Zhuoxu Dong

Crosstalk regulation is widespread in Streptomyces species. Elucidating the influence of a specific regulator on target biosynthetic gene clusters (BGCs) and cell metabolism is crucial for strain improvement through regulatory protein engineering. PteF and PteR are two regulators that control the biosynthesis of filipin, which competes for building blocks with avermectins in Streptomyces avermitilis. However, little is known about the effects of PteF and PteR on avermectin biosynthesis. In this study, we investigated their impact on avermectin biosynthesis and global cell metabolism. The deletion of pteF resulted in a 55.49% avermectin titer improvement, which was 23.08% higher than that observed from pteR deletion, suggesting that PteF plays a more significant role in regulating avermectin biosynthesis, while PteF hardly influences the transcription level of genes in avermectin and other polyketide BGCs. Transcriptome data revealed that PteF exhibited a global regulatory effect. Avermectin production enhancement could be attributed to the repression of the tricarboxylic acid cycle and fatty acid biosynthetic pathway, as well as the enhancement of pathways supplying acyl-CoA precursors. These findings provide new insights into the role of PteF on avermectin biosynthesis and cell metabolism, offering important clues for designing and building efficient metabolic pathways to develop high-yield avermectin-producing strains.

Milbemycins are a group of macrolide pesticides with great potential in the agricultural field owing to their high insecticidal activity and environmental compatibility. Milbemycin A3 and A4 with high bioactivities are the main components of milbemycin-derived products, which require a component ratio A4:A3 of 2.3- to 4.0-fold. Streptomyces bingchenggensis BC04 is a promising milbemycin producer, whereas the component ratio of its products (A4:A3 of 9.0-fold) could not meet the requirement for industrial production. To address this issue, we reconstructed the precursor biosynthetic pathways to fine tune the supply of different acyl-coenzyme A precursors required for milbemycin biosynthesis. Based on an analysis of the intracellular acyl-coenzyme A precursors, we reconstructed stepwise heterogeneous biosynthetic pathways of extender units for milbemycin biosynthesis. Then, we coordinated the supply of milbemycin biosynthetic starter units with temporal promoters. Thanks to these manipulations, we obtained an engineered strain with 39.5% milbemycin titer improvement to 3417.88 mg/L and a qualified component ratio A4:A3 of 3.3-fold. This work demonstrated that coordinating the precursor supply is a simple and effective approach to optimize the component ratio of A4:A3 in milbemycin fermentation products. Moreover, this strategy might also be useful to construct high-yield strains with optimized component ratios of fermentation products in other Streptomyces.