Biosynthesis of bioactive tamarixetin in recombinant Escherichia coli.

Tamarixetin, a monomethylated derivative of quercetin, has been reported to possess many important biological activities. In the present study, a whole cell biotransformation system was used for regiospecific methylation of quercetin to produce 4'-O-methylated quercetin (tamarixetin) using methyltransferase from Streptomyces sp. KCTC 0041BP in Escherichia coli Bl21 (DE3). Its production was enhanced by adding a plasmid containing S-adenosine-l-methionine (SAM) synthase from E. coli K12 (MetK) with subsequent feeding of l-methionine and glycerol in the culture. The best condition produced ∼279 µM (88.2 mg/L) of tamarixetin. The biological activity of tamarixetin was tested and compared with quercetin, 7-O-methylated quercetin, and 3-O-methylated quercetin. Results showed that the growth of all tested cancer cell lines (AGS, B16F10, C6, and HeLa) were inhibited by tamarixetin more effectively than other methylated derivatives of quercetin or quercetin. Tamarixetin also exhibited the best antimelanogenic activity among all compounds tested.

Combinatorial approach for improved cyanidin 3-O-glucoside production in Escherichia coli.

BACKGROUND: Multi-monocistronic and multi-variate vectors were designed, built, and tested for the improved production of cyanidin 3-O-glucoside (C3G) in Escherichia coli BL21 (DE3). The synthetic bio-parts were designed in such a way that multiple genes can be assembled using the bio-brick system, and expressed under different promoters in a single vector. The vectors harbor compatible cloning sites, so that the genes can be shuffled from one vector to another in a single step, and assembled into a single vector. The two required genes: anthocyanidin synthase (PhANS) from Petunia hybrida, and cyanidin 3-O-glucosyltransferase (At3GT) from Arabidopsis thaliana, were individually cloned under PT7, Ptrc, and PlacUV5 promoters. Both PhANS and At3GT were shuffled back and forth, so as to generate a combinatorial system for C3G production. The constructed systems were further coupled with the genes for UDP-D-glucose synthesis, all cloned in a multi-monocistronic fashion under PT7. Finally, the production of C3G was checked and confirmed using the modified M9 media, and analyzed through various chromatography and spectrometric analyses. RESULTS: The engineered strains endowed with newly generated vectors and the genes for C3G biosynthesis and UDP-D-glucose synthesis were fed with 2 mM (+)-catechin and D-glucose for the production of cyanidin, and its subsequent conversion to C3G. One of the engineered strains harboring At3GT and PhANS under Ptrc promoter and UDP-D-glucose biosynthesis genes under PT7 promoter led to the production of ~ 439 mg/L of C3G within 36 h of incubation, when the system was exogenously fed with 5% (w/v) D-glucose. This system did not require exogenous supplementation of UDP-D-glucose. CONCLUSION: A synthetic vector system using different promoters has been developed and used for the synthesis of C3G in E. coli BL21 (DE3) by directing the metabolic flux towards the UDP-D-glucose. This system has the potential of generating better strains for the synthesis of valuable natural products.

Enzymatic synthesis of novel quercetin sialyllactoside derivatives.

Quercetin and its derivatives are important flavonols that show diverse biological activity, such as antioxidant, anticarcinogenic, anti-inflammatory, and antiviral activities. Adding different substituents to quercetin may change the biochemical activity and bioavailability of molecules, when compared to the aglycone. Here, we have synthesised two novel derivatives of quercetin, quercetin-3-O-ß-d-glucopyranosyl, 4''-O-d-galactopyranosyl 3'''-O-α-N-acetyl neuraminic acid i.e. 3'-sialyllactosyl quercetin (3'SL-Q) and quercetin-3-O-ß-d-glucopyranosyl, 4''-O-ß-d-galactopyranosyl 6'''-O-α-N-acetyl neuraminic acid i.e. 6'-sialyllactosyl quercetin (6'SL-Q) with the use of glycosyltransferases and sialyltransferases enzymes. These derivatives of quercetin were characterised by high-resolution quadrupole-time-of-flight electrospray ionisation mass spectrometry (HR-QTOF-ESI/MS) and 1H and 13C nuclear magnetic resonance (NMR) analyses.

One-Pot Multienzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production.

A one-pot multienzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-d-galactose, which was combined with LgtB, a ß-(1,4) galactosyltransferase from Neisseria meningitidis, to modify various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole of UDP-α-d-galactose by consuming two moles of acetylphosphate and one mole of d-galactose in each cycle. The ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The engineered cofactors recycling system with LgtB efficiently added a d-galactose unit to a variety of sugar units such as d-glucose, rutinose, and 2-deoxy-d-glucose. The temperature, pH, incubation time, and divalent metal ions for the OPME-CR system were optimized. The maximum number of UDP-α-d-galactose regeneration cycles (RCmax) was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides efficiently and cost-effectively.

Characterization of regioselective flavonoid O-methyltransferase from the Streptomyces sp. KCTC 0041BP.

A flavonoid comprises polyphenol compounds with pronounced antiviral, antioxidant, anticarcinogenic, and anti-inflammatory effects. The flavonoid modification by methylation provides a greater stability and improved pharmacokinetic properties. The methyltransferase from plants or microorganisms is responsible for such substrate modifications in a regiospecific or a promiscuous manner. GerMIII, originally characterized as a putative methyltransferase in a dihydrochalcomycin biosynthetic gene cluster of the Streptomyces sp. KCTC 0041BP, was tested for the methylation of the substrates of diverse chemical structures. Among the various tested substrates, flavonoids emerged as the favored substrates for methylation. Further, among the flavonoids, quercetin is the most favorable substrate, followed by luteolin, myricetin, quercetin 3-O-ß-D-glucoside, and fisetin, while only a single product was formed in each case. The products were confirmed by HPLC and mass-spectrometry analyses. A detailed NMR spectrometric analysis of the methylated quercetin and luteolin derivatives confirmed the regiospecific methylation at the 4'-OH position. Modeling and molecular docking provided further insight regarding the most favorable mechanism and substrate architecture for the enzymatic catalysis. Accordingly, a double bond between the C2 and the C3 and a single-ring-appended conjugate-hydroxyl group are crucial for the favorable enzymatic conversions of the GerMIII catalysis. Thus, in this study, the enzymatic properties of GerMIII and a mechanistic overview of the regiospecific modification that was implemented for the acceptance of quercetin as the most favorable substrate are presented.