Optimization Of Prenyltransferase NovQ Expression And MK-3 Catalytic Synthesis In Pichia Pastoris

Vitamin K2 is an indispensable vitamin for human beings.Microbial fermentation production of vitamin K2,with less pollution,low energy consumption and high biological activity products,will inevitably become the development trend of vitamin K2 production.Recombinant Pichia pastoris Gpn12,expressing the novQ isopentenyltransferase gene derived from Streptomyces niveus,showed a considerable potential for MK-3 production.In order to make the recombinant strain produce higher yield of MK-3,the conditions of NovQ expression and catalytic synthesis of MK-3 were optimized to obtain the optimal conditions for MK-3 production.This paper explored the effects of initial pH,induction temperature,methanol addition and induction time on NovQ expression.At an initial pH of 7.5,an induction temperature of 25 ? for 96 hours,and 2%methanol was added every 24 h,the expression of NovQ in shake flask increased by about 10 times,while the expression in the 30L fermenter by fed-batch fermentation increased by about 15 times than that of shaking flask before optimization.The effects of initial pH,temperature,methanol addtion,precursors(menadione,isopentenol)addition,catalytic time and cetyltrimethylammonium bromide(CTAB)addition were investigated in the Pichia pastoris whole-cell catalytic synthesis process of vitamin K2 in shaking flask.Catalytic temperature,addtion of menadione and catalytic time were optimized the response surface analysis,and the catalytic conditions were as follows:the catalytic temperature was 31.56 ?,the menadione addition amount was 295.54 mg/L,and the catalytic time was 15.87 h.The experimental results proved that the optimized MK-3 production reached 98.47mg/L in shaking flask,35%higher than that of the control group,which was consistent with the response surface prediction.The catalytic time and the cell catalyst concentration of MK-3 transformation process in 30L fermentor were investigated based on shake flask optimization.The results showed that when the catalyst time was 24 h and the cell catalyst concentration was 220 g/L dry weight,the yield of MK-3 reached 189.67 mg/L in Pichia pastoris in 30 L fermentation tank.In this paper,the expression of NovQ and the MK-3 transformation in Pichia pastoris were optimized,in addtion,the transformation of MK-3 in 30L fermentor was explored primarily,which laid a foundation for scale production of MK-3 of Gpn12.In order to improve the side chain supply in the vitamin K2 synthesis pathway,we also explored the chemical synthesis of vitamin K2 side chain in vitro.The prepared DMAPP and farnesyl pyrophosphate will be directly used in the whole cell catalysis of MK-3.