Data_Sheet_1_A Single-Plasmid Genome Editing System for Metabolic Engineering of Lactobacillus casei.pdf

Genome engineering of Lactobacillus casei, an important industrial microorganism for dairy fermented product, currently relies on inefficient and time-consuming double crossover events. In this study, we developed an easy-to-use genome engineering strategy for metabolic engineering of L. casei for acetoin production. Plasmid pMSP456-Cre, that contains prophage recombinase operon LCABL_13040-50-60 driven by the nisin-controlled inducible expression (NICE) system and the site-specific recombinase gene cre under the control of the promoter of the lactose operon from L. casei, was constructed. Using this plasmid, integration of a hicD3 gene linear donor cassette (up-lox66-cat-lox71-down) was catalyzed by the LCABL_13040-50-60 recombinase and the cat gene was excised by the Cre/lox system with an efficiency of 60%. To demonstrate this system for sequential and iterative knocking out genes in L. casei, another three genes (pflB, ldh and pdhC) related to acetoin production were deleted with the efficiencies of 60, 40, and 60%, respectively. The yielding quadruple mutant could produce a ∼18-fold higher amount of acetoin than the wild-type and converted 59.8% of glucose to acetoin in aerobic. Therefore, these results proved this simple genome engineering strategy have potential in metabolic engineering of L. casei for production of high value-added metabolites.

干酪乳杆菌（Lactobacillus casei）是乳品发酵产业中的重要工业微生物，其当前的基因组工程改造依赖于效率低下且耗时的双交换重组事件。本研究针对用于乙偶姻（acetoin）生产的干酪乳杆菌代谢工程，开发了一套操作简便的基因组工程改造策略。本研究构建了质粒pMSP456-Cre，该质粒包含由乳链菌肽可控诱导表达（Nisin-Controlled Inducible Expression, NICE）系统驱动的前噬菌体重组酶操纵子LCABL_13040-50-60，以及受干酪乳杆菌乳糖操纵子启动子调控的位点特异性重组酶基因cre。利用该质粒，LCABL_13040-50-60重组酶可介导hicD3基因线性供体盒（up-lox66-cat-lox71-down）的整合，随后Cre/lox系统可切除cat基因，切除效率达60%。为验证该系统可用于干酪乳杆菌的连续迭代基因敲除，本研究对另外3个与乙偶姻合成相关的基因（pflB、ldh及pdhC）进行了敲除，敲除效率分别为60%、40%及60%。获得的四突变体菌株的乙偶姻产量较野生型提升约18倍，且在好氧条件下可将59.8%的葡萄糖转化为乙偶姻。综上，本研究结果证明，这套简易基因组工程改造策略在干酪乳杆菌代谢工程用于生产高附加值代谢产物方面具有应用潜力。