CN112646738A

CN112646738A - High-yield glutathione pichia pastoris strain G3-SA and application thereof

Info

Publication number: CN112646738A
Application number: CN202110011525.5A
Authority: CN
Inventors: 许正宏; 史劲松; 徐国强; 高宇豪; 徐建国; 付静; 曹峥; 吴勇杰
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-01-06
Filing date: 2021-01-06
Publication date: 2021-04-13

Abstract

The invention discloses a high-yield glutathione Pichia pastoris strain G3-SA and application thereof, wherein 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 this basis, heterologous expression was derived from the s.cerevisiae adenosine kinase Scadk1, aimed at enhancing energy supply during fermentation. The highest horizontal yield of glutathione in a shake flask can reach (465.50 +/-29.90) mg/L, and the yield of intracellular glutathione is (16.87 +/-0.63) 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 4810mg/L, the yield of the intracellular glutathione is 19.87mg/L/OD, and a new idea is provided for the industrial production of the GSH.

Description

High-yield glutathione pichia pastoris strain G3-SA 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-SA 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-SA 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-producing Pichia pastoris strain G3-SA, which takes Pichia pastoris as a host, heterologously expresses glutathione synthetase Scgsh1 and Scgsh2 genes and adenosine kinase Scadk1 genes from saccharomyces cerevisiae, and the obtained engineering bacterium is named as Pichia pastoris strain G3-SA.

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

Furthermore, the nucleotide sequences of the glutathione synthetase Scgsh1 and Scgsh2 genes and the adenosine kinase Scadk1 gene are respectively shown as SEQ.NO. 01-SEQ.NO. 03.

The invention also aims to provide a construction method of the high-yield glutathione Pichia pastoris strain G3-SA, which comprises the following steps: 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 then G3 is taken as a host, and the adenosine kinase Scadk1(Gene ID:851812) Gene from saccharomyces cerevisiae is expressed in a heterologous way, and the obtained engineering bacterium is named as pichia pastoris strain G3-SA.

Furthermore, the specific method is that pGAPZA is used as an initial plasmid, Bgl II and BamH I are used for enzyme digestion, and a fragment inserted with Xba I is inserted into the enzyme digestion, and the constructed plasmid is named as 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, the resulting fragment was ligated to pGAPZT and linearized BY SacI into G3 strain, and the strain was selected on YPD plates containing 2mg/L Zeocin and named G3-SA.

Furthermore, in the fermentation process of the heterologous expression of the adenosine kinase Scadk1 gene, 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 Pichia glutathione yeast strain G3-SA.

It is another object of the present invention to provide a gene encoding the high-yielding Pichia glutathione yeast strain G3-SA as described above.

The invention also aims to provide a method for preparing glutathione, which adopts the high-yield glutathione Pichia pastoris strain G3-SA or the high-yield glutathione Pichia pastoris strain G3-SA 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-SA 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 this basis, heterologous expression was derived from the s.cerevisiae adenosine kinase Scadk1, aimed at enhancing energy supply during fermentation. At present, the highest yield of the glutathione in a shake flask can reach (465.50 +/-29.90) mg/L, and the yield of the intracellular glutathione is (16.87 +/-0.63) 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 4810mg/L, and the yield of the intracellular glutathione is 19.87mg/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-SA in example 2; wherein, M: DL2000 Marker; 1: g3 genome validation, 2, 3, 4: and (5) carrying out PCR verification on the genome of the engineering bacteria.

FIG. 3 is a graph showing the results of shaking flask fermentation by G3-SA in example 3;

FIG. 4 is a graph of the effect of sodium citrate addition on the intracellular GSH and ATP levels of G3-SA in example 4; wherein, the adding time of the sodium citrate is optimized, the adding concentration of the sodium citrate is optimized, and the change curve of the ATP in the cell is calculated.

FIG. 5 shows the results of fermentation of G3-SA in 5L fermentor in example 5.

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 is heterologously expressed, so that endogenous metabolic transformation is carried out on host cells, and the supply of intracellular ATP is effectively enhanced by combining the addition of exogenous sodium citrate, and the synthesis of intracellular GSH is further enhanced.

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, using GAP-F/GAP-R (sequence shown in Table 1) as primer to amplify P on genome_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. The genome of S.cerevisiae BY4741 is taken as a template, and GSH1F/GSH1R (shown in a sequence table 1) and GSH2F/GSH2R (shown in a sequence table 1) are respectively subjected to PCR amplification to obtain target genes Scgsh1 and Scgsh2 fragments (shown in a gene sequence of SEQ. NO.01 and SEQ. NO. 02) which are respectively connected to pPICKT to form plasmids P1 and P2. And the subsequent construction is carried out after verification by YZGF/YZGR (sequence shown in Table 1). Then, the P on the P1 plasmid was amplified with G1F/G1R (see Table 1 for sequence) and TF/TR (see Table 1 for sequence), respectively_GAPScgsh1 and T_Aox1The fragment is connected into a P2 plasmid by adopting a Novozam C113 multi-fragment homologous recombination and ligation kit to obtain a plasmid pP-P containing Scgsh1 and Scgsh2 expression units_GAPScgsh1-Scgsh2, the 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 containing high copy gene factors, namely the G3 strain, can be obtained after genome verification of the grown larger colonies, as shown in FIG. 1.

Example 2: construction of G3-SA Strain

Construction of pGAPZT-Scadk1 plasmid: in the present study, pGAPZA was used as a starting plasmid, and the starting plasmid was transformed, and the transformed plasmid was named pGAPZT. Firstly, ZXF/ZXR (shown in a sequence table 1) is used as a primer, pGAPZA is used as a template for PCR amplification, so that Xba I is arranged at the front end of a promoter of the recovered product X fragment, and Nhe I enzyme cutting site is arranged at the tail end of a terminator. pGAPZA was cleaved with BglII and BamHI to remove intermediate fragments, and the X fragment was substituted to give a plasmid containing the isocaudarner enzyme, which was designated as p_GAPZX。

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 using the primer YZF/YZR (see Table 1 for sequence). Respectively taking S.cerevisiae BY4741 and genome as templates, performing PCR amplification BY using primers SADKF/SADKR (the sequence is shown in Table 1) to obtain a target gene Scadk1 fragment (the gene sequence is shown in SEQ. NO. 03), and connecting the target gene Scadk1 fragment to pGAPZT BY using a C113 kit to obtain an expression plasmid pGAPZT-Scadk 1. And verified by primers YZ2F/YZ2R (sequences are shown in Table 1) to construct

Construction of G3-SA 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. After finishingThe giberellin GS115 becomes competent cell to obtain Pichia pastoris G3 strain, then Pichia pastoris G3 strain becomes competent cell, and SacI is adopted to linearize the recombinant plasmid p_GAPZT-Scadk1, the plasmid was electrotransferred to Pichia pastoris G3 strain according to the manual (Invitrogen) to obtain G3-SA. 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.

TABLE 1 primer sequences

Example 3: shaking flask fermentation of G3-SA strain

The shake flask level fermentation medium is a YPD medium comprising: 20g/L of glucose, 10g/L of yeast powder and 20g/L 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 220rpm and cultured for 30h with the initial OD of 0.2, and mixed liquor with the concentration of 10mM glutamic acid, 10mM cysteine and 10mM glycine is added in the initial period. 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.

The results of the shake flask fermentation are shown in FIG. 3, from which it can be seen that the expression of the s.cerevisiae-derived adenosine kinase did not significantly affect the growth of the cells. However, when an amino acid was added to a final concentration of 10mM, the expression of adenosine kinase promoted the synthesis of GSH. Compared with G3 strain and the empty plasmid control group G3-ZT strain, the yield of G3-SA is the highest (387.95 +/-10.47) mg/L, and is improved by 20.61 percent compared with the control G3(321.65 +/-8.50 mg/L). And the yield of the cells is correspondingly improved to (15.83 +/-0.29) mg/L/OD.

Example 4: G3-SA strain shake flask fermentation added with sodium citrate and change of intracellular ATP content

The process is the same as in example 3, except that 4g/L sodium citrate is added at the time of fermentation to 12.

The results of shake flask fermentation are shown in fig. 4, in which fig. 4a is the optimization of the sodium citrate addition time, fig. 4b is the optimization of the sodium citrate addition concentration, and fig. 4c is the change curve of intracellular ATP. Increasing energy supply through metabolic engineering has a significant effect on increasing intracellular GSH accumulation. Based on this, we tried endogenous in combination with exogenous to enhance GSH synthesis. The optimization experiment of adding sodium citrate to the G3-SA strain finally shows that the effect of adding sodium citrate with the concentration of 4G/L is best at 12h of fermentation time, the yield can be improved to 465.5 +/-29.90 mg/L and is improved by 16.66 percent compared with the control G3-SA (387.95 +/-10.47 mg/L).

Then, the change of intracellular ATP content of each recombinant strain and after adding sodium citrate was investigated, and as a result, as shown in FIG. 4a below, the intracellular ATP content of each strain after adding sodium citrate in 16-28h was higher than that of the control, because the tricarboxylic acid circulation path was strengthened by the addition of sodium citrate, and the intracellular ATP content was increased. 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. In conclusion, the addition of sodium citrate in shake flasks or the enhancement of intracellular ATP supply by metabolic engineering can effectively relieve the limit of ATP deficiency in the case of excess amino acids.

Example 5: fermentation in G3-SA strain fermentation tank

5L fermenter Medium (g/L): glucose 50, yeast powder 5, (NH4)₂SO₄ 11，K₂HPO₄ 7，MgSO ₄5，CaCl₂0.5，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 5L 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 results of the fermentor are that the biomass of G3-SA strain reached stationary phase in 44h, the biomass OD600 reached 257.0, which was 10.40 times higher than that of the shake flask fermentation (24.71), and glucose was consumed rapidly at 16h, with the residual amount being 0.36G/L. GSH accumulated as a primary metabolite with the growth of the cells and reached 1060mg/L at 44h before the amino acid precursor mixture was fed. 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 4810mg/L at 56h, the yield is 10.33 times higher than that of the highest yield (465.50 +/-29.90) mg/L at the shake flask level, and the yield of the intracellular GSH is 19.87 mg/L/OD.

Comparative example 1: shaking flask fermentation of G3 strain

The procedure is as in example 3, except that the strain added is the G3 strain.

The result of shake flask fermentation is shown in fig. 6, the growth of the control strain GS115 is lower than that of the engineering bacteria, and the transfer of the two genes has almost no influence on the growth, wherein G1 is a blank control group engineering bacteria which is constructed by using pichia pastoris GS115 as a host and transforming and expressing the engineering bacteria of the empty plasmids; 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 GSH of 141.96mg/L at 30h, 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 it was hypothesized that the addition of precursor would make ATP starvation a major limiting factor.

In conclusion, compared with the G3 strain, the saccharomyces cerevisiae adenosine kinase Scadk1 is further heterologously expressed, the purpose of enhancing energy supply in the fermentation process is really realized, compared with the shake flask fermentation level of the G3 strain, the highest shake flask level yield of the glutathione can reach (465.50 +/-29.90) mg/L, and the yield of intracellular glutathione is (16.87 +/-0.63) mg/L/OD. Further carrying out fermentation of the engineering bacteria G3-SA in a 5L fermentation tank, wherein the highest yield of the glutathione in 56h is 4810mg/L, and the yield of the intracellular glutathione is 19.87 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.

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-SA and application thereof

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tctggctcca agaaaattca gcaattgttg acagatgagg gcgtattagg taaatacatc 1020

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

Claims

1. A high-yield glutathione Pichia pastoris strain G3-SA is characterized in that: pichia pastoris is taken as a host, glutathione synthetase Scgsh1 and Scgsh2 genes and adenosine kinase Scadk1 genes from saccharomyces cerevisiae are expressed in a heterologous way, and the obtained engineering bacterium is named as a Pichia pastoris strain G3-SA.

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

3. The high yielding pichia glutathione strain G3-SA according to claim 1, wherein: the nucleotide sequences of the glutathione synthetase Scgsh1 and Scgsh2 genes and the adenosine kinase Scadk1 gene are respectively shown as SEQ.NO. 01-SEQ.NO. 03.

4. The construction method of the high-yield glutathione Pichia pastoris G3-SA 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, the adenosine kinase Scadk1 gene from saccharomyces cerevisiae is expressed in a heterologous way, and the obtained engineering bacterium is named as pichia pastoris strain G3-SA.

5. The construction method of the high-yield glutathione Pichia pastoris G3-SA 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 was obtained BY PCR amplification of S.cerevisiae BY4741 genome, the obtained fragment was ligated to pGAPZT and linearized transformed into G3 strain with SacI, and the obtained strain was screened on YPD plate containing 2mg/L Zeocin to obtain G3-SA.

6. The construction method of the high-yield glutathione Pichia pastoris G3-SA according to claim 4, characterized in that: in the fermentation process of the heterologous expression of the adenosine kinase Scadk1 gene, 4g/L of sodium citrate is exogenously added when the fermentation time is 15-16 h.

7. The high-yield Pichia glutathione yeast strain G3-SA according to claim 4, wherein the plasmid is obtained by the process of the construction method.

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

9. A method for preparing glutathione, which adopts the high-yield Pichia glutathione yeast strain G3-SA as any one of claims 1-3 or the high-yield Pichia glutathione yeast strain G3-SA constructed by the method as any one of claims 4-6 to synthesize glutathione by a fermentation method.

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