Comparative Transcriptomic Profiling of High-Yielding Saccharopolyspora pogona Mutants Generated by Consecutive N-methyl-N-nitro-N-nitrosoguanidine Mutagenesis

Butenyl-spinosyn is a highly effective macrolide insecticide produced by Saccharopolyspora pogona, but its low yield remains a major bottleneck for large-scale industrial application. In this study, a strategy based on consecutive N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagenesis was employed, leading to the isolation of a genetically stable high-yielding mutant strain, ASAGF9-86, which produced 134.8 mg/L of butenyl-spinosyn-a 2.25-fold increase compared to the parental strains. Comparative transcriptomic analysis revealed extensive transcriptional reprogramming potentially associated with the high-yielding phenotype. The butenyl-spinosyn biosynthetic gene cluster was strongly upregulated in ASAGF9-86 at the early fermentation stage, indicating enhanced biosynthetic activity. In addition, genes encoding hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and 6-phosphogluconate dehydratase were markedly upregulated in the glycolysis, oxidative pentose phosphate, and Entner-Doudoroff pathways, which may contribute to improved carbon utilization efficiency and NADPH generation. Concurrently, reduced expression of fatty acid biosynthetic genes and increased transcription of fatty acid degradation genes may favor malonyl-CoA availability, the main precursor for polyketide assembly. Notably, the branched-chain amino acid degradation pathway-particularly the valine and isoleucine branches-was significantly upregulated, potentially enhancing the supply of methylmalonyl-CoA and propionyl-CoA, key precursors for butenyl-spinosyn biosynthesis. These findings demonstrated the effectiveness of NTG mutagenesis in strain development and provide insights into transcriptional regulation that may underlie enhanced precursor availability and biosynthetic capacity. This work offers a theoretical framework for the rational engineering of S. pogona and contributes to the development of high-yielding actinomycete strains for antibiotic production.