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Background  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. 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).    Results  Anthocyanin  O -methyltransferase (AOMT) orthologs from various plant sources were co-expressed in  Escherichia coli  with  Petunia hybrida  anthocyanidin synthase ( Ph ANS) and  Arabidopsis thaliana  anthocyanidin 3- O -glucosyltransferase ( At 3GT).  Vitis vinifera  AOMT ( Vv AOMT1) and fragrant cyclamen ‘Kaori-no-mai’ AOMT ( Ckm OMT2) 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  Vv AOMT1 and  Ckm OMT2 expression conditions, production was further improved to 23 mg/L using  Vv AOMT1. 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.    Conclusions  An  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.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-016-0623-3) contains supplementary material, which is available to authorized users.

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