State Key Laboratory of Development Biology of Freshwater Fish, Hunan Provincial Key Laboratory for Microbial Molecular Biology, College of Life Science, Hunan Normal University, Changsha 410081, China.
J Agric Food Chem. 2020 Dec 9;68(49):14660-14669. doi: 10.1021/acs.jafc.0c04443. Epub 2020 Dec 1.
Spinosyns, the secondary metabolites produced by , are the active ingredients in a family of novel biological insecticides. Although the complete genome sequence of has been published, the transcriptome of remains poorly characterized. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of . Through transcriptomic analysis of different periods of 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 -ΔPEP 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 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.
多杀菌素是由 产生的次生代谢产物，是一类新型生物杀虫剂的有效成分。尽管 已公布其全基因组序列，但 转录组仍未得到充分描述。在本研究中，我们采用高通量 RNA 测序（RNA-seq）技术对 进行转录组分析。通过对 不同生长时期的转录组分析，我们发现了大量差异表达基因，并根据其不同功能进行了分类。基于 RNA-seq 数据，我们利用 CRISPR-Cas9 方法敲除了 PEP 磷酸烯醇式丙酮酸变位酶基因（orf06952-4171）。与野生型相比，-ΔPEP 中多杀菌素 A 和 D 的产量分别提高了 178.91mg/L 和 42.72mg/L，达到野生型的 2.14 倍和 1.76 倍。对突变株的分析也验证了转录组数据的有效性。PEP 磷酸烯醇式丙酮酸变位酶基因的缺失导致丙酮酸含量增加，并影响多杀菌素的生物合成。在 中补充磷酸烯醇丙酮酸为生产多杀菌素提供了底物。我们预计这些转录组分析结果将有助于进一步研究放线菌中的次生代谢产物。
