Engineering S. cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides

Glycosylation of flavonoids is a promising approach to improve the pharmacokinetic properties and pharmacological activities. However, chemical glycosylation remains restricted by such disadvantages as poor regio- and stereoselectivities, and large-scale application of whole-cell glycosylation is still hampered by inefficient UDP-sugar formation. Using a combinatorial approach, an engineered Saccharomyces cerevisiae strain was constructed to enhance the production of flavonoid glucosides.

Fig. 1. Hydrolytic activity to luteolin 7-O-glucoside in strains with the deletion of glucosidases. (a) Biomass (OD600). (b) The remaining rate of luteolin 7-O-glucoside in the liquid medium. The values are presented as the means, and the error bars show the SD (n=3).

Firstly, a suitable glucosyltransferase (SbGT) were obtained from Scutellaria baicalensis Georgi. Then, three glucosidase genes (EXG1, SPR1, YIR007W) were knocked out using homologous integration, and the EXG1 gene was determined to be the decisive gene of S. cerevisiae in the process of hydrolysing flavonoid glucosides. To further enhance the potential glycosylation activity of S. cerevisiae, two genes encoding phosphoglucomutase (PGM2) and UTP-glucose-1-phosphate uridylyltransferase (UGP1) involved in the synthetic system of uridine diphosphate glucose (UDPG) were over-expressed in S. cerevisiae. Finally, an engineered yeast strain was constructed to enhance the production of flavonoid glucosides by combining the expression of SbGT, PGM2, and UGP1 with the deletion of glucosidases.

Fig. 2. Differences in the level of scutellarein 7-O-glucoside produced by strains W303-1b/SbGT34, W303-1b/ES∆/SbGT34 and W303-1b/ES∆/PU/SbGT34 over time. Strains were incubated with 0.2 mM scutellarein. The values are presented as the means, and the error bars show the SD (n=3).

Consequently, the resulted engineered strains were assayed for whole-cell biotransformation of scutellarein, and approximately 4.8 g (1.2 g/L) of scutellarein 7-O-glucoside was produced in 4 L of medium after 54 h of incubation in a 10-L fermenter while being supplied with ~ 3.5 g of scutellarein. Most important, this platform without glucosidase activity could be used to modify a wide range of valued plant secondary metabolites and to explore of their biological functions using whole-cell S. cerevisiae as a biocatalyst.

Huimin Wang 1, Yan Yang 1, Lin Lin 3, Wenlong Zhou 2, Minzhi Liu 2, Kedi Cheng 2, Wei Wang 1
1State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences,
Beijing, China

2Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission,
Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China
3College of Life Science, Qufu Normal University, Qufu, Shandong, China

 

Publication

Engineering Saccharomyces cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides.
Wang H, Yang Y, Lin L, Zhou W, Liu M, Cheng K, Wang W
Microb Cell Fact. 2016 Aug 4

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