An efficient preparation and biocatalytic synthesis of novel C-glycosylflavonols kaempferol 8-C-glucoside and quercetin 8-C-glucoside through using resting cells and macroporous resins

Abstract

Background C-glycosylated flavonoids are a main type of structural modification and can endow flavonoids with greater stability, bioactivity, and bioavailability. Although some C-glycosylated flavonoids have been biosynthesized in vivo or vitro, only a few C-glycosylflavonols have been prepared by these methods. Results In this study, several uridine 5’-diphosphate (UDP)-glucose biosynthesis pathways and Escherichia coli hosts were screened to reconstruct recombinant strains for producing the novel C-glycosylflavonols kaempferol 8-C-glucoside and quercetin 8-C-glucoside. To increase C-glycosylflavonol production, the timing of flavonol addition was adjusted, and glycerol was added to avoid degradation of C-glycosylflavonols. By using resting cell bioconversion, the highest kaempferol 8-C-glucoside and quercetin 8-C-glucoside production reached 16.6 g/L and 12.5 g/L, respectively. Then, ultrasound-assisted adsorption/desorption was used to prepare C-glycosylflavonols by using macroporous resins. Through screening macroporous resins and optimizing the adsorption/desorption conditions, the highest adsorption capacity and desorption capacity for kaempferol 8-C-glucoside on HPD100 reached 28.57 mg/g and 24.15 mg/g, respectively. Finally, kaempferol 8-C-glucoside (15.4 g) with a yield of 93% and quercetin 8-C-glucoside (11.3 g) with a yield of 91% were obtained from 1 L of fermentation broth. Conclusions Kaempferol 8-C-glucoside and quercetin 8-C-glucoside are novel C-glycosylflavonols, which have not been extracted from plants. This study provides an efficient method for the preparation and biocatalytic synthesis of kaempferol 8-C-glucoside and quercetin 8-C-glucoside by metabolic engineering of Escherichia coli. Graphical Abstract

Production of Four FlavonoidC-Glucosides inEscherichia coli

“…19 Additionally, 16.6 g/L kaempferol 8-C-glucoside was synthesized in E. coli expressing TcCGT and engineering the UDP-glucose synthesis pathway. 20 The use of microorganisms for the synthesis of natural compounds via the complete biosynthetic pathway can provide insights into synthesizing the target compound(s) in a more complex system. Biotransformation of flavonoids into their corresponding C-glucosides using E. coli has been studied extensively.…”

“…E. coli harboring WjGT1 produced approximately 172 mg/L isovitexin and 105 mg/L kaempferol 6- C -glucoside upon periodic addition of flavonoids . Additionally, 16.6 g/L kaempferol 8- C -glucoside was synthesized in E. coli expressing TcCGT and engineering the UDP-glucose synthesis pathway …”

Efficient and Economic Utilization of Cellobiose for Glycosylation Modification by Regulating Carbon Source Supply and Metabolic Pathway In Vivo

“…In this work, we utilized the recombinant strain containing Trollius chinensis C-glycosyltransferase (TcCGT) gene and the cellobiose phosphorolysis route as a model to investigate the conversion rate of cellobiose. 12,30 Three genes (pgm, agp, and ushA) were knocked out from the genome of E. coli to relieve the degradation and diversion of the cellobiose phosphorolysis route. Moreover, the conversion rate of cellobiose was improved by regulating the carbon source supply in vivo.…”

“…In nature, the glycosylation modification is catalyzed by glycosyltransferases with UDP-sugar as the donor and natural compounds as acceptors. − The supply of UDP-sugar is important to improving the efficiency of glycosylation modification. UDP-glucose is an important UDP-sugar in the glycosylation modification and can be converted to UDP-glucuronic acid, UDP-galactose, and UDP-rhamnose. , Several routes have been used to biosynthesize UDP-sugar in vivo. ,,, Based on cellobiose phosphorylase, a novel cellobiose phosphorolysis route has been developed to biosynthesize UDP-glucose, UDP-rhamnose, and UDP-galactose in vivo. − Compared to other routes, the cellobiose phosphorolysis route can endow cells more resistant to common inhibitors in the growth environment and express more recombinant enzymes .…”

“…The aim of this work was to develop an economic and efficient utilization of cellobiose for glycosylation modification, while considering the green chemistry of the whole process. In this work, we utilized the recombinant strain containing Trollius chinensis C-glycosyltransferase (TcCGT) gene and the cellobiose phosphorolysis route as a model to investigate the conversion rate of cellobiose. , Three genes ( pgm , agp , and ushA ) were knocked out from the genome of E. coli to relieve the degradation and diversion of the cellobiose phosphorolysis route.…”