小白链霉菌中CRISPR-Cas9基因敲除系统的构建与优化OA北大核心CSTPCD

Streptomyces albulus is the principal strain for the industrial production of ε-polylysine.Traditional breeding approaches that rely on mutagenesis and resistance screening have been employed to enhance the s-polylysine yield of S.albulus.Nevertheless,the di-minishing returns often seen with these traditional methods have become a significant hindrance to progress in breeding techniques.As a re-sult,the application of metabolic engineering strategies has become imperative to augment ε-polylysine production in S.albulus.In this light,this study have developed a CRISPR-Cas9-based gene editing platform.With S.albulus GS114 as the research object and the ε-poly-lysine synthase gene pls as the target gene,the pls gene was successfully knocked out in the genome of S.albulus GS114 with a 100％edi-ting efficiency.To further amplify the array of knock-out strains available,this study meticulously refined the conjugation protocol for S.al-bulus GS114.The optimal conditions identified include a donor-recipient ratio of 1∶1,a magnesium ion concentration of 30 mmol/L,an in-cubation of heat-activated spores at 55 ℃ for 10 minutes,followed by a 20-hour antibiotic coverage post-incubation.Under these optimized conditions,tthe conjugation efficiency reached 2.5 × 10-8 per recipient cell,marking a 78％improvement over the control.This research not only pioneers a pivotal tool for S.albulus metabolic engineering in the future but also offers insightful guidance for constructing CRISPR-Cas systems in other industrial Streptomycetes.