Enabling Targeted Positive Selection in an Anaerobic Lignin-Degrading Bacterial Community

The conversion of plant biomass to biofuel is a promising avenue to reduce the use of fossil fuels. However, the biofuel industry is stymied by the inefficient breakdown of lignin, a complex and uniquely disorganized polymer from plants. While microbial communities present in livestock ruminants and anaerobic digesters may be better adapted to break down biomass more efficiently, controlling these natural communities to maximize degradation and meet industry goals remains a large barrier to this approach. Advancements in microbiome editing have enabled unprecedented access to site-and-species specific targeting of complex microbial communities through the use of RNA-guided CRISPR Cas transposases. However, our ability to enrich edited cells within a population without affecting the surrounding community is limited. In this study, we developed positive selection strategies for anaerobic lignin-degrading microbiomes by searching for broadly indigestible nutrient sources (e.g. C, N, P, metabolites) with complementary small catabolism gene clusters that would be provided on the editing vector. We screened several glucosides for substrate utilization and toxicity when grown in minimal media as the sole carbon source. We performed community amplicon sequencing and metabolite utilization analyses as a proof of principle for the approach. Taken together, this work aims to address a major limitation in our current microbiome editing capabilities by establishing a more universal method for positive selection in lignin-degrading bacterial communities.