Heterologous production of epothilones in Streptomyces albus J1074 via promoter engineering of the biosynthetic gene cluster and precursor supply enhancement

Epothilones, synthesized by hybrid polyketide synthases (PKS)-nonribosomal peptide synthetases (NRPS), are clinically valuable anti-cancer macrolides with potent activity against multidrug-resistant tumors. They are naturally produced by the myxobacterium <i>Sorangium cellulosum</i>. However, their yield in the native host is extremely low. Additionally, the slow growth rate and poor genetic tractability of <i>S. cellulosum</i> have severely hindered efforts to optimize epothilone biosynthesis in this host. Herein, we established a heterologous epothilone production system in <i>Streptomyces albus</i> J1074, a widely employed chassis for secondary metabolite biosynthesis, via two core strategies: (1) refactoring the epothilone PKS/NRPS gene cluster with endogenous strong constitutive promoters to enhance transcription; (2) boosting supply of the acetyl-CoA starter unit and the malonyl-CoA extender unit. When optimizing promoters for epothilone PKS/NRPS genes, we identified a key challenge: EpoD, the largest PKS component, is toxic to <i>Escherichia coli</i> cloning hosts due to induced stress responses and metabolic disruption. To address this, we screened strong constitutive promoters from <i>Streptomyces</i> with low activity in <i>E. coli</i> to optimize the <i>epoD</i> promoter. Supplementation of sodium acetate to the medium increased epothilone production by 4.1-fold, and subsequent overexpression of enzymes catalyzing acetate-to-acetyl-CoA and acetyl-CoA-to-malonyl-CoA conversions boosted yields by an additional 3.3-fold. Ultimately, a maximum epothilone titer of 439.8 μg L^-1 was achieved in <i>S. albus</i> J1074 cultured in 250-mL flasks. To our knowledge, this represents the highest epothilone yield reported in <i>Streptomyces</i> hosts to date. This work provides an efficient heterologous production system for epothilones and insights into optimizing PKS/NRPS-derived natural product biosynthesis.