Metabolic engineering of Escherichia coli for production of bioactive natural products via kynurenine pathway

Abstract

Kynurenine pathway is present in eukaryotes (e.g., human and yeast) and a few actinomycetes, which starts from oxidation of L-tryptophan, and can lead to the biosynthesis of valuable bioactive natural products, including kynurenic acid (dietary supplement), 3-hydroxyanthranilic acid (neuroprotective agent), 3-hydroxy-4-methylanthranilic acid (a precursor of anti-tumors) and actinocin (a precursor of anti-tumors). Due to the availability of well-established molecular tools and metabolic engineering strategies, Escherichia coli has emerged as a cell factory for the enhanced production of various natural products beyond primary metabolites with industrial values. In this study, we developed an E. coli platform strain by constructing kynurenine pathway, which is capable of producing multiple bioactive natural products; here, the E. coli-based production of kynurenic acid, 3-hydroxyanthranilic acid, 3-hydroxy-4-methylanthranilic acid and actinocin are demonstrated. Kynurenine pathway was first constructed in E. coli by exploring relevant biosynthetic genes from multiple species. Metabolic engineering strategies were subsequently implemented to increase the native L-tryptophan pool. Furthermore, the inhibition of a competitive pathway as well as the enhancement of two cofactors, S-adenosyl-L-methionine and pyridoxal 5′-phosphate (PLP), were performed to optimize the reaction fluxes. By expressing additional product-specific biosynthetic genes, the final engineered E. coli strains could produce, for the first time, kynurenic acid, 3-hydroxyanthranilic acid, 3-hydroxy-4-methylanthranilic acid and actinocin accordingly in a minimal medium with glucose as sole carbon source. This study demonstrates that E. coli and the metabolic engineering strategies implemented herein can serve as a robust heterologous production platform for various bioactive natural products.