The combination of glycerol metabolic engineering and drug resistance marker-aided genome shuffling to improve very-high-gravity fermentation performances of industrial Saccharomyces cerevisiae

► Metabolic engineering and genome shuffling were combined for breeding yeast strains. ► Glycerol formation was reduced by hetero-expression GAPN and disrupting FPS1p. ► The ethanol tolerance of engineering strains was improved by novel genome shuffling. ► The mutant produced more ethanol and less byproduct with faster fermentation rate. ► The use of drug-resistance markers improved the efficiency of the breeding strategy.