毕赤酵母全细胞催化合成新型高倍甜味剂莱鲍迪苷A

Pichia pastoris Whole Cell-Catalyzed Synthesis of a Novel High-intensity Sweetener Rebaudioside A

本研究构建经密码子优化后的UDP-糖基转移酶UGT76G1和蔗糖合成酶AtSUS基因的重组毕赤酵母菌株GS115/p PIC9KUGT/p PICZA-At SUS,实现双酶在毕赤酵母中的胞内共表达。利用共表达菌株作为全细胞催化剂在体外进行催化反应,可成功将ST转化为RA,并在此基础上对其最适反应温度、反应p H、底物UDP浓度与底物蔗糖浓度条件进行优化。重组菌株经甲醇诱导,对不同发酵时间菌体的催化能力进行探究,确定发酵120 h菌体催化能力最佳,RA产量为0.58 mg/mL。对共表达菌株催化体系中全细胞催化条件进行优化,优化后的催化体系为:反应pH 7.0,UDP浓度1 mM,蔗糖浓度70 mM,MgCl2浓度3 mM,ST浓度10 mg/mL,将OD600为30的细胞与上述体系混合后在最适温度45℃下,200 r/min反应15 h,重组菌可将10 mg/mL ST转化为7.46 mg/mL RA,为RA酶法生物合成及其产业化应用提供技术支持。

In this study,we constructed a codon-optimized UDP-glycosyltransferase UGT76G1 and a sucrose synthase At SUS gene recombinant Pichia pastoris strain,GS115/pPIC9K-UGT/pPICZA-AtSUS,to achieve intracellular co-expression of the two enzymes in Pichia pastoris.The co-expressed strain was used as a whole-cell catalyst for the in vitro catalytic reaction,to successfully convert ST into RA.On this basis,optimization was performed on reaction temperature,reaction pH,substrate UDP concentration and substrate sucrose concentration.After the recombinant strain was induced with methanol,the catalytic capacity of the cells at different fermentation times was investigated.The catalytic capacity of the cells was the greatest after fermentation for 120 h,with the RA yield as 0.58 mg/m L.After the optimization of the catalytic conditions for the whole cells in the catalytic system of co-expressed,the optimized catalytic conditions were:reaction pH 7.0,UDP concentration 1 mM,sucrose concentration 70 mM,MgCl2 concentration 3 mM,ST concentration 10 mg/mL,with the recombinant bacteria capable of converting 10 mg/mL ST to 7.46 mg/mL RA(if the cells with an OD600 value of 30 were mixed with the above systems and allowed a reaction for 15 h at 200 rpm and an optimum temperature of 45℃).This research provides technical support for the RA enzymatic biosynthesis and its industrialization.