Synthesis of umbelliferone derivatives in Escherichia coli and their biological activities

Abstract

Background: Umbelliferone, also known as 7-hydroxycoumarin, is a phenolic metabolite found in many familiar plants. Its derivatives have been shown to have various pharmacological and chemo-preventive effects on human health. A uridine diphosphate glycosyltransferase YjiC from Bacillus licheniformis DSM 13, a cytochrome P450BM3 (CYP450 BM3) variant namely mutant 13 (M13) from Bacillus megaterium, and an O-methyltransferase from Streptomyces avermitilis (SaOMT2) were used for modifications of umbelliferone.

Results: Three umbelliferone derivatives (esculetin, skimmin, and herniarin) were generated through enzymatic and whole cell catalysis. To improve the efficiencies of biotransformation, different media, incubation time and concentration of substrate were optimized and the production was scaled up using a 3-L fermentor. The maximum yields of esculetin, skimmin, and herniarin were 337.10 μM (67.62%), 995.43 μM (99.54%), and 37.13 μM (37.13%), respectively. The water solubility of esculetin and skimmin were 1.28-folds and 3.98-folds as high as umbelliferone, respectively, whereas herniarin was 1.89-folds less soluble than umbelliferone. Moreover, the antibacterial and anticancer activities of herniarin showed higher than umbelliferone, esculetin and skimmin.

Conclusions: This study proves that both native and engineered enzymes could be employed for the production of precious compounds via whole cell biocatalysis. We successfully produced three molecules herniarin, esculetin and skimmin in practical amounts and their antibacterial and anticancer properties were accessed. One of the newly synthesized molecules the present research suggests that the combinatorial biosynthesis of different biosynthetic enzymes could rapidly promote to a novel secondary metabolite.

Keywords: Glycosylation; Hydroxylation; Methylation; Umbelliferone.

Fig. 1

The chemical structure of the simple coumarins

Fig. 2

a In vitro reaction mixture and b whole cells bioconversion of umbelliferone on the HPLC-PDA analysis. (i) control reaction of umbelliferone using E. coli BL21 (DE3); (ii) hydroxylation with M13 and (iii) CYP450 BM3 at 48 h; (iv) glycosylation with YjiC at 12 h; and (v) methylation with SaOMT2 at 48 h, respectively

Fig. 3

The scale-up of a hydroxylated b glucosylated c methylated umbelliferone and cell growth at OD₆₀₀ nm in 3-L fermentation at different time intervals

Fig. 4

The growth of four cancer cell lines were treated with umbelliferone and its derivatives