Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus.
Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus.
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DOI：
10.3168/jds.2017-14142
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发表时间：
2018-08
影响因子：
3.5
通讯作者：
Z. Xiong;Linghui Kong;Guangqiang Wang;Yongjun Xia;Hui Zhang;B. Yin;Lian-zhong Ai
中科院分区：
文献类型：
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作者：
Z. Xiong;Linghui Kong;Guangqiang Wang;Yongjun Xia;Hui Zhang;B. Yin;Lian-zhong Ai
双功能谷胱甘肽合成酶（GshF）是一种能同时催化ATP依赖的还原型谷胱甘肽（GSH）生物合成的酶。在这项工作中，19个推定的gshF挖掘从20个代表性的乳酸杆菌物种的全基因组测序。为了分析这些推定的GshF的功能，选择来自植物乳杆菌和干酪乳杆菌的GshF，并在大肠杆菌中成功表达。与不表达GshF的对照组相比，E.大肠杆菌GshF基因的表达，证明了乳酸杆菌GshF基因对GSH的生物合成具有功能活性。此外，通过表达L. plantarum在E.以大肠杆菌为例，在优化的诱导条件和前体浓度下，GSH产量（286.5 μM）比未优化条件下的对照提高了177.9%。转录分析表明，外源表达GshF显著抑制了内源GSH代谢和前体生物合成的关键基因，表明外源表达GshF可提高GSH效价。总的来说，我们的研究结果表明，GSHF是丰富的乳酸杆菌和GSHF的异源表达是一种有效的策略，用于改善GSH的生物合成。
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.