Tylosin is an important macrolide antibiotic widely used in veterinary medicine. Efficient heterologous biosynthesis can improve its biosynthesis efficiency and develop analogs with enhanced therapeutic activity. In this study, we successfully cloned an 87 kb tylosin biosynthetic gene cluster (BGC) from Streptomyces fradiae QL100 using ExoCET technology and achieved heterologous expression in three Streptomyces hosts via a two-step assembly strategy. Among these, S. albus J1074 was identified as the optimal host for tylosin production. Fermentation medium optimization through supplementation with 4% soybean oil and 0.8% fish meal increased tylosin yield by 47.8%. Subsequent promoter replacement of key biosynthetic genes (tylGI and tylI) further elevated the yield to 29.35 mg/L, representing a 101.0% increase over the initial level. Additionally, knockout of the regulatory gene tylQ not only increased the yield by 17.4%, but accelerated the biosynthetic onset. This work established a comprehensive strategy for the heterologous production and synthetic biology refactoring of complex natural products, encompassing large, GC-rich gene cluster capture, host selection, and pathway optimization.
Song et al. (Sun,) studied this question.