RESUMEN
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.
RESUMEN
Sugar transporters have significant contributions to regulate metabolic flux towards products and they are general potential targets for engineering of high-yield microbial cell factories. Streptomyces, well-known producers of natural product pharmaceuticals, contain an abundance of sugar transporters, while few of them are well characterized and applied. Here, we report a previously unidentified ATP-binding cassette (ABC) sugar transporter TP6568 found within a Streptomyces avermitilis transposon library, along with its key regulator GM006564. Subsequent in silico molecular docking and genetic experiments demonstrated that TP6568 possessed a broad substrate specificity. It could not only promote uptake of diverse monosaccharides and disaccharides, but also enhance the utilization of industrial carbon sources such as starch, sucrose, and dextrin. Constitutive overexpression of TP6568 resulted in decrease of residual total sugar by 36.16%, 39.04%, 38.40%, and 30.21% in engineered S. avermitilis S0, Streptomyces caniferus NEAU6, Streptomyces bingchenggensis BC-101-4, and Streptomyces roseosporus NRRL 11379 than their individual parent strain, respectively. Production of avermectin B1a, guvermectin, and milbemycin A3/A4 increased by 75.61%, 56.89%, and 41.13%, respectively. We then overexpressed TP6568 in combination with the regulator GM006564 in a high-yield strain S. avermitilis S45, and further fine-tuning of their overexpression levels boosted production of avermectin B1a by 50.97% to 7.02 g/L in the engineering strain. Our work demonstrates that TP6568 as a promising sugar transporter may have broad applications in construction of high-yield Streptomyces microbial cell factories for desirable natural product pharmaceuticals. KEY POINTS: ⢠TP6568 from Streptomyces avermitilis was identified as a sugar transporter ⢠TP6568 enhanced utilization of diverse industrially used sugars in Streptomyces ⢠TP6568 is a useful transporter to construct high-yield Streptomyces cell factories.
