期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 68, 期 49, 页码 14660-14669出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.0c04443
关键词
Saccharopolyspora spinosa; spinosad; RNA-seq; CRISPR-Cas9; replenishment pathway
资金
- National Natural Science Foundation of China [31770106]
- National Basic Research Program (973) of China [2012CB722301]
- Hunan Province college students research learning and innovative experiment project [s201910542045]
- Key Research and Development Program of Hunan Province [2019NK2192]
Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of novel biological insecticides. Although the complete genome sequence of S. spinosa has been published, the transcriptome of S. spinosa remains poorly characterized. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of S. spinosa. Through transcriptomic analysis of different periods of S. spinosa growth, we found large numbers of differentially expressed genes and classified them according to their different functions. Based on the RNA-seq data, the CRISPR-Cas9 method was used to knock out the PEP phosphonomutase gene (orf 06952-4171). The yield of spinosyns A and D in S. spinosa-Delta PEP was 178.91 mg/L and 42.72 mg/L, which was 2.14-fold and 1.76-fold higher than that in the wild type (83.51 and 24.34 mg/L), respectively. The analysis of the mutant strains also verified the validity of the transcriptome data. The deletion of the PEP phosphonomutase gene leads to an increase in pyruvate content and affects the biosynthesis of spinosad. The replenishment of phosphoenol pyruvate in S. spinosa provides the substrate for the production of spinosad. We envision that these transcriptomic analysis results will contribute to the further study of secondary metabolites in actinomycetes.
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