Malolactic Fermentation in Grape Musts by a Genetically Engineered Strain ofSaccharomyces cerevisiae

Abstract

Malate enters Saccharomyces cerevisiae by simple diffusion. Due to the lack of a malate transporter and the low affinity of the S. cerevisiae malic enzyme, this yeast is unable to degrade malate efficiently. We have constructed a malolactic yeast strain by co-expressing the malate permease gene (mae 1) of the fission yeast Schizosaccharomyces pombe and the Lactococcus lactis malolactic gene (mleS) in S. cerevisiae. The recombinant strain of S. cerevisiae transported malate and actively metabolized malate to lactate within three days in Cabernet Sauvignon and Shiraz grape musts at 20°C. The malolactic fermentation in Chardonnay grape must was completed within seven days at 15°C. The efficient degradation of malate in grape musts is important to wineries and the availability of malolactic yeasts will allow the early application of cellar operations for storage and aging of wine.

Constructing recombinantSaccharomyces cerevisiaestrains for malic-to-fumaric acid conversion

“…Both plasmids contain the URA3 marker and the ENO1 promoter and terminator sequences, whilst pBBH4 includes the T. reesei XYNSEC secretion signal upstream of the cloning site (Njokweni et al 2012 ). Plasmid pHV3 (Volschenk et al 1997a ), containing the S. pombe malate permease gene ( mae1 ) and LEU2 selectable marker, was used to promote the active transport for malic acid and fumaric acid, as opposed to passive diffusion. Plasmid YEplac181 served as a LEU2 control.…”