A recursive pathway for isoleucine biosynthesis arises from enzyme promiscuity

Enzyme promiscuity can be the starting point for the evolution of new enzymatic activities and pathways. Previously Cotton et al. (2020) identified underground isoleucine biosynthesis routes that can replace the canonical route in Escherichia coli , after they deleted the enzymes that catalyze the formation of its precursor 2-ketobutyrate. Using this strain and short-term evolution we identify a new pathway for isoleucine biosynthesis based on the promiscuous activity of the native enzyme acetohydroxyacid synthase II. We demonstrate that this enzyme catalyzes the previously unreported condensation of glyoxylate with pyruvate to generate 2-ketobutyrate in vivo. The gene encoding this enzyme, ilvG , is inactivated by a frameshift mutation in the laboratory model strain E. coli K-12 MG1655. Its evolutionary reactivation we report here points to a potential natural role in isoleucine biosynthesis in E. coli . Isoleucine biosynthesis proceeds with a further condensation step of 2-ketobutyrate with pyruvate, again catalyzed by AHAS, giving the proposed pathway the unusual property of recursivity. The discovered enzyme activity uses glyoxylate and pyruvate as direct central metabolic precursors for isoleucine biosynthesis instead of its canonical ‘indirect’ biosynthesis via the amino acid threonine. Unlike previously discovered underground isoleucine routes by Cotton et al., this route is more likely to play a role in natural isoleucine biosynthesis in E. coli due to the use of ubiquitous metabolites and its activity in aerobic conditions. The discovered route further expands the known metabolic space for isoleucine biosynthesis in E. coli and potentially other organisms, and could find applications in biotechnological isoleucine production.