酵母前体饲喂验证植物次生代谢产物功能基因方法学初探

Functional identification of plant secondary metabolite biosynthesis gene in Saccharomyces cerevisiae by cell feeding

目的筛选效率最高的酵母前体饲喂方式,为在酵母中进行基因功能验证提供方法学指导。方法首先构建含PsP6H基因和PsCPR基因重组酵母工程菌,然后用4种不同的方法进行前体饲喂,最后质谱检测下游产物二氢血根碱来确定最优方案。结果最优的前体饲喂方法是将酵母工程菌经过半乳糖诱导31 h后在pH 8.0的Tris EDTA溶液中孵育24 h。结论不同的前体饲喂方式对酵母工程菌下游产物产生不同影响,经优化后的酵母前体饲喂方法较稳定,重复性好。

Objective To screen the most efficient cell feeding way and provide guidance for functional verification of genes in Saccharomyces cerevisiae. Methods Firstly, the recombinant industrial S. cerevisiae strains containing PsCPR and PsP6H genes were constructed. Then four different cell feeding methods were used. At the end, the downstream product dihydrosanguinarine was detected via UPLC Q-TOF MS/MS. Results The best method of cell feeding was that the recombinant industrial strain induced by galactose for 31 h and then incubated in the TE solution buffer（pH = 8.0） for 24 h. Conclusion Different cell feeding ways have different effects on the downstream products of recombinant industrial S. cerevisiae. The optimized cell feeding method shows stability and good repeatability.