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

Microb Cell Fact. 2019 Jan 17;18(1):7. doi: 10.1186/s12934-019-1056-6.

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 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.

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

MeSH terms

  • Anthocyanins / analysis
  • Anthocyanins / biosynthesis*
  • Bioreactors
  • Catechin / metabolism
  • Chromatography, High Pressure Liquid
  • Escherichia coli / metabolism*
  • Glucose / metabolism
  • Glucosides / analysis
  • Glucosides / biosynthesis*
  • Glucosyltransferases / genetics
  • Metabolic Engineering
  • Oxygenases / genetics
  • Plant Proteins / genetics
  • Plasmids / genetics
  • Plasmids / metabolism

Substances

  • Anthocyanins
  • Glucosides
  • Plant Proteins
  • cyanidin-3-O-beta-glucopyranoside
  • cyanidin
  • Catechin
  • Oxygenases
  • anthocyanidin synthase
  • Glucosyltransferases
  • Glucose