Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli

Abstract

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3␤-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.Among the natural pigments in plants, anthocyanins are the largest water-soluble group, found in most fruits, flower petals, and leaves. These fascinating compounds can exist in many structural forms, both simple and complex, governed by physiological regulations and chemical modifications which have profound effects on their stability and colors (13). Among the variety of biological roles, anthocyanins are utilized for the recruitment of pollinators and seed dispersers and in UV protection. Initial interest in the practical applications of brightly colored anthocyanins has stemmed from their potential as replacements for banned dyes because they have no apparent adverse effects on human health (2,5,24). Recently, however, much attention has been drawn to anthocyanin-derived plant products due to their general antioxidant properties (10, 17, 22) and a consistent association between the consumption of diets rich in fruits and vegetables and a lower risk of chronic diseases, including cancer and cardiovascular disease (6, 14). As a result, anthocyanins, currently produced industrially as mixtures from various plant extracts, are becoming attractive targets for fermentation production from well-characterized microbial hosts such as Escherichia coli.Six major classes of anthocyanidins, the aglycon forms of anthocyanins, exist: pelargonidin, cyanidin, delphinidin, p...

Consequences of a Deficit in Vitamin B6 Biosynthesis de Novo for Hormone Homeostasis and Root Development in Arabidopsis

“…The gene encoding dihydroflavonol reductase (At5g42800) was also up-regulated in pdx1.3 leaves compared with the wild type (4.52-fold). The latter is considered to be a rate-limiting step (Das et al, 2012) late in anthocyanin biosynthesis and is a target of PAP1 and PAP2 (Yan et al, 2005). On the other hand, no significant changes in the expression of GL3, TT8, or MYBL2 were observed compared with the wild type in the microarray.…”

Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase

“…In vitro experiments with recombinant LDOX suggested that the enzyme could produce not only its natural product, cyanidin, but depending on the C-4 stereochemistry of the leucocyanidin substrate, possibly also dihydroquercetin and quercetin (Turnbull et al 2003). Also, Xavonols could be observed as a side product in Escherichia coli cells simultaneously expressing recombinant F3H, DFR, LDOX and a Xavonoid 3-O-glucosyltransferase, potentially due to an alternate reaction catalysed by LDOX (Yan et al 2005). The clear reduction of Xavonol glycoside accumulation in the ldox Xs1-2 double mutant compared to the Xs1-2 single mutant (Fig.…”

“…The Wrst assumption has been shown to be correct in this work and by the work of Owens et al (2008a). The second assumption is based mainly on the work of Turnbull et al (2003Turnbull et al ( , 2004 and Yan et al (2005). In vitro experiments with recombinant LDOX suggested that the enzyme could produce not only its natural product, cyanidin, but depending on the C-4 stereochemistry of the leucocyanidin substrate, possibly also dihydroquercetin and quercetin (Turnbull et al 2003).…”

Biosynthesis of Natural Flavanones in Saccharomyces cerevisiae

“…The health-promoting effects of flavonoids have stimulated significant research toward the elucidation of their biosynthetic networks, as well as the development of production platforms using well-characterized hosts (8,19). Within this context, Hwang et al recently described recombinant Escherichia coli strains that lead to the synthesis of flavanones, the common precursors of the vast majority of flavonoids (7).…”