Three-pathway combination for glutathione biosynthesis in Saccharomyces cerevisiae.

Liang Tang,Wenlong Zhou,Minzhi Liu,Kai Cheng,Wei Wang,Weiwei Wang,Yan Yang,Kedi Cheng

doi:10.1186/s12934-015-0327-0

Abstract

BackgroundGlutathione (GSH), a pivotal non-protein thiol, can be biosynthesized through three pathways in different organisms: (1) two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (Gsh1 or GshA) and glutathione synthetase (Gsh2 or GshB); (2) a bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (GshF); (3) an alternative condensation of γ-glutamyl phosphate synthesized by γ-glutamyl kinase (Pro1 or ProB) with cysteine to form γ-glutamylcysteine which was further conjugated to glycine by glutathione synthetase. The Gsh1 and Gsh2 of conventional GSH biosynthetic pathway or the bifunctional GshF reported previously have been independently modulated for GSH production. This study developed a novel three-pathway combination method to improve GSH production in Saccharomyces cerevisiae.ResultsA bifunctional enzyme GshF of Actinobacillus pleuropneumoniae was functionally expressed in S. cerevisiae and Pro1 in proline biosynthetic pathway was exploited for improving GSH yield. Moreover, two fusion proteins Gsh2-Gsh1 and Pro1-GshB were constructed to increase the two-step coupling efficiency of GSH synthesis by mimicking the native domain fusion of GshF. The engineered strain W303-1b/FGP with three biosynthetic pathways presented the highest GSH concentration (216.50 mg/L) and GSH production of W303-1b/FGP was further improved by 61.37 % when amino acid precursors (5 mM glutamic acid, 5 mM cysteine and 5 mM glycine) were fed in shake flask cultures. In batch culture process, the recombinant strain W303-1b/FGP also kept high efficiency in GSH production and reached an intracellular GSH content of 2.27 % after 24-h fermentation.ConclusionsThe engineered strains harbouring three GSH pathways displayed higher GSH producing capacity than those with individually modulated pathways. Three-pathway combinatorial biosynthesis of GSH promises more effective industrial production of GSH using S. cerevisiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0327-0) contains supplementary material, which is available to authorized users.

Highlights

Glutathione (GSH), a pivotal non-protein thiol, can be biosynthesized through three pathways in different organisms: (1) two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (Gsh1 or GshA) and glutathione synthetase (Gsh2 or GshB); (2) a bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (GshF); (3) an alternative condensation of γ-glutamyl phosphate synthesized by γ-glutamyl kinase (Pro1 or ProB) with cysteine to form γ-glutamylcysteine which was further conjugated to glycine by glutathione synthetase
Compensatory pathway for GSH synthesis was characterized in yeast and Escherichia coli that lack Gsh1 and GshA [12, 13]. γ-glutamyl kinase (GK) encoded by PRO1 in eukaryotes or proB in prokaryotes, the first enzyme in proline biosynthetic pathway, catalyzes the formation of intermediate γ-glutamyl phosphate, which if accumulated could partially react with cysteine to generate γ-GC, and Gsh2 adds glycine to γ-GC to form GSH
We focused on direct modulation of the GSH biosynthetic pathway via a method of three-pathway combination

Summary

Introduction

Glutathione (GSH), a pivotal non-protein thiol, can be biosynthesized through three pathways in different organisms: (1) two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (Gsh or GshA) and glutathione synthetase (Gsh or GshB); (2) a bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (GshF); (3) an alternative condensation of γ-glutamyl phosphate synthesized by γ-glutamyl kinase (Pro or ProB) with cysteine to form γ-glutamylcysteine which was further conjugated to glycine by glutathione synthetase. The Gsh and Gsh of conventional GSH biosynthetic pathway or the bifunctional GshF reported previously have been independently modulated for GSH production. Γ-glutamyl kinase (GK) encoded by PRO1 in eukaryotes or proB in prokaryotes, the first enzyme in proline biosynthetic pathway, catalyzes the formation of intermediate γ-glutamyl phosphate, which if accumulated could partially react with cysteine to generate γ-GC, and Gsh adds glycine to γ-GC to form GSH.

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Conclusion