Yeast strains expressing the lactic lacticodeshydrogenase gene a

Details Yeast strains expressing the lactic lacticodeshydrogenase gene a Yeast strains expressing the lactic lacticodeshydrogenase gene a

1994-11-25

1998-01-27

Ketter, James

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

4353201, 536 232, 536 241, C12N 119, C12N 1531, C12N 1581

Patent

active

057121524

DESCRIPTION:

BRIEF SUMMARY
The invention relates to the construction of yeasts expressing an alcoholic/lactic mixed fermentation.
Both of these major types of fermentation of sugars are traditionally employed in the agricultural foodstuffs industry: alcoholic fermentation, for which yeasts of the genus Saccharomyces are responsible, leads chiefly to the formation of ethanol and CO.sub.2 ; and lactic fermentation (lactic bacteria) leads to the formation of lactic acid.
Alcoholic fermentation and lactic fermentation possess metabolic pathways which are almost coincident as far as pyruvate. At this stage, this intermediate is treated differently by two final electron acceptor systems. In alcoholic fermentation, pyruvate is decarboxylated to acetaldehyde, the latter compound being reduced to ethanol by means of an alcohol dehydrogenase; in the lactic fermentation, pyruvate is reduced directly to lactate by means of a lactic dehydrogenase (LDH).
The microorganisms used in agricultural food-stuffs possess one or other of these two metabolic pathways, and accomplish one or other of these two fermentations exclusively.
The inventors undertook to construct a yeast strain capable of accomplishing both alcoholic fermentation and lactic fermentation, resulting in a fermentation having an outcome intermediate between these two types.
The principle of the construction carried out by the inventors consisted in cloning the gene for a lactic dehydrogenase of a GRAS lactic bacterium (Lactobacillus casei), and in achieving expression of this gene in Saccharomyces so as to establish therein, at the end of the carbon flux, an electron acceptor system which has to compete with the wild-type system.
Such a construction had never been proposed in the prior art, since it was generally accepted that three major groups of obstacles could stand in the way of its functioning: and fermentative activity of Saccharomyces, resulting, for example, from a problem of membrane translocation of lactate.
Now, surprisingly, the inventors have managed to obtain a viable and functional system which leads to the diversion of a large part of the carbon flux towards lactate.
The subject of the present invention is yeast strains, which contain at least one copy of a gene coding for a lactic bacterium LDH, under the control of sequences regulating the expression of said gene in yeast.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 summarizes the mutagenesis strategy, showing the 3.5 kb insert contained in plasmid pG4 together with the coding region of LDH gene. The PCR primers are also shown (SEQ ID NOS: 5-8).
FIG. 2 diagrams the steps of reconstruction of the gene.
FIG. 3 shows the introduction of the LDH gene into the multicopy plasmid pVT100-U.
FIG. 4 shows the introduction of the LDH gene into the single-copy plasmid YCp50.
FIGS. 5A and 5B show the growth of transformant V5/pVT100-U-LDH* and a control strain, respectively.
FIGS. 5C and 5D show the lactate and ethanol production of transformant V5/pVT100-U-LDH* and a control strain, respectively.
FIG. 5E shows the specific activity of LDH over time for the transformant V5/pVT100-U-LDH*.
Sequences regulating the expression of a gene" are understood to mean sequences of the promoter and terminator type which are active in yeast. The promoters and terminators of different genes may be used, combined in different combinations. The promoters and terminators, known per se, of the alcohol dehydrogenase I (ADHI), phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDE) genes may be mentioned by way of a non-limiting example.
The invention also comprises the expression cassette obtained by combining said regulatory sequences and the LDH gene; this cassette may be carried by a plasmid, or integrated in the chromosomal DNA of the host yeast.
According to a preferred embodiment of the present invention, said yeasts belong to the genus Saccharomyces.
According to another preferred embodiment of the present invention, the gene expressed is that for Lactobacillus casei LDH.
For the integrated gene to be expressed in y

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