De novo biosynthesis of C-arabinosylated flavones by utilization of indica rice C-glycosyltransferases

Abstract

Flavone C-arabinosides/xylosides are plant-originated glycoconjugates with various bioactivities. However, the potential utility of these molecules is hindered by their low abundance in nature. Engineering biosynthesis pathway in heterologous bacterial chassis provides a sustainable source of these C-glycosides. We previously reported bifunctional C-glucosyl/C-arabinosyltransferases in Oryza sativa japonica and O. sativa indica, which influence the C-glycoside spectrum in different rice varieties. In this study, we proved the C-arabinosyl-transferring activity of rice C-glycosyltransferases (CGTs) on the mono-C-glucoside substrate nothofagin, followed by taking advantage of specific CGTs and introducing heterologous UDP-pentose supply, to realize the production of eight different C-arabinosides/xylosides in recombinant E. coli. Fed-batch fermentation and precursor supplement maximized the titer of rice-originated C-arabinosides to 20–110 mg/L in an E. coli chassis. The optimized final titer of schaftoside and apigenin di-C-arabinoside reached 19.87 and 113.16 mg/L, respectively. We demonstrate here the success of de novo bio-production of C-arabinosylated and C-xylosylated flavones by heterologous pathway reconstitution. These results lay a foundation for further optimal manufacture of complex flavonoid compounds in microbial cell factories.

Identification of a flavonoid C-glycosyltransferase from fern species Stenoloma chusanum and the application in synthesizing flavonoid C-glycosides in Escherichia coli

“…8). The yields were similar to that previously reported [19,20]. For example, Chen et al constructed a recombinant E. coli by introducing two P450 genes (F2H and CPR) and one CGT gene, which resulted in 26.6 mg/L of isoorientin and 23.9 mg/L of orientin [19].…”

“…Furthermore, when UDP-glucose was supplied in vivo, the maximum conversion rate of ScCGT1-P164T toward phloretin reached 99% (about 43 mg/L; Additional file 2: Table S3), and the whole fermentation process only took 18 h. The research on introducing plant F2Hs and CGTs into E. coli to produce flavone C-glycosides are extremely limited. At present, only two relevant studies have been reported, and both of them take a truncated F2H gene (in the transmembrane region) coupled with a P450 reductase (CPR) to co-express in E. coli, which provide the reaction intermediates for the catalysis of CGTs [18,19]. In this study, the soluble protein CjFNS I/F2H and ScCGT1-P164T were combined in E. coli, which led to a high conversion from naringenin to vitexin (20 mg/L) and isovitexin (19 mg/L) (Fig.…”

“…For example, a flavone C-glycoside pathway was reconstructed in yeast by introducing P450 F2H and CGT genes from Oryza sativa [17]. In addition, Chen et al [19] proposed the de novo biosynthesis of flavone di-C-glycosides with arabinosyl or other pentosyl groups in E. coli. To further improve the titer of flavone C-glycosides, one-step whole-cell synthesis in E. coli was employed [20,21].…”