Deployment of a CRISPR-Associated Transposon System to Engineer an Acid Fermenter Microbial Community for Resource Recovery

Acid fermenters are an emerging biotechnology that harnesses microbial communities to process organic wastes into chemical precursors that can replace many of the compounds traditionally produced by the fossil fuel industry. Synthetic biologists have recently developed tools that give us unprecedented, bottom-up control of microbial communities and which are potentially deployable in acid fermenters to manipulate the often-wide ranging profile of simple, organic products. Better control of this profile will reduce downstream processing costs and increase the efficiency of any ensuing valorization process paired with an acid fermenter.

Specifically, we use conjugation to deliver a recently isolated, transposon-encoded CRISPR-Cas system (INTEGRATE) to native recipients in an acid fermenter community. This system interrupts the acetate pathway causing metabolites to funnel down alternative metabolic pathways, namely the butyrate pathway. INTEGRATE will cause interruption by insertion of a beta-glucosidase gene (bglC), into acetate pathway genes. BglC results in a protein that can degrade cellobiose, a rate limiting step in the overall degradation of cellulose. Our approach will reduce acetate production, increase butyrate production, and improve degradation of cellulosic wastes in an acid fermenter community.

We demonstrate this by first isolating and expressing bglC from Thermobifida fusca YX in E. coli. Then we incorporate bglC into INTEGRATE and use it to interrupt acetate pathway genes in E. coli. We next add the customized INTEGRATE system onto a transmissible vector and demonstrate transfer from E. coli and successful expression in Clostridium acetobutylicum, a common microorganism found in acid fermenter communities.