Engineering strain tolerance to inhibitory compounds in biomass hydrolysate feedstocks

Carbon source utilization is more than just efficient conversion of sugars to high-value fermentation products. Optimum performance also often relies on robust cell growth in the presence of unavoidable contaminants in the feedstocks. Second generation biofuels — the two-step conversion of plant cell wall polymers (cellulose and hemicellulose) to their simple sugar constituents, and then of those sugars into valuable fuel molecules through fermentation — provides a classic example of carbon source feedstocks loaded with a complex mixture of additional plant cell wall compounds that can be toxic to the engineered microbial production strain.

How do you engineer cells for a desired phenotype when you may not even know which genes to target? Unlike metabolic engineering programs, where the native or heterologous biosynthetic pathway genes are often well known and understood as gene editing targets, programming tolerance to toxic or inhibitory compounds can be a more nebulous endeavor even in an organism with a thoroughly annotated genome. Classical mutagenesis strategies were once an investigator’s best available option in this scenario. But not any longer.

The Onyx^® platform is here to take the guesswork out of Diversity Generation for obtaining beneficial variants. Without restricting ourselves to a short list of usual suspects such as transporters and stress response genes, or poking around in the dark with random mutagenesis, we pursued a methodical survey of the entire E. coli genome by rapidly generating genomewide knock-out and promoter ladder libraries with precise and controlled gene editing.