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 Sorangium cellulosum. However, their yield in the native host is extremely low. Additionally, the slow growth rate and poor genetic tractability of S. cellulosum have severely hindered efforts to optimize epothilone biosynthesis in this host. Herein, we established a heterologous epothilone production system in Streptomyces albus 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 Escherichia coli cloning hosts due to induced stress responses and metabolic disruption. To address this, we screened strong constitutive promoters from Streptomyces with low activity in E. coli to optimize the epoD 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 S. albus J1074 cultured in 250-mL flasks. To our knowledge, this represents the highest epothilone yield reported in Streptomyces hosts to date. This work provides an efficient heterologous production system for epothilones and insights into optimizing PKS/NRPS-derived natural product biosynthesis.