小白链霉菌(Streptomyces albulus)是工业上生产ε-聚赖氨酸的主要菌株。为提高S.albulus的ε-聚赖氨酸合成能力,基于诱变和抗性筛选的传统育种方法被普遍应用。然而,由于传统育种存在“疲劳效应”,当前S.albulus 的育种陷入了瓶颈。为利用代谢工程方法进一步提高S.albulus的ε-聚赖氨酸合成能力,亟需建立基于CRISPR-Cas9的基因编辑方法。为此,该文以S.albulus GS114为研究对象,以ε-聚赖氨酸合成酶基因pls为靶基因,构建了CRISPR-Cas9基因敲除系统,成功敲除了pls,编辑效率为100%。为进一步提高获得的敲除株数量,对S.albulus GS114结合转移条件进行了系统优化,获得的最优结合转移条件为:供受体比例1∶1,镁离子浓度30 mmol/L,55 ℃热激孢子10 min,培养20 h后覆盖抗生素。在最优转移条件下,结合转移效率达到2.5×10-8,较优化前提高了78%。该研究结果一方面为后续S.albulus的代谢工程改造提供了重要工具,另一方面为其他工业链霉菌构建CRISPR-Cas系统提供了重要参考。
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 ε-polylysine yield of S.albulus.Nevertheless, the diminishing returns often seen with these traditional methods have become a significant hindrance to progress in breeding techniques.As a result, 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-polylysine synthase gene pls as the target gene, the pls gene was successfully knocked out in the genome of S.albulus GS114 with a 100% editing efficiency.To further amplify the array of knock-out strains available, this study meticulously refined the conjugation protocol for S.albulus GS114.The optimal conditions identified include a donor-recipient ratio of 1∶1, a magnesium ion concentration of 30 mmol/L, an incubation 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.
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