP using traditional metabolic engineering is a good strategy for the performance of energy-intensive reactions. It is reported that expression of VHB protein in Pichia pastoris increases oxygen uptake capacity of the bacteria, and further increases ATP supply, resulting in a 19-fold increase in SAM yield compared to the control. Tani et al found that ADK from saccharomyces cerevisiae was the rate-limiting step in the conversion of AMP to ATP, and later confirmed in Harit's study of SAM production using pichia pastoris.

In summary, ATP is a main limiting factor for glutathione synthesis, and how to increase ATP supply and effectively increase glutathione yield is a technical problem that needs to be solved in the field.

Disclosure of Invention

The purpose of the invention is as follows: research shows that ATP addition or ATP metabolism improvement and regeneration promotion have a promoting effect on glutathione synthesis, but the problems of high cost or limited effect exist. In order to overcome the problems and the defects in the prior art, the invention provides the high-yield glutathione Pichia pastoris strain G3-SF and the application thereof, enhances the synthesis of intracellular GSH, and has universal significance for promoting the synthesis of glutathione or the production of other energy-consuming products.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:

the invention aims to provide a high-yield glutathione Pichia pastoris strain G3-SF, which takes Pichia pastoris as a host, heterologously expresses a glutathione synthetase Scgsh1 gene, a Scgsh2 gene, an adenosine kinase Scadk1 gene and a streptococcus sanguis-derived bifunctional enzyme StgshF gene from saccharomyces cerevisiae, and an obtained engineering bacterium is named as Pichia pastoris strain G3-SF.

Further, the Pichia pastoris GS115 is selected from Pichia pastoris, and the strain is purchased from an vast ling plasmid platform.

Further, the nucleotide sequences of the glutathione synthetase Scgsh1 and Scgsh2 genes, the adenosine kinase Scadk1 gene and the bifunctional enzyme StgshF gene are respectively shown as SEQ.NO.01-SEQ.NO. 04.

The invention also aims to provide a construction method of the high-yield glutathione Pichia pastoris strain G3-SF: taking pichia as a host, firstly heterologously expressing glutathione synthetase Scgsh1(Gene ID:853344) and Scgsh2(Gene ID:854108) genes from saccharomyces cerevisiae, and obtaining an engineering bacterium named G3; and G3 is taken as a host, and the adenosine kinase Scadk1(Gene ID:851812) Gene from saccharomyces cerevisiae and the StgshF (Gene ID: GU138096) Gene from streptococcus sanguis with optimized codons are expressed in a heterologous way, so that the obtained engineering bacterium is named as a pichia pastoris strain G3-SF.

Further, the concrete method is that pGAPZA is used as initial plasmid, BglII and BamHI are used for enzyme digestion, and the insert is insertedThe fragment containing Xba I was inserted therein, and the resulting plasmid was named pGAPZT. Inserting P between BglII and Xba I of pGAPZT_AOX1And (4) sequencing. The Scadk1 Gene (Gene ID:851812) was obtained BY S.cerevisiae BY4741 genomic PCR amplification, and the StgshF (Gene ID: GU138096) Gene was synthesized BY Kingzhi Biotechnology Ltd and codon-optimized. The obtained fragments are respectively connected in pGAPZT, and expression cassettes of two genes are obtained. Then, the StgshF gene expression cassette is connected behind the Scadk1 gene expression cassette in series by the principle of ligase, and is linearized and transferred to a G3 strain by SacI, and the obtained strain is screened by a YPD plate containing 2mg/L Zeocin, and the obtained strain is named as G3-SF.

Furthermore, in the fermentation process of the heterologous expression of the adenosine kinase Scadk1 gene and the bifunctional enzyme gene StgshF, 4g/L of sodium citrate is exogenously added when the fermentation time is 15-16 h.

Another objective of the invention is to provide a plasmid obtained in the process of the construction method of the high-yield glutathione Pichia pastoris strain G3-SF.

It is another object of the present invention to provide a gene encoding the high-yielding Pichia glutathione yeast strain G3-SF according to any one of claims 1 to 3.

The invention also aims to provide a method for preparing glutathione, which adopts the high-yield glutathione Pichia pastoris strain G3-SF or the high-yield glutathione Pichia pastoris strain G3-SF constructed by the method to synthesize glutathione by a fermentation method.

Another object of the present invention is to provide glutathione produced by the above method.

Has the advantages that: compared with the prior art, the high-yield glutathione pichia pastoris strain G3-SF and the application thereof provided by the invention have the following advantages: the invention takes pichia pastoris GS115 as a host, heterologously expresses Scgsh1 and Scgsh2 genes from saccharomyces cerevisiae, obtains the overproduction of GSH, and names engineering bacteria as G3; on the basis, heterologous expression is from saccharomyces cerevisiae adenosine kinase Scadk1 and StgshF gene obtained by Streptococcus sanguis after codon optimization, and aims to enhance energy supply and reconstruct metabolic pathway in the fermentation process of engineering bacteria. At present, the highest yield of the glutathione in a shake flask can reach (527.14 +/-17.92) mg/L, and the yield of the intracellular glutathione is (19.96 +/-0.05) mg/L/OD. The constructed engineering bacteria G3-SA are fermented in a 5L fermentation tank, the highest yield of the glutathione in 56h is 5950mg/L, and the yield of the intracellular glutathione is 25.54mg/L/OD, so that a new idea is provided for the industrial production of the GSH.

Drawings

FIG. 1 is an electrophoretogram constructed from the G3 strain in example 1;

wherein, M in a is 5kb marker; 1,2, PCR amplification of Scgsh1 gene;

m in b is 5kb marker; 1,2, PCR amplification of Scgsh2 gene;

m in c is 5kb marker; 1,2, extracting genome verification primer verification for G2 strain (G2 is constructed engineering bacteria which take pichia pastoris GS115 as a host and only express Scgsh1 gene);

m in d is 5kb marker; 1,2, extracting genome verification primer verification for G3 strain.

FIG. 2 is a graph showing the electrophoretic verification of G3-SF in example 2; m: DL10000 Marker; 1.2, 3, 4, 5: and (5) carrying out PCR verification on the genome of the engineering bacteria.

FIG. 3 is a graph showing the results of G3-SF shake flask fermentation in example 3;

FIG. 4 is a graph showing the change in intracellular ATP content in the fermentation of the G3-SF strain in example 3.

FIG. 5 shows the results of fermentation of G3-SF in a 5L fermentor in example 4;

FIG. 6 is a graph showing the results of shake flask fermentation of the G3 strain in comparative example 1; wherein, a-biomass, b-GSH concentration, c-GSH yield, d-strain fermentation result chart when amino acid precursor is added.

FIG. 7 is a schematic diagram of the method of the present invention.

Detailed Description

According to the invention, the synthesis path of glutathione is enhanced by heterologously expressing Scgsh1 and Scgsh2 in Pichia pastoris, the constructed strain is named as G3, and then the Scadk1 gene from Saccharomyces cerevisiae and the StgshF gene from Streptococcus sanguis are heterologously expressed, so that endogenous metabolic modification is carried out on host cells, and the supply of intracellular ATP is effectively enhanced and the GSH synthesis path is reconstructed by combining the addition of exogenous sodium citrate, thereby further enhancing the synthesis of intracellular GSH.

The invention is further described with reference to the following figures and examples.

Examples

The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

Example 1: construction of G3 Strain

Construction of the P3 plasmid: this patent is constructed with P_GAPIs a promoter, T_AOX1Is a constitutive expression strain of terminator. Firstly, amplifying P on genome by using GAP-F/GAP-R as primer_GAPA promoter sequence. pPIC 3.5K was cleaved with SacI and BamHI to remove P_AOX1Fragment combination of P_GAPThe plasmid constitutively expressed was obtained by sequence substitution and named pPICKT. Genome of S.cerevisiae BY4741 is taken as a template, GSH1F/GSH1R and GSH2F/GSH2R are respectively PCR amplified to obtain target gene Scgsh1 and Scgsh2 fragments (the gene sequences are shown as SEQ. NO.01-SEQ. NO. 02) which are respectively connected to pPICKT to form P1 and P2 plasmids, and the construction is carried out after the verification BY YZGF/YZGR. Then G1F/G1R and TF/TR were used to amplify PGAP-Scgsh1 and T on P1 plasmid, respectively_AAnd the ox1 fragment is connected into a P2 plasmid by adopting a Novozam C113 multi-fragment homologous recombination and connection kit to obtain a plasmid pP-PGAP-Scgsh1-Scgsh2 which simultaneously contains expression units of Scgsh1 and Scgsh2, namely a P3 plasmid. And verified by a verified primer YZGGF/YZGGR, the construction can be realized.

Construction of the G3 strain: the plasmid construction processes are all transformed into escherichia coli JM109 competent cells, screening is carried out on an ampicillin (Amp) LB resistance plate containing 100mg/L, and the obtained transformant is verified by colony PCR and sequenced to obtain the correct transformant. After plasmids pPICKT, P1 and P3 were all linearized with SalI, the linearized plasmids were transformed into Pichia pastoris by electrotransformation according to the manual (Invitrogen), spread on MD histidine-containing deficient plates, and incubated at 30 ℃ for 2-3 d. The grown colonies are spotted into high-mass concentration G418(4mg/mL), and the target strain with high copy gene factor can be obtained after the grown larger colonies are subjected to genome verification, as shown in FIG. 1.

Example 2: construction of G3-SF Strain

Construction of pGAPZT-Scadk1-StgshF plasmid: in the research, pGAPZA is used as a starting plasmid, the starting plasmid is modified, the modified plasmid is named as pGAPZT, and the plasmid realizes the tandem expression of multiple genes in a mode of isocaudarner. Firstly, ZXF/ZXR is used as a primer, pGAPZA is used as a template to carry out PCR amplification, so that the front end of a promoter of the recovered product X fragment is provided with Xba I, and the tail end of a terminator is provided with Nhe I enzyme cutting site. pGAPZA was cleaved with Bgl II and BamHI to remove the intermediate fragment, and the X fragment was substituted to obtain a plasmid containing the isocaudarner enzyme, which was designated pGAPZX. Using ZTF/ZTR as upstream and downstream primers and pPICZA as template to perform PCR amplification to obtain P_AOX1Cutting the pGAPZX vector by Bgl II and Xba I and reacting with P_AOX1Fragment ligation to obtain a fragment containing P_AOX1The plasmid of (1), named pGAPZT, which can integrate expression cassettes of single or multiple genes of interest into chromosomal P_AOX1The position was verified by primers YZF/YZR and used. And amplifying the codon-optimized StgshF fragment (the primer sequence is shown as SEQ. NO. 04) by using a GSHFF/GSHFR primer, and connecting the product to PGAPZT by using a Novozan C113 kit to obtain an expression plasmid pGAPZT-StgshF. And respectively taking the genome of S.cerevisiae BY4741 as a template and SADKF/SADKR as a primer to amplify the target gene Scadkk 1 BY PCR, and connecting the target gene Scadkk 1 to PGAPZT BY using a C113 kit to obtain an expression plasmid pGAPZT-Scadk 1. And amplifying by taking SFF/SFR as a primer and pGAPZT-StgshF as a template to obtain an expression cassette containing StgshF, respectively connecting the expression cassette to a pGAPZT-Scadk1 plasmid which is cut by Nhe I and BamHI by utilizing T4 ligase to obtain a pGAPZT-Scadk1-StgshF recombinant plasmid, and verifying by using a primer YZ2F/YZ2R to construct the following plasmid.

Construction of G3-SF Strain: the plasmid construction processes are all transformed into escherichia coli JM109 competent cells, screening is carried out on a low-salt LB resistant plate containing 25 mug/mL Zeocin, and the obtained transformant is subjected to colony PCR verification and Tenglin biotechnology limited company for sequencing, so that the correct transformant is obtained. The G3 strain was made competent cells, the above recombinant plasmid pGAPZT-Scadk1-StgshF was linearized with SacI, and the plasmid was electrotransferred to the Pichia pastoris G3 strain according to the manual (Invitrogen) to obtain G3-SF. And coating the electrotransformation product in a YPDZ culture medium, and incubating for 2-3 days at 30 ℃. The grown colony is spotted in YPD medium containing high-quality Zeocin (2mg/mL), and after genome verification of the grown recombinant strain, the target strain containing high copy gene factor can be obtained, as shown in figure 2.

Example 3: G3-SF strain shake flask fermentation and intracellular ATP content change

The shake flask level fermentation medium is a YPD medium comprising: 20g/ml of glucose, 10g/ml of yeast powder and 20g/ml of peptone.

And (3) shaking flask fermentation: both the seed medium, i.e., YPD medium and the fermentation medium, were YPD medium. The strain is selected from a glycerol tube to be streaked on a YPD plate, the strain is cultured for 2-3 d at 30 ℃, a single colony grown is selected to be a shake flask containing 10mL of seed culture medium, the strain is cultured for 16-18 h at 30 ℃, then the strain is inoculated into a fermentation culture medium at a proper concentration and cultured for 30h at 220rpm, and mixed liquor with the concentrations of 10mM glutamic acid, 10mM cysteine and 10mM glycine is added at the beginning. After fermenting for 30h, the thalli are collected, and the supernatant is used for measuring ethanol, glycerol and glucose. And (3) shaking and incubating for 2h by using 40% ethanol solution at 220rpm, and centrifuging to obtain supernatant containing high-concentration GSH. Another sample is centrifuged and then is subjected to ultrasonic crushing, and the content of intracellular ATP is determined by adopting a Biyuntian ATP detection kit.

The results of shake flask fermentation are shown in FIG. 3, and the fermentation curve of G3-SF is determined, the strain enters logarithmic growth phase within 5h, and enters plateau phase within 20h, and the carbon source is completely exhausted within 20 h. During this time, the levels of glycerol and ethanol by-products were determined, and the glycerol levels were always low during fermentation and therefore not as a by-product of GSH fermentation. Ethanol is accumulated more in the early stage of fermentation, and when the carbon source is exhausted, the ethanol is used as the carbon source to continue fermentation and be exhausted. The yield reached the highest at 30h and then began to decline, probably because the strain activity decreased and the intracellular associated enzyme activity decreased as the fermentation time increased. And GSH degrading enzyme exists in cells to degrade the synthesized GSH. The yield of intracellular GSH is highest at 30h and is (453.26 +/-18.33) mg/L, the yield of intracellular GSH is (18.71 +/-0.43) mg/L/OD, then the change of intracellular ATP content after each recombinant strain and the addition of sodium citrate is researched, and the result is shown in the following figure 4, the intracellular ATP content of the strains within 16-28h after the addition of the sodium citrate is higher than that of a control, because the addition of the sodium citrate and the metabolic modification enhance the regeneration of intracellular ATP, thereby improving the intracellular ATP content. The ATP content reaches the highest value within 20h, and then rapidly decreases, probably because the thallus grows into a plateau stage in the period, and then a large amount of GSH is synthesized, and a large amount of ATP is consumed.

Example 4: fermentation in G3-SF strain fermentation tank

100L fermenter Medium (g/L): glucose 50, yeast powder 5, (NH4)₂SO₄ 11，K₂HPO₄ 7，MgSO ₄ 5，CaCl₂ 0.5，KCl 0.5。

Fermentation in a fermentation tank: the seed culture medium is cultured for 16-18 h, and the seed culture medium is inoculated into a 100L fermentation tank according to the inoculation amount of 10%. When the pH of the culture medium is reduced to 5.5, 50% ammonia water is fed in to maintain the pH of the fermentation process at 5.5 all the time. 10mL of PTM1 was contained in each liter of medium, and when the sugar in the initial medium decreased to 5g/L, 700g/L of glucose was supplemented to keep the ethanol concentration below 3 g/L. When fermenting for 16h, 4g/L sodium citrate is added. When the fermentation time reaches 48h, the mixture of 25mM glutamic acid, 25mM cysteine and 25mM glycine is fed to the final concentration. Sampling at regular time, and measuring the biomass, the glutathione concentration, the glycerol, the reducing sugar and the ethanol concentration.

As shown in FIG. 5, the fermentation result of the fermentor is that the biomass of G3-SF strain is in the late logarithmic growth phase at 40h and reaches the stationary growth phase at 48h, the biomass OD600 reaches 243.0, which is 9.20 times higher than that of biomass OD600(26.4) in the flask fermentation level, and simultaneously the glucose is rapidly consumed at 16h, and the residual amount is 0.33G/L. GSH accumulated as a primary metabolite with the growth of the cells and reached 960mg/L at 44h before the amino acid precursor mixture was dosed. When the amino acid solution is fed at the moment, the synthesis efficiency of the glutathione is rapidly increased, the yield of the GSH reaches 5950mg/L at the maximum at 56h, the yield is increased by 11.28 times compared with the highest yield (527.14 +/-17.92) mg/L at the shake flask level, and the yield of the intracellular GSH is 25.54 mg/L/OD.

Comparative example 1: shaking flask fermentation of G3 strain

The procedure is as in example 4, except that the strain added is the G3 strain.

The result of shake flask fermentation is shown in fig. 6, the growth of a control strain GS115 is lower than that of an engineering bacterium, and the transfer of G1 and G2 genes hardly affects the growth, wherein G1 is a blank control group engineering bacterium which is a constructed engineering bacterium which takes pichia pastoris GS115 as a host and is transformed to express an empty plasmid; g2 is an engineering bacterium which is constructed by taking pichia pastoris GS115 as a host and only expresses Scgsh1 gene. G3 strain had a 30h GSH of 141.96mg/L and the yield was 2.62 fold higher compared to the control. In order to improve the synthesis efficiency of GSH, a mixed solution of glycine, cysteine and glutamic acid with the final concentration of 10mM is added when the fermentation is carried out for 0 h. From FIG. 6(d), it can be seen that the addition of the amino acid precursor has a certain inhibitory effect on the growth of the strain, which may be caused by the toxic effect of cysteine in the precursor on the strain, but the addition of the precursor does greatly improve the ability of the bacteria to synthesize GSH. At 30h of fermentation, GSH production was (302.27. + -. 5.06) mg/L and intracellular yield was (11.79. + -. 0.35) mg/L/OD, but this level was not very high and was suspected to be due to poor synthetase capacity or insufficient intracellular energy supply.

In conclusion, compared with the G3 strain, the invention further heterologously expresses the Scadk1 gene from the saccharomyces cerevisiae and the StgshF gene from the streptococcus sanguis, and really achieves the purpose of enhancing the energy supply in the fermentation process, compared with the shake flask fermentation level of the G3 strain, the highest shake flask level yield of the glutathione of the invention can reach (527.14 +/-17.92) mg/L, and the yield of the intracellular glutathione is (19.96 +/-0.05) mg/L/OD. The constructed engineering bacteria G3-SA are fermented in a 5L fermentation tank, the highest yield of the glutathione in 56h is 5950mg/L, and the yield of the intracellular glutathione is 25.54 mg/L/OD. The results of both shaking flask fermentation and fermentation tank fermentation are far higher than the existing technical level, and especially the fermentation capacity of a 5L fermentation tank is greatly improved, so that a new idea is provided for the industrial production of GSH.

TABLE 1 primer sequences

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Sequence listing

<110> university of south of the Yangtze river

<120> high-yield glutathione pichia pastoris strain G3-SF and application thereof

<141> 2021-01-06

<160> 4

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aaagctgtgg aatcgtataa gtcacaacaa agttcttcta caactagtga tcctattgtc 660

gcattcattg tgcaaagaaa cgagagaaat gtgtttgatc aaaaggtctt ggaattgaat 720

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gtggtttatt acagaacggg ttacacaacc actgattaca cgtccgaaaa ggactgggag 900

gcaagactat tcctcgaaaa aagtttcgca ataaaggccc cagatttact cactcaatta 960

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tccgatgctg agaaaaagag tagtttgtta aaaacttttg tcaaaatata tcccttggat 1080

gatacgaagc ttggcaggga aggcaagagg ctggcattaa gtgagccctc taaatacgtg 1140

ttaaaaccac agcgggaagg tggcggaaac aatgtttata aagaaaatat tcctaatttt 1200

ttgaaaggta tcgaagaacg tcactgggat gcatatattc tcatggagtt gattgaacca 1260

gagttgaatg aaaataatat tatattacgt gataacaaat cttacaacga accaatcatc 1320

agtgaactag gaatttatgg ttgcgttcta tttaacgacg agcaagtttt atcgaacgaa 1380

tttagtggct cattactaag atccaaattt aatacttcaa atgaaggtgg agtggcggca 1440

ggattcggat gtttggacag tattattctt tactag 1476

<210> 3

<211> 669

<212> DNA

<213> Scadk1

<400> 3

atgtctagct cagaatccat tagaatggtc ctaattggcc cacctggtgc cggtaaaggt 60

actcaagctc caaatttgca agagcgtttc catgccgctc acttggccac tggtgacatg 120

ttgagatctc aaatcgcaaa gggcactcaa ttaggtttgg aagcaaagaa aattatggac 180

caaggtggtt tagtctctga tgacattatg gttaacatga tcaaggatga attgaccaac 240

aatccagctt gtaagaatgg gttcatcttg gacggtttcc caagaaccat tcctcaggct 300

gaaaaattgg accaaatgtt gaaagaacaa ggaactcctt tggaaaaagc catcgaattg 360

aaggttgatg atgaattgtt ggttgccaga attaccggta gattaattca cccagcctct 420

ggcagatcct accacaagat ctttaaccca ccaaaggaag acatgaagga tgacgtcacc 480

ggtgaagctt tagttcaaag atctgatgac aatgcagacg ccttgaagaa gagattagct 540

gcttaccatg ctcaaaccga accaattgtt gacttttaca aaaagaccgg tatctgggct 600

ggtgttgatg cttcccaacc tcctgctact gtttgggctg acatcttgaa caagctaggt 660

aaggattaa 669

<210> 4

<211> 2265

<212> DNA

<213> StgshF

<400> 4

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 high-yield glutathione Pichia pastoris strain G3-SF, which is characterized in that: pichia pastoris is taken as a host, and a glutathione synthetase Scgsh1 gene, a Scgsh2 gene, an adenosine kinase Scadk1 gene and a streptococcus sanguis-derived bifunctional enzyme StgshF gene which are derived from saccharomyces cerevisiae are heterologously expressed, so that the obtained engineering bacterium is named as a Pichia pastoris strain G3-SF.

2. The high yielding pichia glutathione strain G3-SF according to claim 1, wherein: the Pichia pastoris GS115 is selected from Pichia pastoris.

3. The high yielding pichia glutathione strain G3-SF according to claim 1, wherein: the nucleotide sequences of the glutathione synthetase Scgsh1 and Scgsh2 genes, the adenosine kinase Scadk1 gene and the bifunctional enzyme StgshF gene are respectively shown as SEQ.NO.01-SEQ.NO. 04.

4. The construction method of the high-yield glutathione Pichia pastoris G3-SF according to claim 1, characterized in that: taking pichia as a host, firstly heterologously expressing glutathione synthetase Scgsh1 and Scgsh2 genes from saccharomyces cerevisiae, and obtaining an engineering bacterium named as G3; and then G3 is taken as a host, an adenosine kinase Scadk1 gene from saccharomyces cerevisiae and a codon-optimized StgshF gene from streptococcus sanguis are heterologously expressed, and the obtained engineering bacteria are named as pichia pastoris strain G3-SF.

5. The construction method of the Pichia pastoris strain G3-SF for high yield of glutathione according to claim 4, characterized in that: the specific method is that pGAPZA is taken as an initial plasmid, Bgl II and BamH I are adopted for enzyme digestion, and a fragment inserted with Xba I is inserted into the initial plasmid, and the constructed plasmid is named as pGAPZT; inserting P between BglII and Xba I of pGAPZT_AOX1A sequence; the Scadk1 gene is obtained after PCR amplification of S.cerevisiae BY4741 genome, and the StgshF gene is synthesized and codon optimized based on the principle of yeast translation optimal codon; the obtained fragments are respectively connected in pGAPZT, expression cassettes of two genes are obtained, then the StgshF gene expression cassette is connected behind the Scadk1 gene expression cassette in series by the principle of isocaudarner connection, SacI is used for linear transformation to a G3 strain, and the obtained strain is screened by a YPD plate containing 2mg/L Zeocin, and the obtained strain is named as G3-SF.

6. The high yield glutathione Pichia pastoris strain G3-SF according to claim 4, which is obtained during the construction method.

7. A gene encoding the high yielding Pichia glutathione yeast strain G3-SF according to any one of claims 1-3.

8. A method for preparing glutathione, which adopts the high-yield Pichia glutathione yeast strain G3-SF according to any one of claims 1-3 or the high-yield Pichia glutathione yeast strain G3-SF constructed according to any one of claims 4-5 to synthesize glutathione by a fermentation method.

9. The method for producing glutathione according to claim 8, wherein: in the fermentation process of the heterologous expression of the adenosine kinase Scadk1 gene and the bifunctional enzyme gene StgshF, 4g/L sodium citrate is exogenously added when the fermentation time is 15-16 h.

10. Glutathione produced according to the method of claim 8 or 9.

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