大肠杆菌中顺式-3-羟脯氨酸羟化酶的定向改造

The directional modification of proline-3-hydroxylase producing cis-3-hydroxyproline

对以L-脯氨酸为底物,产顺式-3-羟脯氨酸(cis-3-hydroxyproline,H3P)的羟化酶基因进行定向改造。从重组菌E.coli BL21(DE3)/p ET21a-ptrp2-H3P出发,通过易错PCR随机突变和定点突变处理,利用多孔板和创建的简易显色法相结合的高通量方法筛选出2株H3P高产菌P-2-H3、P-9-D5。经基因测序后得到5个突变位点R58C、T83I、H89Y、F109Y、Y119I,并在出发菌基础上对此5个位点进行单一定点突变,得到5个突变体TBTR58C、TBT-T83I、TBT-H89Y、TBT-F109Y、TBT-Y119I。发酵24 h测其产量找出3个优势突变位点。以TBT-R58C为出发菌,依次对另外2个位点进行定点突变。得到TBT-R58C-T83I-H89Y突变体。发酵24 h产量达到了1125.3 mg/L。与重组菌相比,TBT-R58C-T83I-H89Y的产量提高了53.6%。

The proline-3-hydroxylase producing cis-3-hydroxyproline （H3P） with proline as substrate was modi- fied. The recombinant strain E. coli BL21 （DE3）/pET21a-ptrp2-H3P was mutagenized by error-prone PCR. Two mutants P-2-H3, P-9-D5 were selected through high throughput screening method using hole plates coupled with sim- ple color developing method. Five mutation sites were found through gene sequencing and five single mutants TBT- R58C, TBT-T83I, TBT-H89Y, TBT-F109Y, and TBT-Y1191 were obtained through site-directed mutagenesis. 3 preferred mutation sites were found based on fermentation of the five single mutants. Based on strain TBT-R58C, the other two sites were site-directed mutated successively to obtain mutant TBT-R58C-T83I-H89Y with yield of 1125.3 mg/L, which increased by 53.6% compared with the original recombinant strain.