Author:

Abstract:

The yeast Saccharomyces cerevisiae is the preferred microorganism to carry out ethanolic fermentations due to its high ethanol tolerance and excellent fermentation capacity. Nevertheless, during the final phases of very high gravity (VHG) fermentations, which are common in brewing and bioethanol production, yeast cells face high ethanol concentrations. This stress can result in slower or arrested fermentations and limits ethanol production. Novel S. cerevisiae strains with superior ethanol tolerance may therefore allow increased yield and efficiency. Insight into the molecular mechanisms underlying ethanol tolerance can potentially guide the development of these stains, yet the polygenic nature of this phenotype has hampered the emergence of a detailed picture about this phenotype, making successful rational strain improvement approaches sparse. Alternatively, global approaches that generate a large pool of genetic variation in a random fashion, including directed evolution, genome-wide mutagenesis, and genome shuffling, have yielded more success. Over the last decade, genome shuffling has emerged as a powerful approach to rapidly enhance complex traits including ethanol tolerance, yet previous efforts have mostly relied on a mutagenized pool of a single strain or only used a limited number of parental strains, which can potentially limit the effectiveness. In this study, we set out to apply different genome shuffling strategies that allow to shuffle the genomes of multiple parental yeasts on an unprecedented scale. We first carried out a screening of 318 different yeasts for ethanol accumulation, sporulation efficiency and genetic relatedness, which yielded eight heterothallic strains that served as parents for genome shuffling. In a first strategy, the parental strains were subjected to multiple consecutive rounds of random genome shuffling with different selection methods, yielding several hybrids that showed increased ethanol tolerance. Interestingly, on average, hybrids from the first generation (F1) showed higher ethanol production than hybrids from the third generation (F3). In a second approach, we applied several successive rounds of robot-assisted targeted genome shuffling, yielding more than 3000 targeted crosses. Hybrids selected for ethanol tolerance showed increased ethanol tolerance and production as compared to unselected hybrids, and F1 hybrids were on average superior to F3 hybrids. In total, 135 individual F1 and F3 hybrids were tested in small-scale very high gravity fermentations. Eight hybrids demonstrated superior fermentation performance over the commercial biofuel strain Ethanol Red, showing a 2 to 7% increase in maximal ethanol accumulation. In a pilot-scale test, the best-performing hybrid fermented 8 L medium containing 32% (w/v) glucose to dryness, yielding 18.7% (v/v) ethanol with a productivity of 0.90 g ethanol/l/h and a yield of 0.45 g ethanol/ g glucose. These novel hybrids are interesting candidate strains for industrial production.