途径工程改造谷氨酸棒杆菌合成L-半胱氨酸

Pathway engineering modification of Corynebacterium glutamicum for L-cysteine synthesis

L-半胱氨酸是一种重要的含硫氨基酸,广泛应用在医药、食品和化妆品等行业。该文以实验室保藏的一株高产L-丝氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)A36为出发菌株,通过加强表达丝氨酸O-乙酰基转移酶(serine O-acetyltransferase,SAT)、O-乙酰基-L-丝氨酸巯基化酶-A和转运蛋白Bcr编码基因强化L-半胱氨酸合成和转运,敲除L-半胱氨酸降解途径关键酶弱化其降解,构建系列重组菌株,其中重组菌S-C-7的L-半胱氨酸产量最高,为286.7 mg/L。进一步通过优化硫源提高菌株S-C-7的L-半胱氨酸产量,结果表明,最佳硫源为硫代硫酸钠,当发酵24 h时添加12 g/L硫代硫酸钠,L-半胱氨酸的产量最高,为581.6 mg/L,较优化前提高1.0倍。最后,在5 L发酵罐对S-C-7进行发酵评价,L-半胱氨酸产量达到1.2 g/L,是目前文献报道谷氨酸棒杆菌产L-半胱氨酸的最高产量;为实现谷氨酸棒杆菌发酵生产L-半胱氨酸奠定了基础。

L-cysteine is an important sulfur-containing amino acid,which is widely used in pharmaceutical,food and cosmetic industries.In this study,C.glutamicum A36 was engineered to efficiently produce L-cysteine from sugar,and showed highest L-serine titer comparing with its’parent strain.In order to produce L-cysteine,two types of the serine O-acetyltransferase(gene encoded by cysE)were overexpressed in strain A36 respectively,strain S-C-1 and S-C-2 had been constructed,L-cysteine titer was 115.8 mg/L and 105.8 mg/L,respectively,and the parent strain A36 couldn’t produce L-cysteine.Subsequently,in order to increase the titer of L-cysteine,several metabolic engineering strategies were performed,including overexpression of the OASS-A(gene encoded by cysK)and the L-cysteine exporter Bcr(gene encoded by bcr),and the deletion of the degradation pathway.A series recombinant strain had been constructed,and among all strains,S-C-7 showed the highest L-cysteine titer of 286.7 mg/L.The sulfur source was optimized to increase L-cysteine titer further,the addition of 12 g/L sodium thiosulfate at 24 h showed the highest L-cysteine titer of 581.6 mg/L,which was two times of that before optimization.Finally,in 5 L fermenter,the L-cysteine titer of strain S-C-7 could reach 1.2 g/L,which was the highest titer of L-cysteine produced by C.glutamicum up to now.It laid a foundation for the L-cysteine production by C.glutamicum.