Effects Of Large Polyketide Synthase Translation On Spinosad Biosynthesis

With the development of life sciences,the research of biopesticides in agriculture is quite important,and it promotes the sustainable development of agriculture with its advantages of high safety and low pollution.Spinosad is a natural product discovered from Saccharopolyspora spinosa,which is a safe and efficient green biopesticide with a unique insecticidal mechanism and it has been widely used in human life.Due to the lack of technologies in China,the research on spinosad is in a backward state compared to Dow AgroSciences LLC,and there is an urgent need to independently develop technologies to improve production for spinosad.Our team used the self-developed ExoCET direct cloning and multi segment assembly technology to construct an artificial gene cluster reconstructed with spinosyn in 2019,by placing its biosynthetic genes under 7 strong promoters.It was successfully heterologously expressed in Streptomyces albus J1074 with a yield of 1.12 mg/L.However,there is still a significant gap between this level and industrial production requirements.Spinosad is a typical polyketide compound with complex structures.It is a type of enzyme or enzyme complex with multiple domains.The polyketide synthase genes are encoded by spnA-E,with lengths ranging from 6 to 17 kb.Bacteria may experience early termination of transcription or mRNA degradation during the transcription process,resulting in incomplete mRNA production.Due to the "transcription and translation" of prokaryote,these truncated mRNA will also be translated,inevitably producing non functional peptides,causing waste of cell resources and increasing the metabolic burden of the organism.In 2014,Yin Peng’s team developed a Toehold Switch system that can sensitively regulate gene expression.The system includes Switch RNA and Trigger RNA,which are paired to initiate RBS translation in specific regions.Sang Woo Seo’s team designed a system called the Protein Quality Control system(ProQC system)to improve the quality of protein synthesis in prokaryotes based on the Toehold Switch system.This system can only be translated when complete mRNA is transcribed,and truncated mRNA cannot serve as a template for translation,thereby improving the efficiency of protein expression.This study increased the synthesis of bacterial full-length proteins by 2.5 times.The large polyketide synthase may produce more truncated mRNAs,by using the ProQC system,it’s possible to increase the proportion of full-length proteins,thereby improving the efficiency of effective protein synthesis.In our study,we analyzed the transcriptional level of the largest spnE gene of 16767 bp in the polyketide synthase genes expressed in Streptomyces albus J1074.The results showed that the transcriptional level gradually decreased from the 5 ’end to the 3’ end.The longer the gene,the greater the possibility of producing truncated mRNA,and the lower the level of expressing full-length mRNA.The polyketide synthase genes are too large that produces a large number of incomplete mRNA,leading to the synthesis of useless proteins.It is possible to reduce the efficiency of spinosyn biosynthesis.This study plans to construct a synthetic protein quality control system for polyketide synthase to regulate the spinosad PKS genes,selectively translate full-length mRNA,increase the quantity of full-length proteins to increase the production of spinosad.In our study,the ProQC system was applied to the biosynthesis gene cluster of spinosad,regulating the gene spnE and its polyketide synthase modules 8,9 and spnD to heterologous expression in S.albus J1074 and S.coelicolor CH999.The experiment first used ProQC system to regulate the expression of green fluorescent protein gene mNeonGreen to verify that the system can be successfully applied to Streptomyces,and then used our genome editing technology to regulate spnE through the system,which increased the production of spinosad in S.albus J1074 by 2.4 times and S.coelicolor CH999 by 13.8 times;The system continues to regulate extension modules 8,8 and 9 in spnE on the basis of regulating spnE,found that the production of spinosyn has been increased,which indicated that individual modules could also participate in the synthesis of spinosyn.It may be related to the structure of the polyketide compounds;continued to regulate spnD through the system,there was no significant increase in the production of spinosyn.The possible reason is the mutual interference between the ProQC elements that regulate spnD and spnE.Therefore,it is necessary to develop more different ProQC elements to simultaneously regulate the expression of multiple polyketide synthases.The efficient biosynthesis of spinosad is a major challenge.In this study,the ProQC system was used to regulate the polyketide synthase gene of spinosad,successfully increasing the production.This protein quality control system provides a new technological means for achieving efficient biosynthesis of natural products,and it is of great significance for the study of more natural product biosynthesis pathways.