Plant flavonoid polyphenols continue to find increasing pharmaceutical and nutraceutical applications; however their isolation, especially of pure compounds, from plant material remains an underlying challenge. In the past Escherichia coli, one of the most well-characterized microorganisms, has been utilized as a recombinant host for protein expression and heterologous biosynthesis of small molecules. However, in many cases the expressed protein activities and biosynthetic efficiency are greatly limited by the host cellular properties, such as precursor and cofactor availability and protein or product tolerance. In the present work, we developed E. coli strains capable of high-level flavonoid synthesis through traditional metabolic engineering techniques. In addition to grafting the plant biosynthetic pathways, the methods included engineering of an alternative carbon assimilation pathway and the inhibition of competitive reaction pathways in order to increase intracellular flavonoid backbone precursors and cofactors. With this strategy, we report the production of plant-specific flavanones up to 700 mg/L and anthocyanins up to 113 mg/L from phenylpropanoic acid and flavan-3-ol precursors, respectively. These results demonstrated the efficient and scalable production of plant flavonoids from E. coli for pharmaceutical and nutraceutical applications.

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirA Pl ) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirA Ec ) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirA Ec and the C terminus of BirA Pl . In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.Many plant-derived natural products are important sources for the discovery of new drugs and nutraceuticals (3, 12). However, drug development from natural compounds faces important challenges that discourage the pursuit of such bioactive materials (33). One major limitation is the low concentration of these metabolites in the host organisms, which causes limited supply for rapid research, development, and clinical trials (6). Synthetic or semisynthetic routes are often employed to remedy this problem (30). However, the complicated structure of many natural molecules often results in lengthy chemical procedures that are impractical for large-scale production. Another approach to drug discovery relies on combinatorial chemistry (5). Even though this method can generate large libraries of compounds with common core structures, only a small fraction of bioactive molecules are identified through high-throughput bioassays (33). Biochemical synthesis presents a promising alternative not only for increasing the availability of important natural products but also for diversifying their chemistry. To serve this purpose, engineered microbes are often employed as living biocatalysts (24).Flavonoids are plant-derived drug candidates and nutraceuticals whose biosynthesis has recently been engineered in recombinant microorganisms (reviewed in references 11 and 24).In plants, flavonoi...