Probiotic organisms benefit human health via many mechanisms. For example, Lactococcus lactis has been shown to have probiotic benefits in both the agricultural and health industries, as an anti-inflammatory preventive treatment for bovine mastitis and as the first organism genetically modified to produce therapeutic protein in the human gut. Many of the above uses require tolerance to chemical (e.g., low pH/high acid), ecological (e.g., competition and survival in the gut or udder microbiome), and antimicrobial (e.g., phage susceptibility in fermentations) environments. Tolerance phenotypes are often complex, requiring many genetic factors which are often unknown and/or not intuitive. One approach to study and improve complex phenotypes such as these is genome-scale engineering, in which mutations are made across the genome to determine the effect of genotype on phenotype. The purpose of this study is to leverage CRISPR-based bacterial genome engineering tools for engineering of probiotic E. coli and Lactococcus lactiswith sufficient efficacy that genome-scale libraries of the organism might be created. Once these libraries are established, they can be used to study the effects of many different selective pressures on engineered probiotics. Applications include, but are not limited to acid tolerance, phage tolerance, protein secretion, survival in the gut environment, and production of exopolysaccharides. Particular emphasis is placed on the effect of various genotypes on the ecological niche of these organisms in complex microbial communities.

Genome-scale engineering in probiotic organisms

251st National Meeting (2016)

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