S13: Evolutionary engineering for hydrolysates tolerance in yeast

Inhibitory compounds are generally formed during the pretreatment and hydrolysis of lignocellulosic biomass.  These inhibitors interfere with microbial growth and reduce productivity.  Thus, the development of more robust biocatalysts is necessary for the efficient use of hydrolysates as feedstock for the production of chemicals and fuels.  Using evolutionary engineering, we evolved Saccharomyces cerevisiae for enhanced tolerance to hydrolysates.  Using the Visualizing Evolution in Real-Time (VERT) method, we ramped-up the selective in a more rational manner and isolated and characterized adaptive mutants from one evolving population.  Growth kinetic analyses of the mutants in individual and combinations of common inhibitors present in hydrolysates (acetic acid, furfural, and hydroxymethylfurfural) showed differential levels of resistance to different inhibitors, with enhanced growth rates up to 57%, 12%, 22%, and 24% in hydrolysates, acetic acid, HMF and furfural, respectively.  Interestingly, some of the adaptive mutants exhibited reduced fitness in the presence of individual inhibitors, but showed enhanced fitness in the presence of combinations of inhibitors compared to the parental strains.  Subsequent transcriptomic analysis revealed different mechanisms for resistance to hydrolysates and a potential cross adaptation between oxidative stress and hydrolysates tolerance in several of the mutants.