【摘要】 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(rpsl^K88R)、W44(rpsl^R86P)、W45(rpsl^K88E)和W46(rpsl^K43N)，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rpsl^K88R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，RpsL^K88R突变体结构与RpsL相比，其侧链发生较大的偏转，影响了与m RNA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。更多还原

【Abstract】 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(rpsl^K88R)、W44(rpsl^R86P)、W45(rpsl^K88E)和W46(rpsl^K43N)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 rps and rpsl^K88R 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 RpsL^K88R mutant structure had a larger deflection of the side chain compared with RpsL which affected the binding effect to m RNA 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.更多还原