Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system

Abstract

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ‐glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate‐limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram‐positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co‐expression of Group‐I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L‐1 glutathione with glutathione content and yield of 4.3% and 25.6 mgglutathione/gglucose, respectively. These values were 6.6‐, 4.9‐, and 4.3‐fold higher than the corresponding values of the wild‐type strain. In a glucose‐limited fed‐batch fermentation, the SKSC222 strain produced 2.0 g L‐1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value‐added chemicals by engineered strains of S. cerevisiae.

The Glutathione System: A Journey from Cyanobacteria to Higher Eukaryotes

“…The N-terminal sequence of GshF is similar to that of E. coli GshA, but the C-terminal sequence is more similar to the D-Ala, D-Ala ligase than to any known GshB [ 125 ]. Interestingly, GSH inhibits neither the GshA activity nor the GshB activity of the GshF, and the GshA activity of GshF is higher compared with that in other organisms, and it is not inhibited by GSH [ 127 ]. More than 20 bacteria, mostly Gram-positive, possess a gshF -like gene.…”

“…More than 20 bacteria, mostly Gram-positive, possess a gshF -like gene. Recently, the S. thermophilus gshF gene was overexpressed in tobacco plants, E. coli and yeast cells to increase their production of GSH, an objective of biotechnological interest [ 126 , 127 , 128 , 129 , 130 ].…”

“…So far, GSH is commercially produced mainly by Saccharomyces cerevisiae strains, which have generally been recognized as safe. Previous studies have focused on overproducing GshA and GshB, but the production yield and titer of GSH in such Saccharomyces cerevisiae strains remain low due to the feedback inhibition on GshA [ 127 ]. To overcome this limitation, the GshF bifunctional enzyme from Gram-positive bacteria was produced in S. cerevisiae , as GshF is insensitive to feedback inhibition.…”

“…To overcome this limitation, the GshF bifunctional enzyme from Gram-positive bacteria was produced in S. cerevisiae , as GshF is insensitive to feedback inhibition. The resulting strain produced 240 mg L −1 GSH with GSH content and yield of 4.3% and 25.6 mg glutathione /g glucose , respectively [ 127 ]. However, this production of GSH by S. cerevisiae competes with glucose demands of other industries and results in high production costs [ 131 ].…”

“…Due to its importance for the food, cosmetic and pharmaceutical industries [ 131 ], GSH is commercially produced mainly by fermentation of Saccharomyces cerevisiae overproducing both GshA and GshB [ 127 ], or the more active GshF bifunctional enzyme [ 127 ]. However, this GSH production competes with glucose demands of other industries [ 131 ].…”