AUTHOR=Xin Yongping , Guo Tingting , Mu Yingli , Kong Jian 

TITLE=A Single-Plasmid Genome Editing System for Metabolic Engineering of Lactobacillus casei

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.03024

DOI=10.3389/fmicb.2018.03024

ISSN=1664-302X

ABSTRACT=<p>Genome engineering of <italic>Lactobacillus casei</italic>, 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 <italic>L. casei</italic> for acetoin production. Plasmid pMSP456-Cre, that contains prophage recombinase operon <italic>LCABL_13040-50-60</italic> driven by the nisin-controlled inducible expression (NICE) system and the site-specific recombinase gene <italic>cre</italic> under the control of the promoter of the lactose operon from <italic>L. casei</italic>, was constructed. Using this plasmid, integration of a <italic>hicD3</italic> gene linear donor cassette (up-<italic>lox66</italic>-cat-<italic>lox71</italic>-down) was catalyzed by the LCABL_13040-50-60 recombinase and the <italic>cat</italic> 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 <italic>L. casei</italic>, another three genes (<italic>pflB, ldh</italic> and <italic>pdhC</italic>) 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 <italic>L. casei</italic> for production of high value-added metabolites.</p>