The Transcriptomics Analysis And Mining Of Key Genes Affecting Spinosad Biosynthesis In Saccharopolyspora Spinosa

Saccharopolyspora spinosa can produce a series of macrolide compounds spinosyns.These secondary metabolites had good insecticidal activity and a broad insecticidal spectrum,so this promising biological pesticide was widely studied all over the world.However,the low spinosyn production of the wild-type S.spinosa,the long fermentation time and high cost,can't meet the requirements of industrial need with large-scale production.At present,the complete genome map and gene annotations of S.spinosa genome have completed.However,the current data is difficult to select suitable target genes for genetic modification to increase the yield of spinosyn.In order to solve this problem,this research sequenced the transcriptome of S.spinosa at three different stages and conducted bioinformatics analysis.Fully understanding the genetic background and gene function annotation,we explored the genes related to spinosyn biosynthesis and functional genes involved in the metabolic networks and analyzed the impact of spinosyn biosynthesis and potential key functional genes.Through the q RT-PCR verification of differentially expressed genes found in transcriptomics,PEP phosphonomutase was selected for genetic modification by using CRISPR/Cas9 method,the key role of this gene in affecting the biosynthesis of spinosyn and related metabolic pathways of S.spinosa was analyzed.In addition,36 biosynthetic gene clusters were predicted by the online prediction software antiSMASH.Five competitive polyketide biosynthetic gene clusters(cluster 1,cluster 14,cluster 19,cluster 28,and cluster 35)were selected for the gene cluster disruption analysis.The CRISPR/Cas9 method was also used to disrupt these five gene clusters to seek an operating platform to disrupt large fragments of S.spinosa.The research achievements are as follows:1.The transcriptomics analysis of S.spinosa at three different stagesBased on the genome of S.spinosa,the total RNA of S.spinosa in three different stages(logarithmic phase T1,pre-stationary phase T2 and end of stationary phase T3)was analyzed by transcriptomics.About 1.40 GB data were produced for each sample,and the average comparison rate with the reference genome was 84.47%.The prediction of transcript samples showed that a total of 34 new transcripts were detected.More than 2700 differentially expressed genes were compared and analyzed.The q RT-PCR analysis of spn family genes related to spinosad biosynthesis confirmed the correction of transcriptome data.Meanwhile,a large number of differentially expressed genes including PEP phosphonomutase gene were screened.2.The effects of PEP phosphonomutase differentially expressed gene on strain growth and spinosad biosynthesis of S.spinosaOn the basis of transcriptomics research,the PEP phosphonomutase gene was selected for genetic modification.This gene was knocked out and overexpressed by using CRISPR/Cas9 method and an overexpression vector based on p OJ260.The yields of spinosyn A and D in S.spinosa-?PEP were 178.91mg/L and 42.72mg/L,respectively,which were 2.14 times and 1.76 times compared with wild-type strain(the yields of spinosyn A and D were 83.51mg/L and 24.34mg/L,respectively).In addition,the spinosyns A and D of the overexpression strain S.spinosa-PEP reached 61.67mg/L and 19.38mg/L.The metabolic pathways affected by this gene were also analyzed.Through the detection of pyruvate content and the q RT-PCR results of related genes,the knockout of PEP phosphonomutase affected the content of pyruvate,further regulated the biosynthesis of precursors such as acetyl-Co A.Ultimately affected the biosynthesis of spinosyns.3.The effect of disruption of five competing polyketide biosynthetic gene clusters on the growth and the production of spinosad in S.spinosaFirstly,antiSMASH online prediction software was used to predict the polyketide biosynthetic gene cluster in the genome of S.spinosa.Using CRISPR/Cas9 method to disrupt the five competing polyketide biosynthetic gene clusters of S.spinosa,five mutant strains were successfully constructed.Through the spinosad production analysis of these five mutant strains,the increase of spinosad biosynthesis was only caused by the mutant strain S.spinosa-?clu28.The cluster 28 has 75%similarity with the flavomycin biosynthesis gene cluster.Meanwhile,the disruption of clu28 caused great changes in the growth of the bacteria,glucose consumption,mycelial morphology,sporulation,insecticidal activity,and spinosad biosynthesis rate.It showed that disrupting the competitive polyketide biosynthesis gene cluster was an important technical strategy to improve spinosad biosynthesis.4.The optimization and screening of phosphate addition concentration in the synthetic fermentation medium of S.spinosaSince the addition of phosphate has an important influence on the biosynthesis of spinosyns in S.spinosa,it participates in the pyruvate metabolism pathway and energy metabolism process in strain.The concentration of phosphate in the synthetic fermentation medium was optimized and screened in S.spinosa-?PEP.Through fermentation experiments,the optimal phosphate concentration was determined to be10 mmol/L.This result provides data support for improving the yield of spinosad and optimizing the inorganic salt composition of fermentation medium.In conclusion,large numbers of differential expression genes and transcriptional regulatory factors were found through transcriptomic analysis of S.spinosa in different phases,which not only provided an important reference for the construction of biosynthetic and metabolic regulatory network of S.spinosa,but also laid an important foundation for the mining of key regulatory genes of spinosad biosynthesis.This study not only deeply studied the physiological metabolism and spinosad biosynthesis of S.spinosa,but also provided a reference basis for the regulation of secondary metabolite biosynthesis and network optimization in actinomycetes.