Enhanced spinosad production in Saccharopolyspora spinosa by employing mannose as an extracellular carbon reservoir and optimizing acetyl-CoA supply pathway

Spinosad, a potent broad-spectrum insecticidal polyketide produced by <i>Saccharopolyspora spinosa</i>, faces limitations in industrial-scale production due to inherent inefficiencies in its native biosynthetic pathways. To overcome this constraint, we devised and implemented a systematic, stepwise combinatorial strategy integrating classical strain improvement, fermentation optimization, and rational genetic engineering to augment spinosad titres. The approach commenced with iterative UV mutagenesis, yielding a stable high-producing mutant, U7, which demonstrated a significant elevation in spinosad production compared to the original strain. Subsequent fermentation optimization via single-factor shake-flask experiments identified mannose as a superior extracellular carbon source over glucose, markedly enhancing mutant U7 spinosad titres. To further augment metabolic efficiency, we employed rational genetic engineering, demonstrating that deletion of <i>manB</i> (beta-mannosidase) and <i>leuA</i> (2-isopropylmalate synthase) coupled with overexpression of <i>pdhC</i> (pyruvate dehydrogenase subunit) synergistically boosted spinosad biosynthesis. By integrating these modifications into mutant U7, we achieved a final spinosad titer of 537.6 mg/L-a 6.1-fold increase over the wild-type strain. This study presents a combinatorial metabolic engineering approach that not only significantly improves spinosad production but also provides a generalizable framework for optimizing polyketide biosynthesis in <i>Streptomyces</i> and related actinomycetes. Critically, the identification of mannose as the preferred carbon source not only directly enhanced precursor supply but also proved pivotal in redirecting metabolic flux through the glycolytic pathway, thereby generating elevated levels of acetyl-CoA. The acetyl-CoA pathway engineering strategy developed here can be readily adapted to enhance the production of other high-value polyketides.