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

Abstract

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.

Keywords: Anthocyanin; Cyanidin 3-O-glucoside; Multi-monocistronic; UDP-D-glucose.

Fig. 1

Engineered pathway for cyanidin-3-O-glucoside (C3G) biosynthesis from (+)-catechin using anthocyanidin synthase (PhANS) and 3-O-glycosyltransferase (At3GT) in E. coli. Also shown in the overexpression of the sugar biosynthesis genes

Fig. 2

Functionality assay of newly constructed piBRTrc and piBRUV5 vectors (a) SDS-PAGE analysis showing the expression of apr^r in piBRTrc-apr^r, piBRUV5-apr^r, piBR181-apr^r, and control (C) with empty pET28a (+) vector. b Visualization of gfp expression in piBR181-gfp, piBRTrc-gfp, piBRUV5-gfp harboring E. coli BL21 (DE3), and control (C) under blue light emission

Fig. 3

Spectroscopic analysis showing biosynthesis of cyanidin-3-O-glucoside (C3G) (a) UHPLC-PDA chromatogram, and b UV/VIS absorbance of (i) (+)- catechin, (ii) cyanidin and C3G, and (iii) C3G standard. c HR-QTOF-ESI/MS analysis confirming the production of C3G, along with the production of intermediate compound cyanidin (i) (+)-catechin, (ii) cyanidin, and (iii) C3G

Fig. 4

Isolation, extraction, and analysis of both extracellular and intracellular cyanidin-3-O-glucoside (C3G) in E. coli culture in modified M9 minimal medium after 36 h of incubation. a UHPLC-PDA profile of C3G. b Extraction of intracellular C3G by sonication, followed by centrifugation. The vials 1, 2, 3, 4, 5, and 6 show supernatant after each extraction. c The remaining cell pellets of different C3G producing strains after final extraction

Fig. 5

Cyanidin-3-O-glucoside (C3G) production comparison of twelve different recombinants showing the combinations of the genes, along with their respective promoters, in monocistronic fashion. Both the extracellular, intracellular and the total C3G titer of each strain has been shown. Error bars represent standard deviations. (NS) indicates not significant difference p > 0.05, and (*) denotes significant difference in C3G yield between the compared strain with p < 0.05

Fig. 6

Time-dependent production profile of cyanidin-3-O-glucoside (C3G) using the recombinant strain S12 in 48 h cultivation time showing both extracellular, intracellular and the total C3G yield. The sample was taken at a 12 h time interval, and the recombinants were cultured in modified M9 minimal media. Error bars represent standard deviations

Fig. 7

Effect of different concentration of glucose of (1, 2, 3, 4, 5, 10, and 15)% on cyanidin-3-O-glucoside (C3G) production using the recombinant strain S₁₂ after 36 h of cultivation showing both extracellular, intracellular and the total C3G yield. Maximum production of C3G was achieved while 5% glucose was supplemented in the medium. Error bars represent standard deviations