基于碳硫模块平衡策略的大肠杆菌高产L-半胱氨酸菌株构建

Construction of an l-cysteine hyper-producing strain of Escherichia coli based on a balanced carbon and sulfur module strategy

摘要:

l-半胱氨酸是一种重要的含硫氨基酸，因其多样的生理功能，l-半胱氨酸在医药、化妆品和食品工业中有着广泛的应用。模块化代谢工程策略在细胞工厂的构建中具有极大的潜力。本研究利用碳硫模块协同表达策略进行大肠杆菌的l-半胱氨酸合成途径构建，构建了一株l-半胱氨酸合成基因工程菌。首先，通过增强l-半胱氨酸前体物质l-丝氨酸(serA^f、serB和serC^Cg)的生物合成以及转录调控因子CysB的表达，l-半胱氨酸的产量由0提高到(0.38±0.02)g/L。然后，通过促进l-半胱氨酸转运和无机硫源的吸收同化、减弱l-半胱氨酸和l-丝氨酸的降解以及异源表达cysE^f和cysB^St，l-半胱氨酸的产量提升至(3.82±0.01)g/L。最后，为了优化碳模块和硫模块的代谢通量，协同表达硫酸盐同化途径与硫代硫酸盐同化途径的基因cysM、nrdH、cysK以及cysIJ，得到l-半胱氨酸高产菌株。在500mL摇瓶和2L发酵罐中分别实现了(4.17±0.07)g/L和(11.94±0.1)g/L的l-半胱氨酸积累。研究结果表明，在细胞内通过对硫碳模块间代谢通量的协调控制，可以实现l-半胱氨酸的高效生物合成。研究结果为微生物发酵生产l-半胱氨酸的产业化奠定了基础。

Abstract:

l-cysteine is an important sulfur-containing α-amino acid. It exhibits multiple physiological functions with diverse applications in pharmaceutical cosmetics and food industry. Here, a strategy of coordinated gene expression between carbon and sulfur modules in Escherichia coli was proposed and conducted for the production of l-cysteine. Initially, the titer of l-cysteine was improved to (0.38±0.02) g/L from zero by enhancing the biosynthesis of l-serine module (serA^f, serB and serC^Cg) and overexpression of CysB. Then, promotion of l-cysteine transporter, increased assimilation of sulfur, reduction or deletion of l-cysteine and l-serine degradation pathway and enhanced expression of cysE^f (encoding serine acetyltransferase) and cysB^St (encoding transcriptional dual regulator CysB) were achieved, resulting in an improved l-cysteine titer (3.82±0.01) g/L. Subsequently, expressions of cysM, nrdH, cysK and cysIJ genes that were involved in sulfur module were regulated synergistically with carbon module combined with utilization of sulfate and thiosulfate, resulting in a strain producing (4.17±0.07) g/L l-cysteine in flask shake and (11.94±0.1) g/L l-cysteine in 2 L bioreactor. Our results indicated that efficient biosynthesis of l-cysteine could be achieved by a proportional supply of sulfur and carbon in vivo. This study would facilitate the commercial bioproduction of l-cysteine.