Preliminary Application Of Ribosome Engineering Technology In Saccharopolyspora Pogona

The butenyl-spinosyns,also called pogonins,structurally related to the ?classical? spinosyns is a novel class of macrocyclic secondary metabolites of aerobic fermentation produced by soil actinomycetes Saccharopolyspora pogona.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.Actually,all of them have touch and feeding poison effect of target pests.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 was difficult to achieve industrial production and large-scale applications.In order to improve the yield of butenyl-spinosyns produced by S.pogona and acquire pogonins-like mutant,we used ribosome engineering technology to screen the streptomycin resistant mutants.In a high inhibitory streptomycin concentration,we isolated 28 spontaneous streptomycin resistant mutants.The yields of butenyl-spinosyns were then analyzed and compared with parent strain.We found a effectively different ability of production pogonins between original strain and mutants.Among plenty of mutants,S13 displayed the greatest increase in the yield of butenyl-spinosyns,which was 1.79 fold higher than that in parent strain.Further analysis of the metabolite profile of S13 by mass spectrometry lead to the discovery of Spinosyn ?1,which was absent from the parent strain.DNA sequencing showed that there existed two point mutations in the conserved regions of rps L gene which encodes ribosomal protein S12 in S13.The mutations occurred a C to A and a C to T transversion mutations occurred at nucleotide pair 314 and 320 respectively,which resulted in the mutations of Proline(105)to Glutamine and Alanine(107)to Valine.It has been proposed that the mutation of rps L alters the conformation of ribosomal protein S12,which resulted in a higher efficiency of protein translation activity at later growth phase,thus cause the change of secondary metabolites production capacity.In addition,Site-Directed Mutagenesis of the rps L gene and genetic recombination further expansion of the ribosome engineering application in strain improvement.In this study,we firstly disrupt rim P gene,encoding a ribosome assembly cofactor in S.pogona,which enhances the production of pogonins at 1.16 times while the ability of rim P gene in the metabolic network of butenyl-spinosyns and the mechanism remains to be elucidated.Streptomycin resistance screening was first application in S.pogona,and successfully identified mutation sites in mutant S13.Meanwhile the target secondary metabolites is also activated by molecular genetic manipulation.This study lays a foundation for ribosome engineering breeding of S.pogona and makes sure that ribosome engineering technology has a significant potential application value.