A non-carboxylative route for the efficient synthesis of central metabolite malonyl-CoA and its derived products

Abstract

While acetyl-CoA carboxylation is the canonical route for endogenous malonyl-CoA formation found in nature, this pathway suffers from slow kinetics, carbon and energy inefficiencies, tight regulations, complicated architecture and severe cellular toxicity, which limit flux towards malonyl-CoA and become a decisive bottleneck limiting the biosynthesis of all malonyl-CoA-derived products (MDPs). In this study, we built the first non-natural route for malonyl-CoA biosynthesis. The designed non-carboxylative malonyl-CoA (NCM) pathway adopts an “acetyl-CoA-independent” mode, thus avoiding carbon loss, ATP consumption and problematic cross-talk with native metabolic networks. The feasibility of this design was demonstrated by mining and recruiting β-alanine-pyruvate transaminase and malonyl-CoA reductase. The NCM catalysis displays a fast kinetics, with the rate being 103-fold higher than that of natural pathway, representing the fastest malonyl-CoA formation rate to the best of our knowledge. Moreover, the NCM catalysis circumvents both multiple tight regulations and extremely complicated architecture associated with natural pathway, with the mass of comprising enzymes reducing by 93%~97%. In addition, the NCM pathway can substitute natural pathway for malonyl-CoA formation within cells, and does not lead to severe cellular toxicity, but rather improved cell’s robustness to a broad range of challenges. Furthermore, introduction of this pathway into multiple microbes including Escherichia coli, Streptomyces gilvosporeus and Saccharopolyspora spinosa greatly improved production of a variety of highly valuable MDPs including short-chain fatty acid and representative phenol, quinone, alkene, aminoglycoside and macrolide polyketide families, with the production of spinosad in S. spinosa reaching an unprecedented level. In summary, this new-to-nature malonyl-CoA formation pathway circumvents nearly all intrinsic inefficiencies of natural pathway, and can serve as a novel and versatile platform for targeting a repertoire of MDPs with applications as fuels, fine chemicals and pharmaceuticals.