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.

Keywords: Adsorption/desorption; Kaempferol 8-C-glucoside; Metabolic engineering; Quercetin 8-C-glucoside; Resting cell bioconversion; UDP-glucose biosynthesis pathway.

Fig. 1

Effects of different UDP-glucose biosynthesis pathways and E. coli hosts on kaempferol 8-C-glucoside production. TcCGT represented hosts carrying pCDFDuet-TcCGT. TcCGT-I represented hosts carrying pCDFDuet-TcCGT and pACYCDuet-cscB-Cep-UgpA. TcCGT-II represented hosts carrying pCDFDuet-TcCGT and pACYCDuet-cscB-Basp-UgpA

Fig. 2

Optimization of the bioconversion conditions for kaempferol 8-C-glucoside production in BL-TcCGT-I. a Effects of different media on kaempferol 8-C-glucoside production. b Effects of temperature on kaempferol 8-C-glucoside production. c Effects of IPTG on kaempferol 8-C-glucoside production. d Effects of cellobiose on kaempferol 8-C-glucoside production. e Effects of DMSO on kaempferol 8-C-glucoside production. f Effects of glycerol on kaempferol 8-C-glucoside production. A total of 2 g/L kaempferol, 5 g/L cellobiose, and 0.1 mM IPTG were added to the recombinant strains, and the fermentation broth was incubated at 30 °C and 180 rpm for 24 h

Fig. 3

Effects of kaempferol and the timing of kaempferol addition on kaempferol 8-C-glucoside production. a Effects of kaempferol on kaempferol 8-C-glucoside production. A total of kaempferol (0.8, 1.6, 2.4, 3, and 3.6 g/L) was added to the recombinant strains. b Effects of the timing of kaempferol addition on kaempferol 8-C-glucoside production. 0.02 mM IPTG was added to the recombinant strains, and the recombinant strains were incubated at 20 °C and 180 rpm for (0, 2, 4, 6, 8, 10, and 12 h)

Fig. 4

The time courses for flavonol consumption and C-glycosylflavonol production in BL-TcCGT-I. a The time courses for kaempferol consumption and kaempferol 8-C-glucoside production, b the time courses for quercetin consumption and quercetin 8-C-glucoside production, c the time courses for quercetin consumption and quercetin 8-C-glucoside production with the addition of glycerol. A total of 3 g/L kaempferol or quercetin, 10 g/L cellobiose, and 0.1 mM IPTG were added to the recombinant strains

Fig. 5

The effects of cell concentration on kaempferol 8-C-glucoside production with LB medium a, TB-Gly medium b, and M9-Gly medium c. Different concentrations of recombinant strains were incubated by using the broth (5 mL) in tubes at 30 °C for 24 h. A total of 3 g/L kaempferol or quercetin and 10 g/L cellobiose were added to the recombinant strains

Fig. 6

The time courses for kaempferol 8-C-glucoside production in M9-Gly medium. A total of 6 g/L kaempferol and 10 g/L cellobiose were added to the recombinant strains

Fig. 7

The time courses for C-glycosylflavonol production using fed-batch in M9-Gly medium. a The time courses for kaempferol 8-C-glucoside production by adding fresh substrates and glycerol, b the time courses for quercetin 8-C-glucoside production by adding fresh substrates and glycerol. In the fermentation process, 4 g/L kaempferol or quercetin and 10 g/L cellobiose were added to the broth at 24 h and 72 h, and 10 g/L glycerol was added to the broth at 24 h, 48 h, 72 h, and 96 h

Fig. 8

Absorption kinetics and desorption of kaempferol 8-C-glucoside on HPD100 under different conditions. a Absorption kinetics of kaempferol 8-C-glucoside on HPD100 under different ultrasound powers, b desorption of kaempferol 8-C-glucoside on HPD100 under different ultrasound powers with 40% ethanol, c desorption of kaempferol 8-C-glucoside on HPD100 under ultrasound power (240 W) with different ethanol concentrations

Fig. 9

¹H-NMR and ¹³C-NMR spectra of kaempferol 8-C-glucoside and quercetin 8-C-glucoside (400 MHz for ¹H-NMR and 101 MHz for ¹³C-NMR, DMSO-d₆, δ in ppm). a ¹H-NMR for kaempferol 8-C-glucoside, b ¹³C-NMR for kaempferol 8-C-glucoside, c ¹H-NMR for quercetin 8-C-glucoside, d ¹³C-NMR for quercetin 8-C-glucoside