Cloning and expression of the gene of the malolactic enzyme of L

Details Cloning and expression of the gene of the malolactic enzyme of L Cloning and expression of the gene of the malolactic enzyme of L

1995-01-17

1996-12-24

Patterson, Jr., Charles L.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

4353201, 43525233, 4352542, 43525421, 435189, 536232, C12P 750, C12N 1553, C12N 1570, C12N 1581

Patent

active

055873049

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to the cloning of the structural gene for the malolactic enzyme of Lactococcus lactis, to its complete nucleotide sequence and to the deduced protein sequence, as well as to DNA fragments carrying the sequence encoding this gene. It also relates to the expression of this gene in various prokaryotic or eukaryotic hosts, and in particular in Saccharomyces and Schizosaccharomyces.
Malolactic fermentation, a secondary fermentation which is observed during the process of winemaking in addition to the alcoholic fermentation performed by yeasts, consists in the degradation, by certain lactic acid bacteria, of the malic acid present in wines, to lactic acid and CO.sub.2. The bacterial species naturally present in the fermentative flora and which are responsible for the malolactic fermentation belong to the genera Lactobacillus, Leuconostoc and Pediococcus.
This reaction has two main advantages: (L-malate) to a monoacid (L-lactate), the consequences of which are advantageous at the organoleptic level, for the majority of red wines and some white wines, better preservation, by using a fermentable substrate, which prevents subsequent fermentations by other strains of undesirable microorganisms. Finally, malolactic fermentation can have an influence on the quality of the wine from the aromatic point of view.
This reaction poses, however, a certain number of problems because of its slowness and its uncontrollable character.
Malolactic fermentation occurs spontaneously only at the end of the natural wine-making process; indeed, the pH, the ethanol content and the presence of SO.sub.2 in the wine considerably slow down the growth of the lactic acid bacteria naturally present in the fermenting mass and which are responsible for the malolactic fermentation. In order to try to accelerate this fermentation, the technique most commonly used lies in the addition, to the fermenting medium, of an inoculum of lactic acid bacteria.
However, this technique does not always resolve the problems of implantation of the lactic acid bacteria because of the difficulty which they have in adapting to the wine medium. Moreover, the introduction of lactic acid bacteria amounts to adding, in addition to the malolactic enzyme whose activity is desired, numerous other enzymes whose action on the numerous substrates present in the wine can result in a development of the organoleptic qualities of the product which is very difficult to control.
It would therefore be desirable to have the possibility of carrying out the malolactic fermentation so as to arrive rapidly, and while removing uncontrollable variables, at an improvement of the product obtained.
With the aim of better controlling the malolactic fermentation, it has been proposed to clone the genes for the malolactic enzymes of lactic acid bacteria, and to introduce and express them in the microorganism which is normally involved in the manufacture of wine, such as S. Cerevisiae.
WILLIAMS et al. [App. Environ. Microbiol. 47: 288-293 (1984)], as well as European Application 103300 describe the cloning of a 5 kb DNA fragment carrying the gene for the malolactic enzyme L. delbrueckii into S. Cerevisiae and into E. coli.
The gene for the malolactic enzyme of L. oenos has also been cloned into E. Coli [LAUTENACH et al., Microbiol., 39:29-39 (1984)].
However, the strains of microorganisms transformed express the gene only at a very low level, insufficient to permit its use in the manufacture of wine.
In addition, in both cases, an instability of the DNA cloned into E. Coli was observed, even going as far as bringing about the complete loss of the gene.
Moreover, a malolactic enzyme, having an activity of a level comparable to that of the malolactic enzyme of bacteria of the genera Leuconostoc, Pediococcus and Lactobacillus mentioned above, have recently been purified from Lactococcus lactis. It is a protein of about 230 kDa, consisting of subunits of about 56 kDa. Its pHi. is about 4.3, and its Km for malic acid is about 10 to 12 mM. Its N-terminal sequence has also b

REFERENCES:
Ansanay, V., et al. (1993) FEBS Letts. 332 (1,2), 74-80.
Danayrolles, M., et al. (1994) FEMS Microbiol. Letts. 116, 79-86.
Renault, P., et al. (1990) Biotech. Abs. 92-03363.
Renault, P., et al. (1987) Biosis Previews Database, 83085283.

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