Acrolein-stressed threshold adaptation alters the molecular and metabolic bases of an engineered Saccharomyces cerevisiae to improve glutathione production

Wenlong Zhou,Yan Yang,Wei Wang,Changkun Li,Kai Cheng,Huimin Wang,Minzhi Liu,Liang Tang

doi:10.1038/s41598-018-22836-2

Abstract

Acrolein (Acr) was used as a selection agent to improve the glutathione (GSH) overproduction of the prototrophic strain W303-1b/FGPPT. After two rounds of adaptive laboratory evolution (ALE), an unexpected result was obtained wherein identical GSH production was observed in the selected isolates. Then, a threshold selection mechanism of Acr-stressed adaption was clarified based on the formation of an Acr-GSH adduct, and a diffusion coefficient (0.36 ± 0.02 μmol·min−1·OD600−1) was calculated. Metabolomic analysis was carried out to reveal the molecular bases that triggered GSH overproduction. The results indicated that all three precursors (glutamic acid (Glu), glycine (Gly) and cysteine (Cys)) needed for GSH synthesis were at a relativity higher concentration in the evolved strain and that the accumulation of homocysteine (Hcy) and cystathionine might promote Cys synthesis and then improve GSH production. In addition to GSH and Cys, it was observed that other non-protein thiols and molecules related to ATP generation were at obviously different levels. To divert the accumulated thiols to GSH biosynthesis, combinatorial strategies, including deletion of cystathionine β-lyase (STR3), overexpression of cystathionine γ-lyase (CYS3) and cystathionine β-synthase (CYS4), and reduction of the unfolded protein response (UPR) through up-regulation of protein disulphide isomerase (PDI), were also investigated.

Highlights

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH), which is synthesized from glutamic acid (Glu), cysteine (Cys) and glycine (Gly), is the most abundant non-protein thiol compound in almost all organisms
To ensure that the engineered strain W303-1b/FGP could be employed in WMVIII medium, its five auxotrophic genes were reversed using the CRISPR/Cas9-mediated gene editing method[12,13], generating the prototrophic W303-1b/FGPPT strain
The results showed that the prototrophic strain has the same capacity for glutathione production as the auxotrophic strain and was more suitable for evolution

Summary

Introduction

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH), which is synthesized from glutamic acid (Glu), cysteine (Cys) and glycine (Gly), is the most abundant non-protein thiol compound in almost all organisms. GSH, a native scavenger, has been demonstrated to play a prime role in the cellular defence against Acr[10]. This observation indicates that Acr has a tight relationship with GSH. The regulation of GSH biosynthesis is far more than pathway engineering, as the GSH level is strictly controlled by a complex regulatory system. To further improve the GSH production of the engineered strain, an adaptive laboratory evolution (ALE) experiment was carried out. A threshold selection mechanism was clarified, and a metabolomic analysis of the evolved strain was performed to elucidate the augmented thiol compounds involved in the enhancement of GSH levels, guiding the re-engineering of the GSH biosynthetic pathway

Methods

Results

Conclusion