Guo Chao, Liu Yuchun, Lv Bo, Li Xinying, Wang Qingrong, Han Wei, Wang Chao
Academy of National Food and Strategic Reserves Administration, 11 Baiwanzhuang Street, Xicheng District, Beijing, 100037, China.
Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, 100081, China.
Curr Microbiol. 2025 Jun 17;82(8):337. doi: 10.1007/s00284-025-04317-8.
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.
核糖体工程是一种利用抗生素抗性突变来调节核糖体功能的策略，已成为提高微生物代谢产物产量的有力方法。在本研究中，在链霉素选择下，将核糖体工程应用于多刺糖多孢菌ASAGF2-G4以提高丁烯基多杀菌素的产量。在2至20μg/mL的浓度范围内筛选链霉素抗性突变体，分离出58个突变体，其中27.6%的突变体丁烯基多杀菌素产量增加。其中，22个突变体在rpsL基因中存在六个不同的突变，导致核糖体蛋白S12中五个氨基酸取代：赖氨酸43突变为精氨酸、赖氨酸43突变为苏氨酸、赖氨酸43突变为天冬酰胺、赖氨酸88突变为谷氨酸、赖氨酸88突变为精氨酸。在链霉素浓度为15μg/mL时，观察到rpsL突变体分离的频率最高。表型特征显示突变体之间的生长动态、pH值变化和葡萄糖利用发生改变，K88R和K43R变体的丁烯基多杀菌素产量显著增加，分别比亲本菌株高1.78倍和1.64倍。定量PCR分析表明，K88R突变体中翻译相关基因（rpsL和frr）、生长相关基因（whiA和bldD）以及关键的丁烯基多杀菌素生物合成基因（busA、busF和busI）显著上调，表明K88R取代通过调节核糖体功能和相关代谢网络增强了目标化合物的生物合成。未来的研究应探索组合方法，包括开发多抗生素抗性突变体和提高核糖体基因的表达，以最大限度地提高丁烯基多杀菌素产量。本研究强调了核糖体工程作为提高丁烯基多杀菌素产量平台的潜力，并为后续工业规模应用提供了基础。
