title: Engineering Saccharomyces cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides.
abstract: BACKGROUND
  Glycosylation of flavonoids is a promising approach to improve the pharmacokinetic properties and biological activities of flavonoids. Recently, many efforts such as enzymatic biocatalysis and the engineered Escherichia coli biotransformation have increased the production of flavonoid glucosides. However, the low yield of flavonoid glucosides can not meet the increasing demand for human medical and dietary needs. Saccharomyces cerevisiae is a generally regarded as safe (GRAS) organism that has several attractive characteristics as a metabolic engineering platform for the production of flavonoid glucosides. However, endogenous glucosidases of S. cerevisiae as a whole-cell biocatalyst reversibly hydrolyse the glucosidic bond and hinder the biosynthesis of the desired products. In this study, a model flavonoid, scutellarein, was used to exploit how to enhance the production of flavonoid glucosides in the engineered S. cerevisiae.
  
  
  RESULTS
  To produce flavonoid glucosides, three flavonoid glucosyltransferases (SbGTs) from Scutellaria baicalensis Georgi were successfully expressed in E. coli, and their biochemical characterizations were identified. In addition, to synthesize the flavonoid glucosides in whole-cell S. cerevisiae, SbGT34 was selected for constructing the engineering yeast. 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 and UTP-glucose-1-phosphate uridylyltransferase involved in the synthetic system of uridine diphosphate glucose were over-expressed in S. cerevisiae. Consequently, approximately 4.8 g (1.2 g/L) of scutellarein 7-O-glucoside (S7G) 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.
  
  
  CONCLUSIONS
  The engineered yeast harbouring SbGT with a deletion of glucosidases produced more flavonoid glucosides than strains without a deletion of glucosidases. 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.
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., Beijing, 100050, China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., Beijing, 100050, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: College of Life Science, Qufu Normal University, Qufu, 273165, Shandong, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.affiliation: Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., Beijing, 100050, China.
authors.forename: Wenlong
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., Beijing, 100050, China.
authors.forename: Minzhi
authors.initials: M
authors.lastname: Liu
authors.affiliation: Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., Beijing, 100050, China.
authors.forename: Kedi
authors.initials: K
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., Beijing, 100050, China. wwang@imm.ac.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
mesh_terms: D047310:Apigenin / Q000378:metabolism; D003470:Culture Media; D005285:Fermentation; D005419:Flavonoids / Q000096:biosynthesis*; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D005959:Glucosidases / Q000235:genetics*; D005960:Glucosides / Q000096:biosynthesis*; D005965:Glucuronates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D060847:Metabolic Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D003470:Culture Media; D005419:Flavonoids; D005960:Glucosides; D005965:Glucuronates; D011994:Recombinant Proteins; C484876:scutellarin; D047310:Apigenin; D005959:Glucosidases; D005947:Glucose
keywords: Glucosidase; Glucosyltransferase; Saccharomyces cerevisiae; Scutellarein 7-O-glucoside