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

Abstract

BackgroundOxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes.ResultsWe improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 S32A gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene.ConclusionsThis is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0658-0) contains supplementary material, which is available to authorized users.

Metabolic engineering of the l-serine biosynthetic pathway improves glutathione production in Saccharomyces cerevisiae

“…These results may imply that surplus l -Ser production or decrease of 3PG by enhanced l -Ser biosynthesis exerts unfavorable effects on growth and glutathione production of host cells. The increase of GSSG ratio in the GCI/ SERs strain also showed that the strain was under strong stress because GSSG ratio showed index of oxidative stress of cells [ 12 – 14 ]. However, the strains overexpressing SER3 also showed high GSSG ratio compared with the control strain.…”

“…However, the strains overexpressing SER3 also showed high GSSG ratio compared with the control strain. In some cases, production of GSSG help to avoid a negative feedback regulation by Gsh1, and consequently increase total glutathione production [12][13][14]. The overexpression of SER3 may lead modest stress of the cells for increased glutathione production.…”

“…GS catalyzes the reaction to form glutathione from γ-GC and Gly. Previously, we obtained a strain overexpressing multicopy of GSH1 and GSH2 genes (GCI strain) that showed increased glutathione production [11], and have used this GCI strain as a host [12][13][14][15].…”