Improvement of oxidized glutathione fermentation by thiol redox metabolism engineering in Saccharomyces cerevisiae

Abstract

Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

Enzymatic improvement of mitochondrial thiol oxidase Erv1 for oxidized glutathione fermentation by Saccharomyces cerevisiae

“…These results suggest that the Erv1 protein directly oxidized GSH in S. cerevisiae cells. The suppression of GSSG production by the simultaneous over-expression of ERV1 and MIA40 genes in our previous study [15] and the much lower oxidation activity of Mia40 for GSH [25] also support this idea.…”

“…In a series of studies, the active residues, cofactor binding residues, and three-dimensional structures of Erv1 have been revealed [23, 27, 28]. In our previous study, we first focused on the ERV1 gene for biomaterial production, and revealed that over-expression of the ERV1 gene enhanced GSSG and total glutathione production in S. cerevisiae cells [15]. In this study, we also first improved Erv1 for biomaterial production and revealed that S32 and N34 residues are critical for oxidation of GSH and protein stability.…”

“…In our previous study, deletion of the GLR1 gene and over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for generating high GSSG production among three types of different thiol oxidase genes in S. cerevisiae cells [15]. Therefore, in this study, we improved the enzymatic activity of Erv1 for GSH oxidation by amino acid replacements, and consequently enhanced GSSG production in S. cerevisiae by using mutant ERV1 genes.…”

“…In industrial mass production of glutathione, GSSG is preferable due to its higher stability. Furthermore, enhancements of GSSG production help to avoid a negative feedback regulation by GSH1 , and consequently increase total glutathione production by fermentation with S. cerevisiae [14, 15]. GSSG also has advantages in utilization; it has the same extent of antioxidant activity in the intestines after dietary intake as GSH [16], and promotes plant growth more potently than GSH [17, 18].…”

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

“…Glutathione Overexpression of YAP1 [58] Manipulation of the sulphate assimilation pathway by overexpressing MET14 and MET16 [59] Improved oxidized glutathione production by overexpression of GSH1, GSH2, and ERV1 and the deletion of GLR1 [60] Adaptive laboratory evolution in the presence of increasing levels of acrolein and screening for enhanced glutathione production [61] Whole-genome engineering via genome shuling and screening for enhanced glutathione production [62] Artemisinin/artemisinic acid Reconstruction of the complete biosynthetic pathway of artemisinic acid, including the three-step oxidation of amorphadiene to artemisinic acid by expression of CYP71AV1, CPR1, CYB5, ADH1 and ALDH1 from Artemisia annua [48] Taxol/taxadiene Expression of a truncated version of the endogenous tHMG1 and GGPPS from Taxus chinensis or Sulfolobus acidocaldarius together with TDC1 from T. chinensis [66] Prediction of the eiciency of diferent GGPPS enzymes via computer aided protein modelling [67] Forskolin Expression of a promiscuous cytochrome P450 from Salvia pomifera [68] Polyketides Heterologous expression of 6-MSA synthase gene from Penicillium patulum together with PPTases from either Bacillus subtilis or Aspergillus nidulans [69] Construction of polyketide precursor pathways by expressing prpE from Salmonella typhimurium and PCC pathway from Streptomyces coelicolor [70] Enhanced cofactor supply by expressing 2-PS from Gerbera hybrida [71] Resveratrol Reconstruction of a de novo pathway by expressing TAL from Herpetosiphon aurantiacus, 4-CL1 from Arabidopsis thaliana and VST1 from Vitis vinifera [49] Expression of 4CL1 from A. thaliana and STS from Arachis hypogaea [73] Expression of PAL from Rhodosporidium toruloides, C4H and 4-CL1 from A. thaliana, and STS from A. hypogaea [74] Expression of 4-coumaroyl-coenzyme A ligase (4CL1) from A. thaliana and stilbene synthase (STS) from V. vinifera [75] Overexpression of the resveratrol biosynthesis pathway, enhancement of P450 activity, increasing the precursor supply for resveratrol synthesis via phenylalanine pathway [76] Dihydrochalcones Expression of the heterologous pathway genes in a TSC13-overexpressing S. cerevisiae strain [78] Alkaloids Expression of 14 monoterpene indole alkaloid pathway genes from Catharanthus roseus and enhanced secondary metabolism to produce strictosidine de novo [79] Construction of the complete de novo biosynthetic pathway to norcoclaurine by expressing a mammalian TyrH enzyme and DODC from Pseudomonas putida, along with four genes required for biosynthesis of its electron carrier cosubstrate …”