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 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. 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 P(trc) promoter and UDP-d-glucose biosynthesis genes under P(T7) 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. [Image: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-019-1056-6) contains supplementary material, which is available to authorized users.