Metabolic Engineering using Computer Simulation Predictions and Protein Expression Studies towards Anthocyanin Production in Escherichia coli

Anthocyanin is a water-soluble plant pigment in the flavonoid family that has recently drawn significant interest for its potential health benefits. This high-valued compound can be produced in Escherichia coli by heterologous expression of the plant proteins anthocyanidin synthase (ANS) and UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT), when supplemented with the substrate (+)-catechin. Metabolic engineering strategies are implemented to improve anthocyanin production in E. coli.;Two computer models were used to predict metabolic interventions (gene overexpressions and knockouts) that may direct carbon flux away from non-essential pathways and towards anthocyanin production. The cipher for evolutionary design (CiED) model predicted gene knockouts that primarily aimed at increased bioavailability of 2-oxoglutarate, a substrate of ANS. The OptForce model predicted overexpressions and knockouts that primarily aimed to improve the bioavailability of UDP-glucose, used by 3GT to glycosylate the anthocyanin. Expression studies were performed to investigate ANS and 3GT expression in E. coli.;Anthocyanin production fermentations using the single-deletion strains predicted by the two computer models did not significantly improve anthocyanin production when compared to the control strain. Expression studies were performed using SDS-PAGE and western blot analysis. When fermentation samples were separated into soluble and insoluble protein fractions, 3GT was found to be mostly insoluble. The insolubility leads to incorrect protein folding, which likely leads to reduction or loss of activity. The low levels of soluble protein expression may explain the fact that increased precursor bioavailability did not improve anthocyanin production yields.