Abstract
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 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.ResultsThe 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.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.
Highlights
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)
We developed a multivariate multi-monocistronic vector system based on a BglBricks system, and used three different promoters PT7, Ptrc, and PlacUV5, in order to clone a total of six different genes, including the genes for uridine diphosphate (UDP)-d-glucose production and C3G production, in a combinatorial approach
Design and construction of expression vector piBRTrc and piBRUV5 Two multi-monocistronic operon system were constructed for this study using the same strategy of BglBricks, in which the bio-parts can be excised all at once, with a self-designing genetic circuit
Summary
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 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. Anthocyanins, the most important subclass of flavonoids and a major part of phenolic compounds, are highly colored plant pigments that are widely distributed in fruits and vegetables [5, 6], and are the products of the phenylpropanoid metabolism pathway. The antioxidant property of anthocyanins is associated with their ability to serve as free radical scavengers, which is attributed to their catechol ring backbones, which play important roles in their bioactivity [14]
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