Transferable genetic toolsets for nonmodel gut Clostridia enable in vivo reversible control of metabolite production.

Gut Clostridia species, including commensal members of the Clostridiaceae and Lachnospiraceae families, maintain microbiota homeostasis and influence human health and disease; however, adequate genetic toolsets to study abundant but nonmodel gut Clostridia are lacking. Here we present a set of transferable and modular genetic toolsets that function broadly across phylogenetically diverse gut Clostridia. We first identify a panel of strong constitutive promoters that drive robust gene expression across diverse clostridial strains. We then develop an inducible promoter system that enables precise, tunable gene regulation and facilitates the implementation of CRISPR-Cas gene-deletion systems. We apply this system for targeted and reversible control of trimethylamine and deoxycholic acid production, two microbiota-derived metabolites implicated in host lipid metabolism and diseases, in mice. This robust genetic toolkit for nonmodel gut Clostridia enables functional studies to causally link microbiota genes to host physiology and disease, paving the way for therapeutic genetic engineering of microbiota.