Efficient metabolic pathway modification in various strains of lactic acid bacteria using CRISPR/Cas9 system for elevated synthesis of antimicrobial compounds.

Lactic acid bacteria (LAB) are known to exhibit various beneficial roles in fermentation, serving as probiotics, and producing a plethora of valuable compounds including antimicrobial activity such as bacteriocin-like inhibitory substance (BLIS) that can be used as biopreservative to improve food safety and quality. However, the yield of BLIS is often limited, which poses a challenge to be commercially competitive with the current preservation practice. Therefore, the present work aimed to establish an optimised two-plasmid CRISPR/Cas9 system to redirect the carbon flux away from lactate towards compounds with antimicrobial activity by disrupting lactate dehydrogenase gene (ldh) on various strains of LAB. The lactic acid-deficient (ldhΔ) strains caused a metabolic shift resulting in increased inhibitory activity against selected foodborne pathogens up to 78 % than the wild-type (WT) strain. The most significant effect was depicted by Enterococcus faecalis-ldh∆ which displayed prominent bactericidal effects against all foodborne pathogens as compared to the WT that showed no antimicrobial activity. The present work provided a framework model for economically important LAB and other beneficial bacteria to synthesise and increase the yield of valuable food and industrial compounds. The present work reported for the first time that the metabolism of selected LAB can be manipulated by modifying ldh to attain metabolites with higher antimicrobial activity. • The development of a new mutant lactic acid bacteria (LAB) strains was attained by genetically disrupt lactate dehydrogenase (ldh) gene, to eliminate the production of lactic acid. • The deletion of the ldh was achieved by introducing an efficient two-plasmid CRISPR system. • The growth rate of the mutant LAB strains show significant changes due to the deletion of ldh. • The effects of metabolic alteration on Bacteriocin-Like Inhibitory Substance (BLIS) was significantly increase up to two-fold resulted from re-routing of the carbon flow towards BLIS pathway. [ABSTRACT FROM AUTHOR]