酵母UDP-葡萄糖合成途径的优化及黄酮葡萄糖苷的合成

Optimization of biosynthetic pathway of uridine diphosphate glucose in yeast for production of flavonoid glucosides

目的研究在酿酒酵母细胞内高表达大肠杆菌UDP-葡萄糖合成途径的2个关键酶对工程酵母细胞的葡萄糖苷化反应活性的影响。方法利用PCR方法获得大肠杆菌磷酸葡萄糖变位酶(PGM)和UDP-葡萄糖焦磷酸化酶(Gal U)基因,构建整合型表达载体,转化酵母;同时共表达具有催化黄酮葡萄糖苷化的UDP-葡萄糖转移酶,研究摇瓶培养条件下PGM和Gal U基因的表达对工程细胞生物催化黄酮类化合物葡萄糖苷化的影响。结果在酵母细胞内表达大肠杆菌UDP-葡萄糖合成途径的2个关键酶PGM和Gal U,提高了组合表达UDP-葡萄糖转移酶的工程菌生物催化合成野黄芩素-7-O-葡萄糖苷的效率,表明PGM和Gal U基因的高表达能促进UDP-葡萄糖的合成;所构建的工程菌也能催化木犀草素、柚皮素、白杨素、汉黄芩素、木蝴蝶素A、黄芩素、槲皮素以及大豆黄酮等黄酮类化合物的葡萄糖苷化。结论组合表达大肠杆菌PGM和Gal U基因能促进酵母细胞内UDP-葡萄糖的合成,进而提高了工程细胞催化合成黄酮葡萄糖苷的效率。

Objective To investigate the effect of expression of phosphoglucomutase and UTP-glucose-1-phosphate uridylyltransferase [two key enzymes in the pathway of synthesizing uracil diphosphate glucose（UDP-glucose）] from Escherichia coli on glucosylation capacity of engineering yeast.Methods In order to up-regulate the biosynthetic pathway of UDP-glucose of Saccharomyces cerevisiae, the two genes encoding phosphoglucomutase（PGM） and UTP-glucose-1-phosphate uridylyltransferase（Gal U） of E. coli were amplified by PCR and then inserted into the yeast integration vectors. The resulting plasmids were linearized by restriction enzyme Not I and transformed into the engineering strain. The integration expression plasmid pδGAPg-Sb GT34 with a UDP-glucose flavonoid glucosyltransferase gene cloned from Scutellaria baicalensis Georgi was also constructed and further integrated into the basic engineering strain W303-1b/ES/Pe G. The effect of high expression of PGM and Gal U on production of flavonoid glucosides was investigated using the engineered strain W303-1b/ES/Pe G/Sb GT34 as whole-cell biocatalyst under the flask culture condition. Results The engineering strain W303-1b/ES/Pe G/Sb GT34 with the high expression of PGM and Gal U genes involving the biosynthesis of UDP-glucose together with the glucosyltransferase was successfully constructed. Its production of scutellarein 7-O-glucoside was further improved in comparison with that of strain W303-1b/ES/Sb GT34. It is indicated that the expression of heterologous PGM and Gal U genes of E. coli contributes to the synthesis of endogenous UDP-glucose of engineering strain. The resulting strain W303-1b/ES/Pe G/Sb GT34 also glucosylated the flavonoids such as luteolin, naringenin, chrysin, wogonin, orohylin A, baicalein, quercetin and daidzein. Conclusion The synthesis of endogenous UDP-glucose of yeast was boosted through the co-expression of PGM and Gal U genes isolated from E. coli, which improves the glycosylation capacity of the engineered strain along with expression of a UDP-glucose falvonoid glucosyltransferase.