CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production

Brady F Cress,Robert J Linhardt,Daniel C Kim,Mattheos A G Koffas,Jon Y Suzuki,Teresita D Amore,Quentin D Leitz

doi:10.1186/s12934-016-0623-3

Abstract

BackgroundAnthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-l-methionine (SAM or AdoMet).ResultsAnthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen ‘Kaori-no-mai’ AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3′-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall.ConclusionsAn E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions.

Highlights

Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue
This affords the opportunity for one-step biotransformation of the cheap and abundant precursor (+)-catechin into the unstable compound cyanidin, which can be significantly stabilized by glycosylation with UDP-glucose: 3-O-glycosyltransferase (3GT) to form cyanidin 3-O-glucoside
CRISPRi‐mediated deregulation of methionine and S‐adenosyl methionine biosynthesis improves peonidin 3-O-glucoside (P3G) titers While we have previously shown that endogenous pools of UDP-glucose are limiting for production of the anthocyanins cyanidin 3-O-glucoside (C3G) and pelargonidin 3-O-glucoside from their corresponding aglycones in E. coli [4], we sought in this work to explore the potential limitation of SAM availability for O-methylation of anthocyanins

Summary

Introduction

Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Complicated syntheses suffer from low yield due to the presence of multiple stereocenters, and, offer little hope as a viable alternative to extraction [2] Despite these difficulties, the brightly colored plant pigments known as anthocyanins are intriguing candidates for continued investigation due to their antioxidant properties, reported health benefits, and high potential for use as natural food and beverage colorants [3] since they already pervade most diets. That Escherichia coli expressing ANS are able to convert the flavan-3-ols afzelechin and catechin into the anthocyanidins pelargonidin and cyanidin, respectively [4] This affords the opportunity for one-step biotransformation of the cheap and abundant precursor (+)-catechin into the unstable compound cyanidin, which can be significantly stabilized by glycosylation with UDP-glucose: 3-O-glycosyltransferase (3GT) to form cyanidin 3-O-glucoside. Such a strategy enables extension and exploration of the heterologous microbial anthocyanin biosynthetic pathway in the genetically tractable host, E. coli

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Conclusion