Refining genetic circuit design for enhanced type II polyketide biosynthesis in Escherichia coli

Polyketides represent a broad class of structurally diverse natural products with potent antibacterial and anticancer activities. Type II polyketide synthases (T2PKS) are multi-subunit complexes responsible for condensing acylated intermediates, including acetyl-CoA and malonyl-CoA, into complex fused-ring aromatic natural products. While type II polyketides have traditionally been associated with gram-positive actinomycetes, more recent discoveries have identified PKS systems in gram-negative bacteria. These findings have provided new genetic tools and opened new pathways for producing polyketides in the versatile chassis organism Escherichia coli . In this study, we altered the genetic circuit of the Photorhabdus luminescens PKS for octaketide production in E. coli . Promoter engineering and ribosome-binding-site optimization of the antDEFBG minimal PKS (mPKS) in M9 minimal media resulted in SEK4 and SEK4b production titers of 36 mg/L and 78 mg/L in strain E. coli sAJM.1506/pANT7, respectively. Further refinement of downstream ketoreductase, aromatase, and cyclase expression enabled the biosynthesis of native polyketide AQ-256 and naphthopyrone ( S )-DNPA reaching a highest measured titer of 141.3 ± 9.2 mg/L after 24 h of shake-flask cultivation. Overexpression of the P. luminescens malonyl-CoA acyl carrier protein transacylase and acetyl-CoA carboxylase complex further improved production by 50%. This work further illustrates the potential of E. coli as a versatile host for the biosynthesis of complex polyketides, paving the way for the scalable production of valuable natural products. By optimizing genetic circuits and pathway expression, this study provides empirical design guidance for engineering microbial systems.