| Summary: | Bafilomycin A<sub>1</sub> is the representative compound of the plecomacrolide natural product family. This 16-membered ring plecomacrolide has potent antifungal and vacuolar H<sup>+</sup>-ATPase inhibitory activities. In our previous work, we identified a bafilomycin biosynthetic gene cluster (<i>baf</i>) from the marine bacterium <i>Streptomyces lohii</i> ATCC BAA-1276, wherein a <i>luxR</i> family regulatory gene <i>orf1</i> and an <i>afsR</i> family regulatory gene <i>bafG</i> were revealed based on bioinformatics analysis. In this study, the positive regulatory roles of <i>orf1</i> and <i>bafG</i> for bafilomycin biosynthesis are characterized through gene inactivation and overexpression. Compared to the wild-type <i>S. lohii</i> strain, the knockout of either <i>orf1</i> or <i>bafG</i> completely abolished the production of bafilomycins. The overexpression of <i>orf1</i> or <i>bafG</i> led to 1.3- and 0.5-fold increased production of bafilomycins, respectively. A genetically engineered <i>S. lohii</i> strain (SLO-08) with <i>orf1</i> overexpression and inactivation of the biosynthetic genes <i>orf2</i> and <i>orf3</i>, solely produced bafilomycin A<sub>1</sub> with the titer of 535.1 ± 25.0 mg/L in an optimized fermentation medium in shaking flasks. This recombinant strain holds considerable application potential in large-scale production of bafilomycin A<sub>1</sub> for new drug development.
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