|CRESS, BRADY - Rensselaer Polytechnic Institute|
|LEITZ, QUENTIN - Rensselaer Polytechnic Institute|
|KIM, DANIEL - Rensselaer Polytechnic Institute|
|AMORE, TERESITA - University Of Hawaii|
|LINHARDT, ROBERT - Rensselaer Polytechnic Institute|
|KOFFAS, MATTHEOS - Rensselaer Polytechnic Institute|
Submitted to: Microbial Cell Factories
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2016
Publication Date: 1/17/2017
Citation: Cress, B.F., Leitz, Q.D., Kim, D.C., Amore, T.D., Suzuki, J.Y., Linhardt, R.J., Koffas, M.A. 2017. CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production. Microbial Cell Factories. 16:10. doi:10.1186/s12934-016-0623-3.
Interpretive Summary: A method to produce the plant pigment peonidin using metabolic engineering was developed. Results from this work demonstrate that natural plant compounds of pharmaceutical or commercial value can be synthesized efficiently in the laboratory using a non-plant, biological system to support basic scientific research as well as commercial applications.
Technical Abstract: Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower, fruit, and vegetable host tissues with hues of predominantly red, 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 distinct pharmacological properties. In this work, we report for the first time, the extension of the 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 methionine (SAM). Specifically, anthocyanin O-methyltransferases (AOMTs) orthologs from various plant sources were co-expressed in E. 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). Following modulation of gene copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved and found to be best using VvAOMT1. Finally, CRISPRi was utilized to repress 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 increased titer over the non-targeting CRISPRi control strain. 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 this strain will also support the ornamental plant industry as a platform for rapidly evaluating putative anthocyanin methyltransferases capable of tailoring flower pigmentation.