Project description:Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools. Here, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs (PAMs) based on bioinformatics analysis combined with plasmid interference assay in S. spinosa. By reprogramming this endogenous CRISPR-Cas system, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the Restriction-Modification (RM) systems was systematically disarmed. We showed that by disarming one type I RM system and two type II RM systems simultaneously, the transformation efficiency of foreign plasmids was significantly increased. Based on the engineered strain with simultaneous deletion of these three RM genes, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster as well as gene insertion at high efficiency. Collectively, we developed a reliable and high-efficiency genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.
Project description:A DNA microarray was designed and constructed using the genome sequence of Saccharopolyspora erythraea strain NRRL 2338. Following growth in liquid medium, we analysed the expression of 6494 ORFs along the time course. The results indicated that the 404 genes, whose expression significatively correlated with the time course, identify three distinct growth phases: a rapid growth until 32 h (phase A); a growth slowdown until 52 h (phase B); another rapid growth phase from 56 h to 72 h (phase C) before entering the stationary phase. We experimentally determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, but also strand specific patterns of expression, and the behavior of major functional classes. Temporal expression of all the gene clusters for secondary metabolism was analyzed, confirming ery cluster up-regulation during the first growth phase, and finding out six secondary metabolism clusters that are clearly regulated during growth. The use of a DNA microarray, specifically designed on the Sac. erythraea genome sequence, improved specificity and sensitivity of gene expression analysis, giving a global and at the same time detailed picture of how Sac. erythraea genes are modulated. Keywords: time course
Project description:We report the high-throughput profiling of saccharopolyspora erythraea including a industrial strain HL3168 E3 and a wild-type strain NRRL23338. The aim was to evaluate the difference in expression of sRNA predicted in silico related to secondary metabolites in Saccharopolyspora erythraea.
