Metabolic engineering of Saccharomyces cerevisiae to enhance resistance toward microbial toxicity in agave extracts.

Agave, which thrives in arid regions of North Americas, is a promising alternative feedstock for biofuel production due to its high sugar content and drought tolerance. A major challenge to using agave as a feedstock for bioprocessing is its toxicity. Saponins, natively produced glycosides, disrupt cell membranes and are highly toxic to Saccharomyces cerevisiae even at low concentrations. To enable agave as a feedstock, we combined rational engineering with adaptive laboratory evolution (ALE) to create new S. cerevisiae strains with high tolerance to agave extracts. Heterologous overexpression of tomatinase from Fusarium oxysporum improved tolerance up to 10% agave extracts. In parallel, 27 serial passages of ALE resulted in evolved strains that can grow in 4% extracts and produce ethanol equivalent to the starting strains. Whole genome sequencing revealed mutations in coding sequences of multidrug efflux pumps (PDR5 and PDR1) and a non-coding sequence adjacent to α-glucosidase gene (IMA3), suggesting roles in resistance to saponins. Combining both approaches - tomatinase overexpression and evolved strains - yielded a high-tolerant strain that produced ethanol in 20% agave extracts. Our dual approach to strain engineering demonstrates an effective multi-level strategy of developing microbial hosts with increased resistances to toxicity feedstock components, ultimately enabling new bioprocesses.