title: Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance.
abstract: BACKGROUND
  During the final phases of bioethanol fermentation, yeast cells face high ethanol concentrations. This stress results in slower or arrested fermentations and limits ethanol production. Novel Saccharomyces cerevisiae strains with superior ethanol tolerance may therefore allow increased yield and efficiency. 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, which can potentially limit the effectiveness. Here, we explore novel robot-assisted strategies that allow to shuffle the genomes of multiple parental yeasts on an unprecedented scale.
  
  
  RESULTS
  Screening of 318 different yeasts for ethanol accumulation, sporulation efficiency, and genetic relatedness yielded eight heterothallic strains that served as parents for genome shuffling. In a first approach, 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 3,000 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 an 8-l pilot-scale test, the best-performing hybrid fermented 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.
  
  
  CONCLUSIONS
  We report the use of several different large-scale genome shuffling strategies to obtain novel hybrids with increased ethanol tolerance and fermentation capacity. Several of the novel hybrids show best-parent heterosis and outperform the commonly used bioethanol strain Ethanol Red, making them interesting candidate strains for industrial production.
authors.affiliation: Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium.|Laboratory for Systems Biology, VIB, Bio-Incubator, Gaston Geenslaan 1, 3001 Leuven, Belgium.
authors.forename: Tim
authors.initials: T
authors.lastname: Snoek
authors.affiliation: Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium.|Laboratory for Systems Biology, VIB, Bio-Incubator, Gaston Geenslaan 1, 3001 Leuven, Belgium.
authors.forename: Martina
authors.initials: M
authors.lastname: Picca Nicolino
authors.affiliation: Laboratory of Enzyme, Fermentation and Brewing Technology, KU Leuven technologiecampus Ghent, Gebroeders De Smetstraat 1, 9000 Ghent, Belgium.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Van den Bremt
authors.affiliation: Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium.|Laboratory for Systems Biology, VIB, Bio-Incubator, Gaston Geenslaan 1, 3001 Leuven, Belgium.
authors.forename: Stijn
authors.initials: S
authors.lastname: Mertens
authors.affiliation: Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium.|Laboratory for Systems Biology, VIB, Bio-Incubator, Gaston Geenslaan 1, 3001 Leuven, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Saels
authors.affiliation: Laboratory of Enzyme, Fermentation and Brewing Technology, KU Leuven technologiecampus Ghent, Gebroeders De Smetstraat 1, 9000 Ghent, Belgium.
authors.forename: Alex
authors.initials: A
authors.lastname: Verplaetse
authors.affiliation: Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium.|Laboratory for Systems Biology, VIB, Bio-Incubator, Gaston Geenslaan 1, 3001 Leuven, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Steensels
authors.affiliation: Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium.|Laboratory for Systems Biology, VIB, Bio-Incubator, Gaston Geenslaan 1, 3001 Leuven, Belgium.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Verstrepen
keywords: Bioethanol; Breeding; Complex phenotype; Ethanol tolerance; Evolutionary engineering; Fermentation; Genome shuffling; Heterosis; Hybridization; Very high gravity