Engineering Central Metabolic Pathways for High-Level Flavonoid Production in Escherichia coli

Abstract

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...

OPINIONS Acetyl-coenzyme A carboxylase – an attractive enzyme for biotechnology

“…In this study (2S)-naringenin and (2S)-pinocembrin were synthesized in E. coli by employing an artificial biosynthetic cluster of genes that included phenylalanine ammonia lyase from yeast, cinnamate/coumarate:CoA ligase from the actinomycete Streptomyces coelicolor, chalcone synthase from a licorice plant, and chalcone isomerase from a Pueraria plant. A similar effect, related to an increased availability of malonyl-CoA in the cell, was observed during the production of five different plant flavonones accompanied by expression of Photorhabdus luminescens ACCase gene (Leonard et al, 2007). In this case, the increased ACCase activity present in E. coli cells was additionally supported by overexpression of bacterial gene coding for Acs.…”

Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli

“…4 Converting renewable resources into monoterpene products by engineered microorganisms was interesting technology and developed quickly recently year, which have the advantages of fast growth, no need for land during their growth and sustainable development. 5,6 Geraniol is likely to be synthesized from geranyl diphosphate (GPP), the universal precursor of all monoterpenes, which can be produced from both the methylerythritol 4-phosphate pathway and the mevalonate (MVA) pathway in plants. 7,8 Although many microorganisms carry out the methylerythritol 4-phosphate pathway or MVA pathway to supply the intermediates dimethylallyl pyrophosphate and isopentenyl pyrophosphate, they are unable to produce the monoterpenes because of the absence of monoterpene synthase.…”

Heterologous Production of Curcuminoids

“…It is believed that the biosynthetic production of flavonoids, stilbenoids, and polyketides is controlled by the limited intracellular pool of malonyl-CoA (116,137,188). In these pathways, three molecules of malonyl-CoA are needed to obtain the final product, whereas for curcuminoid production, only one molecule is needed.…”

“…In the last decade, several metabolic engineering approaches were developed to increase the intracellular pool of malonyl-CoA. One of them is the overexpression of acetyl-CoA carboxylase (ACC), which converts acetyl-CoA into malonyl-CoA (106,116,188,(190)(191)(192) (Fig. 6).…”