Engineering controllable alteration of malonyl-CoA levels to enhance polyketide production and versatility in E. coli

<title>Abstract</title> Engineering the heterologous expression of polyketide synthases (PKSs) in <italic>Escherichia coli</italic> has led to the production of various valuable natural and unnatural products. However, the limited availability of malonyl-CoA (M-CoA) in <italic>E. coli</italic> remains a significant impediment to efficient polyketide overproduction. In this study, engineered <italic>E. coli</italic> strains were developed to augment M-CoA levels for enhanced polyketide production by introducing a malonate transport and CoA ligation pathway. Additionally, control over the M-CoA pool was achieved by disrupting the endogenous M-CoA pathway and reconstituting it through malonate supplementation. The engineered strains exhibited increased M-CoA, fatty acid and polyketide titers, improved substrate selectivity of PKSs, and successful production of &gt; 99% uniformly labeled ¹³C polyketides. Adaptive laboratory evolution of these strains provided valuable insights into pathways governing the M-CoA pools and the accumulation of mutations that resulted in significantly increased M-CoA and polyketide levels. This approach not only enhances the utility of <italic>E. coli</italic> as a heterologous host for polyketides but also illuminates the intricate dynamics of M-CoA modulation in these systems.