Cloning the Gene for the Malolactic Fermentation of Wine from Lactobacillus delbrueckii in Escherichia coli and Yeasts

Abstract

The gene responsible for the malolactic fermentation of wine was cloned from the bacterium Lactobacillus delbrueckii into Escherichia coli and the yeast Saccharomyces cerevisiae. This gene codes for the malolactic enzyme which catalyzes the conversion of L-malate to L-lactate. A genetically engineered yeast strain with this enzymatic capability would be of considerable value to winemakers. L. delbrueckii DNA was cloned in E. coli on the plasmid pBR322, and two E. colt clones able to colnVert L-malate to L-lactate were selected. Both clones contained the same 5-kilobase segment of L. delbrueckii DNA. The DNA segment was transferred to E. coli-yeast shuttle vectors, and gene expression was analyzed in both hosts by using enzymatic assays for L-lactate and L-malate. When grown nonaerobically for 5 days, E. coli cells harboring the malolactic gene converted about 10% of the L-malate in the medium to L-lactate. The best expression in S. cerevisiae was attained by transfer of the gene to a shuttle vector coitaining both a yeast 2-pLm plasmid and yeast chromosomal origin of DNA replication. When yeast cells harboring this plasmid were grown nonaerobically for 5 days, ca. 1.0% of the L-malate present in the medium was converted to Llactate. The L. delbrueckii controls grown under these same conditions converted about 25%. A laboratory yeast strain containing the cloned malolactic gene was used to make wine in a trial fermentation, and about 1.5% of the L-malate in the grape must was converted to L-lactatt. Increased expression of the malolactic gene in wine yeast will be required for its use in winemaking. This will require itl Increased understihding of the factors governing the expression of this gene in yeasts.

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids

“…Strains of Saccharomyces cerevisiae can only metabolize malate to a limited extent (see Radler, 1993 for a review) and strains of Leuconostoc oenos are currently used to deacidify wine. Several attempts have been made to introduce the bacterial malolactic gene into S. cerevisiae (Ansanay et al, 1993;Denayrolles et al, 1995;Williams et al, 1984). However, the recombinant yeast strains were unable to degrade malate effectively to L-lactate.…”

Microbiology of the malolactic fermentation: Molecular aspects

“…The first attempt was the cloning of the gene for malolactic activity of Lactobacillus delbrueckii into S. cereuisiae by Williams et al [24]. Only 1% of L-malate was converted into L-lactate.…”

Some roles of malic acid in the malolactic fermentation in wine making

“…The first attempt, by Williams et al [88], to clone the malolactic gene was in its own way successful. This work was reported in 1984 before much understanding of the molecular biology of the malolactic bacteria, even before much notion as to whether plasmids might be involved or not.…”