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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

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. [ABSTRACT FROM AUTHOR]

The prenylation of flavonoids is a main type of structural modification and can endow flavonoids with greater bioactivity and bioavailability. A soluble prenyltransferase (NgFPT) gene from Nocardiopsis gilva was cloned, expressed and characterized in Escherichia coli. The optimal activity of NgFPT was at pH 7.5 and 30 °C. The activity of NgFPT was significantly enhanced by Ca2+, Al3+, and DMSO. NgFPT showed high selectivity to prenylate flavanones at 3′-C to generate 3′-C-prenyl-flavanones. The Kcat and Km of recombinant NgFPT for naringenin were 0.001 s−1 and 0.045 mM, respectively. Then, recombinant strains were reconstructed by introducing NgFPT gene and the isopentenol utilization pathway. Escherichia coli hosts and fusion tags were screened to improve the yield of 3′-C-prenyl-naringenin in vivo , resulting in maximal 3′-C-prenyl-naringenin production at 3.5 mg/L. By optimizing biotransformation conditions and adopting the resting cell bioconversion, maximum 3′-C-prenyl-naringenin production reached 10.3 mg/L with a specific productivity of 0.21 mg/L/h after 48 h incubation. Thus, the article provides a regiospecific soluble prenyltransferase and a method for the production of 3′-C-prenyl-naringenin by metabolic engineering. [Display omitted] • A regiospecific prenyltransferase was cloned and expressed. • NgFPT exhibited high selectivity to prenylate flavanones at 3′-C. • Escherichia coli hosts and fusion tags were screened to improve the production. • Recombinant strains were reconstructed to biosynthesize 3′-C-prenyl-naringenin. • The article provides a method for the production of 3′-C-prenyl-naringenin. [ABSTRACT FROM AUTHOR]

The steroid metabolites 9-hydroxy-androstenedione (9-OH-AD), androstadienedione (ADD) and androstenedione (AD) are important steroidal pharmaceuticals. In order to raise the production of steroid metabolites, an efficient resting cell phytosterol bioconversion process was developed to produce 9-OH-AD in the presence of hydroxypropyl-b-cyclodextrin (HP-β-CD). Cell growth medium containing phytosterol as an inducer positively improved cell activity. Under aerobic conditions, bioconversion proceeded at 70 g L-1 phytosterol in the presence of HP-β-CD (the optimized molar ratio of HP-β-CD/phytosterol was 1:1) with 30 g L-1 resting Mycobacterium neoaurum NwIB-yV cells (cell dry mass) in a 5-L bioreactor, where 9-OH-AD production and space-time yield reached 36.4 g L-1 and 9.1 g L-1 d-1, respectively. The recycling of cells and HP-β-CD enables cost-saving and industrial applications. This bioprocess was also applied for the production of ADD and AD. The production of these steroid metabolites was much higher than that reported in previous studies. [ABSTRACT FROM AUTHOR]