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

A technology of G3-SF and yeast strains, which is applied to the high-yield glutathione Pichia strain G3-SF and its application fields, which can solve the problems of unrealistic, expensive, and inability to increase production needs in large-scale production, and achieve enhanced energy supply Effect

Active Publication Date: 2021-05-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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-SF and application thereof
  • High-yield glutathione pichia pastoris strain G3-SF and application thereof
  • High-yield glutathione pichia pastoris strain G3-SF 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 as primers to amplify the P 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, GSH2F / GSH2R PCR was used to amplify target gene Scgsh1 and Scgsh2 fragments (gene sequences as shown in SEQ.NO.01-SEQ.NO.02) were respectively connected to In pPICKT, construct P1 and P2 plasmids, and use YZGF / YZGR to verify and do the next construction. Then use G1F / G1R and TF / TR to amplify PGAP-Scgsh1 and T on P1 plasmid respectively A The ox1 fragment was then ligated into the P2 plasmid using the Novizan C113 multi-fragment homologous recombination ligat...

Embodiment 2

[0041] Embodiment 2: G3-SF strain construction

[0042] Construction of pGAPZT-Scadk1-StgshF plasmid: In this study, we used pGAPZA as the starting plasmid and modified it, and named the modified plasmid pGAPZT. This plasmid realizes the tandem expression of multiple genes by means of isotailase. First, use ZXF / ZXR as primers and pGAPZA as a template for PCR amplification, so that the recovered product X fragment has an XbaI at the front of the promoter and a NheI restriction site at the end of the terminator. The pGAPZA was cut with BglⅡ and BamHI to remove the middle fragment, and the X fragment was replaced to obtain a plasmid containing the same tail enzyme, which was named pGAPZX. 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 ...

Embodiment 3

[0044] Embodiment 3: G3-SF bacterial strain shakes flask fermentation and intracellular ATP content change

[0045] The fermentation medium at shake flask level is YPD medium, including: glucose 20g / ml, yeast powder 10g / ml, peptone 20g / ml.

[0046] Shake flask fermentation: the seed culture medium is YPD medium and the fermentation medium is YPD medium. Pick the strain from the glycerol tube and streak it on the YPD plate, culture at 30°C for 2-3 days, pick the grown single colony into a shaker flask containing 10mL seed medium, culture at 30°C for 16-18h, and inoculate at an appropriate concentration Cultivate in the fermentation medium at 220 rpm for 30 h, and initially add 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, t...

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Abstract

The invention discloses a high-yield glutathione pichia pastoris strain G3-SF and an application thereof. Pichia pastoris GS115 is taken as a host, Scgsh1 and Scgsh2 genes from saccharomyces cerevisiae are subjected to heterologous expression, 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 streptococcus sanguis derived from a codon optimized StgshF gene, and aims to enhance energy supply in an engineering bacterium fermentation process and reconstruct a synthesis path of GSH. The maximum yield of glutathione at a shake flask level can reach (527.14+/-17.92) mg/L, and the intracellular glutathione yield is (19.96+/-0.05) mg/L/OD. The constructed engineering bacterium G3-SA is fermented in a 5L fermentation tank, the highest yield of glutathione in 56h is 5950mg/L, the intracellular glutathione yield is 25.54 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-SF 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): C12N1/19C12N15/81C12N15/54C12N15/52C07K5/037C12R1/84
CPCC12N9/1205C12N9/93C12N15/52C12N15/815C07K5/0215C12Y207/0102C12Y603/02002C12Y603/02003C12N2800/22
Inventor 徐国强许正宏高宇豪刘娜史劲松
Owner JIANGNAN UNIV
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