须糖多孢菌(Saccharopolyspora pogona)发酵产生的次级代谢物丁烯基多杀菌素(butenyl-spinosyn)是一种绿色生物杀虫剂.但由于发酵产量低不能满足工业生产需求,选育高生产性能的菌株是实现工业化生产的关键.本研究以须糖多孢菌为底盘细胞分别构建了核糖体蛋白S12(ribosomal protein S12,RpsL)及RpsL突变体的基因过表达工程菌株Q8(rpsl)、W43(rpslK88R)、W44(rpslR86P)、W45(rpslK88E)和 W46(rpslK43N),通过摇瓶发酵验证及RpsL蛋白结构比对,分析RpsL对丁烯基多杀菌素合成的影响.结果表明,工程菌株Q8、W43、W44、W45和W46分别使丁烯基多杀菌素的产量提高至275％、146％、111％、122％和降低至75％.进一步研究表明,rpsl和rpslK88R基因的过表达提高了合成途径基因的转录水平,从而使丁烯基多杀菌素的产量显著提高.对二者进行同源建模发现,RpsLK88R突变体结构与RpsL相比,其侧链发生较大的偏转,影响了与mRNA的结合作用进而降低了核糖体翻译效率,因此其提产效果略低于RpsL.本研究为蛋白质工程改造RpsL提高丁烯基多杀菌素的产量提供参考.
Ribosomal Protein RpsL Regulates Butenyl-spinosyn Synthesis in Saccharopolyspora pogona
Butenyl-spinosyn,a secondary metabolite produced by fermentation of Saccharopolyspora pogona,is a green bio-insecticide.However,due to the low yield of fermentation and can not meet the needs of industrial production,the breeding of strains with high production performance of butenyl-spinosyn is the key to realize industrial production.In this study,ribosomal protein S12(RpsL)and RpsL mutant gene overexpression engineering strains Q8(rpsl)、W43(rpslK88R)、W44(rpslR86P)、W45(rpslK88E)和 W46(rpslK43N)were constructed were constructed using Saccharopolyspora pogona as the base cells.The effect of RpsL on the synthesis of butenyl-spinosyn was studied by shaking flask fermentation and comparison of RpsL protein structure.The results showed that the engineered strains Q8,W43,W44,W45,W46 increased the yield of butenyl-spinosyn to 275％,146％,111％,122％and reduced to 75％,respectively.The overexpression of rpsl and rpsslK88R genes increased the transcription level of the synthetic pathway genes,and thus significantly increased the yield of butenyl-spinosyn.At the same time,homologous modeling of the two was conducted,and it was found that RpsLK88R mutant structure had a larger deflection of the side chain compared with RpsL,which affected the binding effect to mRNA and thus reduced the ribosome translation efficiency,so its yield extraction effect was slightly lower than RpsL.This study provides a reference for protein engineering RpsL to improve the yield of butenyl-spinosyn.
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