A PyrR Regulating the Pyrimidine Biosynthesis Lost Antibacterial Ability Using CRISPR/Cas9-Mediated Knock-out for Metabolic Engineering at Lactobacillus casei

Abstract

Background Lactobacillus casei had four possible probiotic roles, such as antimicrobial antagonism, competitional adhesion, immunoregulation and inhibition of bacterial toxin. The complex environment of gut microbe blocked to explore the antimicrobial scheme through genetics, metabolomics, proteomics, and signal transduction wth Lactobacillus casei. The genome editing Lactobacillus and germ-free animal provided the similar studying model of cell signal pathways to fully elucidate the probiotic mechanisms in animal. Results To delineate the metabolic reactions of nucleotides from L. casei associated with mechanisms of inhibiting pathogens and immunoregulation, we report the PyrR gene which was an important regulon to promote the pyrimidine biosynthesis, and PyrR deficient L. casei strain was successfully constructed with CRISPR-Cas9D10A tool. There were some changes with the basic biological characterization, such as decreasing the growth, auxotroph and morphological damage in the PyrR deficient strain. The metabolic profiles of supernatant between PyrR deficient and wild strain revealed a complementary regulation from other metabolic pathways within decreasing pyrimidine biosynthesis. Besides, the PyrR deficient strain significantly lost the character of inhibiting the growth of pathogens. We further identify PyrR regulating pyrimidine biosynthesis, which further improved the internalization and colocalization with the development of macrophages. Conclusions Evidence shows that PyrR gene is an active key for regulating pyrimidine biosynthesis in L. casei supernatant against a wide range of pathogens. The complementary regulation for the PyrR deficient could no inhibit the growth of pathogens, and immune regulation in the macrophages from mice. Thus, the deletion of PyrR in L. casei lost the probiotic character of antimicrobial antagonism and immunoregulation.