PSV-8 Development of genetically engineered microbes as probiotics to enhance ruminant productivity

Abstract Alternatives to antibiotics (especially metaphylactic use of antibiotics in livestock) are critically needed to ensure animal and human health while meeting the increasing food demand of the growing human population. One of the proposed alternatives to antibiotic use that is gaining momentum is the use of probiotics or direct-fed microbials (DFMs), which are live microbes that have specialized functions that improve gastrointestinal health and performance. However, to date, most probiotics or DFMs tested as alternative to antibiotics to reduce pathogen colonization have shown limited effectiveness with inconsistent results. This is due to the fact that only, generally accepted as safe (GRAS) bacterial strains are being used as DFMs or probiotics in the animal industry. However, metagenome studies have clearly shown the presence of unique biosynthetic gene clusters and other beneficial genes within gut environments in livestock species in non-GRAS organisms. As such, with advances in gene editing techniques, tools are now available to strategically integrate beneficial genes found in non-GRAS approved microbial strains into GRAS approved strains to develop genetically engineered microbes with beneficial functions. As a first step towards developing genetically engineered microbes to improve animal health and performance, we engineered a biosynthetic gene cassette identified from rumen microbes into GRAS approved Bacillus strains. We utilized chasis-independent recombinase-assisted genome engineering (CRAGE) and random transposon mutagenesis to insert biosynthetic genes into GRAS approved strains. Furthermore, we performed functional screening experiments to identify strains containing the engineered gene cassette and confirmed using sequencing. This approach can be used to engineer beneficial genes and generate personalized probiotics to improve animal health and productivity.