| Spinosad,polyketide-derived macrolides produced in the actinomycete Saccharopolyspora spinosa,has been developed as a broad-spectrum and effective insecticide with no toxicity on nontarget insects and mammals.The spinosad-producing strain ATCC 49460 was isolated from the soil of a waste winery by a scientist from the Eli Lilly Company in 1982.After several years of traditional mutagenesis breeding,the yield of its derivative strain ASAGF73 has been greatly increased,which provides an excellent research material for revealing the high yield mechanism of spinosad by comparative omics studies.In this study,the genomic sequence of the high-yielding strain ASAGF73 was determined.The length of its chromosome is 8778227 bp,which contains 9167 coding frames.Compared with ATCC 49460,there are three major fragment deletions on the ASAGF73 chromosome,with the sizes of 30.01 kb,78.52 kb and 33.08 kb,respectively.In addition,there are 5761 InDel(insertion/deletion)and 2423 SNV(single nucleotide variations)sites in ASAGF73 genome,affecting at least 290 gene coding frames.Analysis of the transcriptional profiling of the two strains revealed that the genetic alteration of ASAGF73 selectively regulates genomic transcription and changes the expression of key enzymes in primary metabolism,increasing the metabolic flux of the fatty acid degradation pathway,the glycolysis pathway,and the branched-chain amino acid metabolism pathway,reducing the metabolic rate of tricarboxylic acid cycle,which is benificial to the supply of precursor for spinosad production.The enhanced expression of ribosomal protein synthesis and oxidative phosphorylation-related genes also contribute to the efficient antibiotic synthesis.In addition,the transcription of the spinosad biosynthetic gene cluster was significantly up-regulated in ASAGF73.Based on the results of omics analysis,we focus on the active fatty acid metabolism pathway in ASAGF73 and carry out further studies aimed at improving the production of spinosad by fermentation optimization and genetic modification.The spinosad production increased significantly with the addition of strawberry seed oil(511.64 mg/1)and camellia oil(520.07 mg/1),compared to the control group without oil(285.76 mg/l)and soybean oil group(398.11 mg/l).These oils stimulated the expression of the genes coding for acyl-CoA synthases and acetyl-CoA carboxylase,which increased the supply of spinosad synthesis precursor malonyl-CoA,compared to soybean oil.We observed that H2O2 produced during fatty acid ?-oxidation was involved in improving spinosad production.Increased titer of spinosad was achieved using the strain ASAGF46 in which the fatty acid degradation pathway was strengthened via inserting a copy of the fadD1 and fadE genes of Streptomyces coelicolor in the genome of ASAGF73.The spinosad production of genetically engineered strain reached 784.72 mg/l in the medium containing camellia oil.The higher spinosad production level(843.40 mg/l)was detected with the addition of 0.01 mM thiourea and up to 1203.26 mg/1 in the fermentor.Therefore,this study provides practicable information that manipulation targeting fatty acid metabolic pathway can be served as a feasible strategy to boost spinosad biosynthesis.In summary,the present study reveals the molecular mechanism of high yield in ASAGF73 by comparing genome and transcriptome analysis.Based on these analyses,we performed initial attempts for targeted engineering for spinosad overproduction,which is also defined as forward engineering.These studies will pave the way for spinosad productivity improvement and industrial bioprocess optimization.At the same time,the strategy applied here also has certain reference significance for studying the mechanism of high yield of other antibiotics and forward engineering. |
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