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 at two key fermentation stages revealed extensive transcriptional reprogramming associated with the high-yielding phenotype. The butenyl-spinosyn biosynthetic gene cluster exhibited strong and early transcriptional activation in ASAGF9-86, 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, suggesting altered central carbon metabolism and increased NADPH-generating potential. Concurrently, transcriptional changes were also detected in the fatty acid metabolism and branched-chain amino acid degradation pathways, which may be associated with precursor supply for polyketide biosynthesis. These findings demonstrate the effectiveness of NTG mutagenesis in strain development and provide transcriptomic insights that may inform future rational engineering of S. pogona and related actinomycetes.