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

BackgroundMulti-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 P-T7, P-trc, and P-lacUV5 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 P-T7. Finally, the production of C3G was checked and confirmed using the modified M9 media, and analyzed through various chromatography and spectrometric analyses.ResultsThe engineered strains endowedwith newly generated vectors andthe genes for C3G biosynthesis and UDP-d-glucose synthesis were fed with 2mM (+)-catechin and d-glucose for the production of cyanidin, and itssubsequent conversion to C3G. One of the engineered strains harboring At3GT and PhANS under P-trc promoter and UDP-d-glucose biosynthesis genes under P-T7 promoter led to the production of 439mg/L of C3G within 36h 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.ConclusionA 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.