Production of anthocyanins in metabolically engineered microorganisms: Current status and perspectives

Abstract

Microbial production of plant-derived natural products by engineered microorganisms has achieved great success thanks to large extend to metabolic engineering and synthetic biology. Anthocyanins, the water-soluble colored pigments found in terrestrial plants that are responsible for the red, blue and purple coloration of many flowers and fruits, are extensively used in food and cosmetics industry; however, their current supply heavily relies on complex extraction from plant-based materials. A promising alternative is their sustainable production in metabolically engineered microbes. Here, we review the recent progress on anthocyanin biosynthesis in engineered bacteria, with a special focus on the systematic engineering modifications such as selection and engineering of biosynthetic enzymes, engineering of transportation, regulation of UDP-glucose supply, as well as process optimization. These promising engineering strategies will facilitate successful microbial production of anthocyanins in industry in the near future.

Keywords: 4CL, 4-coumaroyl-CoA ligase; ANS, anthocyanidin synthase; Anthocyanin; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; DSSC, dye-sensitized solar cell; Enzyme engineering; F3GT, flavonoid 3-O-glucosyltransferase; F3H, flavanone 3-hydroxylase; F3′5′H, flavonoid 3′, 5′-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FGT, flavonoid glucosyltransferase; Metabolic engineering; Microbial production; UV, ultraviolet.

Fig. 1

The structure of anthocyanins. Modifications (such as glycosylation, hydroxylation, methylation, and acylation) at C3’ (R1), C4’ (R2), C5’ (R3), C3 (R4), C5 (R5) and C7 (R6) generate structural analogs. R1-R5 are functional groups derived from glycosyl, hydroxyl, methyl, and acyl units. Representative anthocyanins and their structures are listed.

Fig. 2

The pathway of anthocyanin biosynthesis in plants. The general precursor phenylalanine or tyrosine derived from the shikimate pathway is converted to 4-coumaroyl-CoA through the phenylpropanoid pathway or the combined effect of TAL and 4CL, respectively. One molecule of 4-coumaroyl-CoA is condensed with three molecules of malonyl-CoA to form one molecule of naringenin chalcone, which is subsequently converted to naringenin by CHI. Naringenin, the major intermediate compound, undergoes various hydroxylation to form diverse anthocyanidins. Further glycosylation and other decorations act on the anthocyanidin compounds to generate anthocyanins. Abbreviations: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CPR, cytochrome P450 reductase; TAL, tyrosine ammonia lyase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′, 5′-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; FGT, flavonoid glucosyltransferase; OMT, O-methyltransferase; ACT, anthocyanin acyltransferase.

Fig. 3

The strategies applied in metabolic engineering of E. coli for the biosynthesis of anthocyanins. The modifications of the anthocyanin-producing strains focus on the enzymes in the metabolic pathways, the transport of substrates and products, and the supply of cosubstrate UDP-glucose. The current production process is based on dividing the whole biocatalysis into two stages, i.e., cell growth and enzyme production at normal pH in the first stage, and anthocyanin production and accumulation at a lower pH in the second stage.