Ribosome Engineering for Enhanced Butenyl-Spinosyn Production in Saccharopolyspora pogona.

Ribosome engineering, a strategy that utilizes antibiotic resistance mutations to modulate ribosomal function, has emerged as a powerful approach for enhancing microbial metabolite production. In this study, ribosome engineering was applied to Saccharopolyspora pogona ASAGF2-G4 under streptomycin selection to improve butenyl-spinosyn production. Screening for streptomycin-resistant mutants at concentrations ranging from 2 to 20 µg/mL resulted in the isolation of 58 mutants, of which 27.6% exhibited increased butenyl-spinosyn production. Among these, 22 mutants harbored six distinct mutations in the rpsL gene, resulting in five amino acid substitutions in the ribosomal protein S12: Lys43 to Arg, Lys43 to Thr, Lys43 to Asn, Lys88 to Glu, and Lys88 to Arg. The highest frequency of rpsL mutant isolation was observed at a streptomycin concentration of 15 µg/mL. Phenotypic characterization revealed altered growth dynamics, pH shifts, and glucose utilization among the mutants, with the K88R and K43R variants exhibiting significantly increased butenyl-spinosyn production-1.78-fold and 1.64-fold higher than that of the parental strain, respectively. Quantitative PCR analysis showed significant upregulation of translation-related genes (rpsL and frr), growth-related genes (whiA and bldD), and key butenyl-spinosyn biosynthetic genes (busA, busF, and busI) in the K88R mutant, suggesting that the K88R substitution enhances target compound biosynthesis by modulating ribosomal function and associated metabolic networks. Future research should explore combinatorial approaches, including the development of multi-antibiotic-resistant mutants and elevated expression of ribosomal genes, to maximize butenyl-spinosyn yields. This study underscores the potential of ribosome engineering as a platform for improving butenyl-spinosyn production and provides a foundation for subsequent industrial-scale applications.