基于低拷贝质粒的高产紫色杆菌素谷氨酸棒杆菌的构建和发酵

Construction and Fermentation of Efficient Violacein-producing Corynebacterium glutamicum Strains using Low Copy Number Plasmids

该研究以公认安全(Generally Recognized as Safe,GRAS)的谷氨酸棒杆菌(Corynebacterium glutamicum)为宿主,构建高产紫色杆菌素的重组菌株。利用谷氨酸棒杆菌天然大质粒pTET3的复制与分配元件,构建了低拷贝质粒pOK12CG1,该质粒在谷氨酸棒杆菌中的拷贝数约为6拷贝/基因组,且与谷氨酸棒杆菌常用质粒pEC-XK99E和pXMJ19兼容。以低拷贝质粒pOK12CG1为骨架构建了携带紫色杆菌素合成操纵子(vioABCDE)的质粒pCGvio,并分别以谷氨酸棒杆菌标准株ATCC 13032和插入序列(Insertion Sequence,IS)元件删除株为宿主,构建了7株合成紫色杆菌素的重组菌株。通过初步筛选,发现基于低拷贝质粒的重组菌株ATCC 13032/pCGvio,其紫色杆菌素产量(508.24 mg/L)高于基于中高拷贝质粒的重组菌株ATCC 13032/pECvio(376.16 mg/L),而基于低拷贝质粒的IS元件删除重组菌株ISDM023/pCGvio紫色杆菌素产量达到了610.13 mg/L。进一步采用正交实验设计对重组菌株ISDM023/pCGvio进行培养基体积比(V_(LB):V_(BHIS))、诱导时间和IPTG诱导剂浓度这3个因素的发酵条件优化。结果表明,在VLB:VBHIS为1:2、诱导时间为18 h、IPTG浓度为0.75 mmol/L的优化条件下,紫色杆菌素的摇瓶发酵产量可达了1007.47 mg/L。该研究成功构建了紫色杆菌素的谷氨酸棒状杆菌重组菌株ISDM023/pCGvio,为谷氨酸棒状杆菌高效合成紫色杆菌素奠定了实验基础,也为其它产物的高效合成提供参考。

For high violacein production,a recombinant strain was constructed using Corynebacterium glutamicum[generally recognized as safe(GRAS)]as the host cell.The low copy number plasmid pOK12CG1 was constructed using the replication and partition elements from pTET3,a natural large plasmid of C.glutamicum.The plasmid pOK12CG1,maintained at~6 copies per genome,was compatible with common plasmids pEC-XK99E and pXMJ19 of C.glutamicum.The violacein synthesis operon(vioABCDE)-carrying plasmid pCGvio was constructed using the low copy number plasmid pOK12CG1 as backbone.Seven violacein-producing recombinant strains were developed using the standard strain of C.glutamicum ATCC 13032 and the deletion strain of the insertion sequence(IS)element as host cells.Initial screening indicated that the recombinant strain ATCC 13032/pCGvio harboring low copy number plasmids produced more violacein(508.24 mg/L)than the recombinant strain ATCC13032/pECvio harboring medium and high copy number plasmids(376.16 mg/L).Violacein production by the IS element deletion recombinant strain ISDM023/pCGvio reached 610.13 mg/L.Fermentation conditions for the recombinant strain ISDM023/pCGvio were further optimized by orthogonal experimental design to optimize three factors:medium volume ratio(V_(LB):V_(BHIS)),induction time,and IPTG inducer concentration.Under optimal conditions(V_(LB):V_(BHIS)=1:2,induction time=18 h,and IPTG concentration=0.75 mmol/L)the final production yield of violacein in shake flasks was 1007.47 mg/L.Herein,the recombinant strain ISDM023/pCGvio of C.glutamicum was successfully constructed,providing a biological system for efficient production of violacein in C.glutamicum and serving as a reference for the efficient production of other high-value compounds.