Coupling fatty acid biosynthesis to β-oxidation enhances the biosynthesis of malonyl-CoA-dependent polyketides.

Malonyl-CoA is a major extender unit in polyketide biosynthesis; however, in Escherichia coli, it is predominantly consumed by fatty acid biosynthesis, limiting its availability for heterologous polyketide production. Here, we engineered a fatty acid biosynthesis-β-oxidation carbon-recycling pathway (FABOX pathway) that redirects fatty acid biosynthetic flux toward free fatty acid formation while reinforcing β-oxidation, thereby recycling carbon through acetyl-CoA to support malonyl-CoA-dependent polyketide biosynthesis. Application of this strategy to three malonyl-CoA-dependent polyketides, 1,3,8-trihydroxyanthraquinone (AQ256), phloroglucinol, and flaviolin, increased AQ256 and phloroglucinol production, whereas no clear improvement was observed for flaviolin. 13C-malonate tracing was consistent with β-oxidation-associated recycling of malonate-derived carbon into acetyl-CoA and extensive incorporation of malonate-derived carbon into AQ256. Here, we show that, although its effectiveness depends on the product and cultivation conditions, the FABOX pathway provides a mechanistically distinct flux-reconfiguration strategy for enhancing malonyl-CoA-dependent polyketide biosynthesis, complementing conventional precursor-supply-based approaches.