Shanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China.
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
J Dairy Sci. 2020 Jan;103(1):161-165. doi: 10.3168/jds.2019-17346. Epub 2019 Nov 14.
Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter P was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.
乳球菌（Lactococcus lactis）是最重要的益生菌乳酸菌（LAB）之一，被广泛应用于乳制品工业中，作为生产重组蛋白的细胞工厂。目前，使用的是一种乳链菌肽（nisin）控制的诱导表达系统，这是 LAB 中唯一的商业表达系统。然而，可用于调节乳球菌基因表达的遗传修饰方法相当有限。在这里，我们开发了一种用于乳球菌 NZ9000 基因转录抑制的 2 质粒系统，该系统使用了诱导型簇状规律间隔短回文重复序列（CRISPR）-dCas9。一个诱导型启动子 P 被用于驱动来自酿脓链球菌的 dCas9 的表达，而一个强组成型启动子 P 则驱动单个或多个靶基因的单指导 RNA 表达。dCas9 能够实现单或多个靶基因的 CRISPR 干扰介导的沉默，基因表达显著降低，最高可达 99%。此外，使用该系统鉴定出了 NZ9000 中的一个假定青霉素酰胺酶（LLNZ_07335）为胆盐水解酶，用于胆盐抗性。据我们所知，这是首次在乳球菌中发现与胆盐耐受性相关的功能性基因。总的来说，我们的工作为乳球菌或其他 LAB 的各种应用引入了一种新的基因抑制工具。
