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

A technology of G3-SA, yeast strain, applied in application, peptide, fermentation and other directions, can solve the problems of unrealistic production scale, can not increase production needs, expensive and other problems, and achieve the effect of enhancing energy supply

Pending Publication Date: 2021-04-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when Cys is added in excess, ATP, which acts as an energy carrier, becomes the limiting factor for GSH production
To solve this problem, Liang et al. increased the production of GSH by 11% by directly adding ATP. ATP is an expensive raw material, and scale-up production is unrealistic.
Another example is that Wang et al. used citrate as a co-energy substrate to promote the production of NADH, and then strengthened the production of ATP through the electron transport chain, which increased the yield of strain SAM and GSH and their co-production by 27.5%. However, this method could not improve Sufficient ATP to meet high GSH production needs

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1: G3 strain construction

[0039] P3 plasmid construction: this patent is constructed with P GAP for the promoter, T AOX1 A constitutive expression strain for the terminator. First, use GAP-F / GAP-R (see Table 1 for the sequence) as primers to amplify the P gene on the genome. GAP promoter sequence. Digest pPIC 3.5K with SacI and BamHI to remove P AOX1 fragment and use P GAP Sequence substitution resulted in a constitutively expressed plasmid, which was named pPICKT. Using the genome of S.cerevisiae BY4741 as a template, GSH1F / GSH1R (see Table 1 for the sequence) and GSH2F / GSH2R (see Table 1 for the sequence) were used to amplify the target gene Scgsh1 and Scgsh2 fragments by PCR respectively (the gene sequence is shown in SEQ.NO.01 , shown in SEQ.NO.02) were respectively connected to pPICKT to form P1 and P2 plasmids. And use YZGF / YZGR (see Table 1 for the sequence) to do the next construction after verification. Then use G1F / G1R (see Table 1 for the...

Embodiment 2

[0041] Embodiment 2: G3-SA strain construction

[0042] Construction of pGAPZT-Scadk1 plasmid: In this study, we used pGAPZA as the starting plasmid, modified it, and named the transformed plasmid pGAPZT. First, use ZXF / ZXR (see Table 1 for the sequence) as primers and pGAPZA as a template to carry out PCR amplification, so that the recovered product X fragment has an XbaI at the front end of the promoter and an NheI restriction site at the end of the terminator. Use BglⅡ and BamHI to digest pGAPZA to remove the middle fragment, and replace it with the X fragment to obtain a plasmid containing the same tail enzyme, which is named p GAP ZX.

[0043] Using ZTF / ZTR as upstream and downstream primers and pPICZA as a template for PCR amplification to obtain P AOX1 Fragment, use BglⅡ and XbaΙ to digest the pGAPZX vector and combine with P AOX1 Fragments ligated to obtain a AOX1 The plasmid, named pGAPZT, can integrate the expression cassette of single or multiple target genes in...

Embodiment 3

[0048] Embodiment 3: G3-SA bacterial strain shakes flask fermentation

[0049] The fermentation medium at shake flask level is YPD medium, including: glucose 20g / L, yeast powder 10g / L, peptone 20g / L.

[0050] Shake flask fermentation: the seed culture medium is YPD medium and the fermentation medium is YPD medium. Pick the strain from the glycerol tube to the YPD plate, culture at 30°C for 2-3 days, pick the grown single colony into a shake flask containing 10mL seed medium, culture at 30°C for 16-18h, and then set the initial OD as 0.2 Inoculated into the fermentation medium and cultured at 220 rpm for 30 h, and initially added a mixed solution with concentrations of 10 mM glutamic acid, 10 mM cysteine, and 10 mM glycine. After 30 hours of fermentation, the bacteria were collected, and the supernatant was used to measure ethanol, glycerol and glucose. Incubate with 40% ethanol solution at 220rpm for 2h. After centrifugation, the supernatant contains high concentration of GS...

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Abstract

The invention discloses a high-yield glutathione pichia pastoris strain G3-SA and application thereof. Pichia pastoris GS115 is used as a host, Scgsh1 and Scgsh2 genes derived from saccharomyces cerevisiae are heterologously expressed, GSH overproduction is obtained, and an engineering bacterium is named as G3; and on the basis, adenosine kinase Scadk1 derived from saccharomyces cerevisiae are heterologously expressed to enhance energy supply in the fermentation process. The highest 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 engineering bacterium G3-SA is subjected to fermentation in a 5L fermentation tank, the highest yield of glutathione is 4810 mg / L within 56 h, the yield of intracellular glutathione is 19.87 mg / L / OD, and a new thought is provided for industrial production of GSH.

Description

technical field [0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a high-yield glutathione Pichia strain G3-SA and its application. Background technique [0002] Glutathione is a tripeptide active substance, which widely exists in animals, plants and microorganisms, and has the function of protecting and regulating the redox balance in cells. Its molecular weight is 307.33, and it is composed of glutamic acid, cysteine ​​and glycine. The content in various organisms is different, and the content in yeast and animal offal is relatively high. GSH also plays a wide range of roles in the clinical field, and has significant curative effects on liver diseases, kidney diseases and cardiovascular diseases. [0003] The main characteristics of GSH are due to its having a gamma amide bond and a thiol group. In most prokaryotes and eukaryotes, it is produced by a two-step reaction. The first step is to generate glutamylcysteine ​​from g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/52C12N15/54C12N15/81C12N15/66C12P21/02C07K5/037C12R1/84
CPCC12N9/93C12N9/1205C12N15/815C12N15/66C07K5/0215C12Y603/02003C12Y207/0102
Inventor 许正宏史劲松徐国强高宇豪徐建国付静曹峥吴勇杰
Owner JIANGNAN UNIV
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