Pichia pastoris Whole Cell-Catalyzed Synthesis of a Novel High-intensity Sweetener Rebaudioside A
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Abstract:
In this study, we constructed a codon-optimized UDP-glycosyltransferase UGT76G1 and a sucrose synthase AtSUS 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/mL. 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.
