Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus.

Bifunctional glutathione synthetase (GshF) has recently been reported to simultaneously catalyze the 2-step ATP-dependent biosynthesis of reduced glutathione (GSH). In this work, 19 putative gshF were mined from the complete sequenced genome of 20 representative Lactobacillus species. To functionally analyze these putative GshF, GshF from Lactobacillus plantarum and Lactobacillus casei were selected and successfully expressed in Escherichia coli. Compared with the control without expressing GshF, GSH titers were enhanced significantly in E. coli with overexpression of GshF, demonstrating that putative GshF from Lactobacillus have functional activities on GSH biosynthesis. Moreover, with the expression of GshF from L. plantarum in E. coli as a paradigm, GSH yield (286.5 μM) was strongly improved by 177.9% with optimized induced conditions and precursor concentration compared with the control under unoptimized conditions. Transcriptional analysis showed that key genes of endogenous GSH metabolism and precursor biosynthesis were remarkably suppressed by GshF expression, indicating that the increase of GSH titer was attributed to heterologous expression of GshF. Overall, our results suggested that gshF is enriched in Lactobacillus and that heterologous expression of GshF is an efficient strategy for improving GSH biosynthesis.