Improvement Of Saccharopolyspora Pogona Strain By Ribosome Engineering And Analysis Of The Metabolites

Butenyl-spinosyns are a family of new macrocyclic secondary metabolites produced by soil actinomycetes Saccharopolyspora pogona.The butenyl-spinosyns are similar to spinosads.Compared to spinosads,the butenyl-spinosyns have a wider insecticidal spectrum and better insecticidal activities which is similar to the second-generation spinosyn called spinetoram.This characters make it could be a new generation high-efficiency and environment-friendly insecticide.However,the wild-type S.pogona is imperfect on account of its weak biosynthesis ability of butenyl-spinosyns and very long fenmation period.In the meantime,international studies on butenyl-spinosyns develop slowly.Therefore,it is not yet required to reach the level of industrial fermentation.In order to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona,we used ribosome engineering technology to screen the paromomycin resistant mutants.In 10×MIC paromomycin concentration,we isolated 54 spontaneous paromomycin resistant mutants.By a preliminary study on metabolites changes,we found that pseudoaglycone(PSA),a component of butenyl-spinosyns,was overexpressed in the resistant mutants.There were 6 mutants in which the yields were higher than the original strain,about 20%were positive and 45%were negative mutants.The mutant P-7 has the largest increase rate of butenyl-spinosyns yield,which improved 2.2-fold compared to the original strain.DNA sequence analysis showed that two point mutations occurred in the conserved regions of rpsL gene encoding ribosomal protein S12,the 314th by C mutation for A,the corresponding proline mutated to glutamine.The 320th by C mutation for T,the corresponding alanine mutated to valine.The rpsL gene mutations may cause the S12 ribosomal protein space conformation and the activity of protein translation changes,leading to the variation of secondary metabolites produced capability.To further address the problem of conversion from pseudoaglycone to butenyl-spinosyns,we duplicated the six forosamine biosynthesis genes,busS,busR,busQ,busP,busO and busN,according to biosynthetic pathway of butenyl-spinosyns.The pOJ260-Forosamine was constructed stepwise by restriction digestion and ligation,and imported into S.pogona by conjugational transfer.LC results showed that the combinant P7-Forosamine could convert excessively accumulated intermediates to butenyl-spinosyns.In this study,we firstly applied ribosome engineering in Saccharopolyspora pogona,screened mutant strains in which production of butenyl-spinosyns increased,and successfully identified the mutation sites.The conversion from excessively accumulated intermediates to butenyl-spinosyns was achieved by overexpression of forosamine biosynthetic genes.This study provides significant guides for ribosome engineering breeding of microbial strains and genetical improvment.