Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-28
2001-08-14
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S254110, C435S254210, C435S254200, C435S069100, C435S320100, C530S350000, C536S023700, C536S023740
Reexamination Certificate
active
06274311
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method and nucleotide sequence for transforming microorganisms. More particularly, the invention relates to a recombinant DNA molecule, to a gene, to a polypeptide, to a transformed yeast strain, to a method of transforming a yeast strain, to a method of producing a desired polypeptide, and to a fermentation method.
BACKGROUND OF THE INVENTION
The transport of L-malic acid across the plasma membrane and its degradation in microorganisms is of considerable interest in many fields, particularly those involving fermentation by yeasts. L-malic acid may be used as a sole carbon and energy source by the yeasts
Candida sphaerica
(Corte-Real et al., 1989),
Hansenula anomala
(Corte-Real and Leao, 1990) and
Candida utilis
(Cassio and Leao, 1993). The dissociated form of malate is transported across the plasma membrane by proton symports which are inducible and subjected to glucose repression. However, in
Zygosaccharaomyces bailii
(Rodriquez and Thornton, 1990) and
Schizosaccharomyces pombe
(
S. pombe
) (Sousa et al., 1992), L-malic acid can only be metabolized in the presence of an assimilable carbon source (Osothsilp and Subden, 1986). L-malic acid is actively transported in the dissociated form whereas the undissociated acid enters the cell via simple diffusion (Baranowski and Radler, 1984; Osothsilp and Subden, 1986; Sousa et al., 1992). Competitive inhibition of initial uptake rates of L-malic acid by succinic acid, D-malic acid, faumaric acid, oxaloacetic acid, &agr;-ketoglutaric acid, maleic acid and malonic acid strongly suggests that these acids are transported by the same carrier in
S. pombe
(Sousa et al, 1992).
Malic acid degradation is of particular interest to wineries. Wine yeast strains of
Saccharomyces cerevisiae
(
S. cerevisiae
) cannot metabolize malate in grape must efficiently and changes in the total acidity of the wine during vinification are therefore insignificant (Gao, 1995). Production of well-balanced wines requires the controlled reduction of excess malic acid, particularly in the colder viticultural regions of the world.
Chemical deacidification has been used to reduce the total acidity of wine. Chemical deacidification is typically carried out by (a) amelioration—which is essentially dilution of the malic acid with sugar water; (b) precipitation—the addition of calcium, potassium or other cations to produce an insoluble salt; or (c) masking—adding grape juice or sucrose to the finished wine to mask the sour taste of malic acid. All these methods result in residual malate which can support malolactic fermentation by contaminating bacteria unless treated with elevated doses of sulfites.
Malolactic fermentation methods for malic acid degradation rely on the conversion of L-malic acid to L-lactic acid and CO
2
by malolactic bacteria, for example, species of Leuconostoc, Lactobacillus, and Pediococcus. The malolactic bacteria may be found on grapes which become part of the winery microflora, or commercially available frozen or freeze-dried cultures of the bacteria may be introduced into the wine. Malolactic fermentation methods have a number of disadvantages; for example, the malolactic bacteria ferment terpenes which change the character of the wine. Control of malolactic fermentations is often difficult resulting in incomplete malolactic fermentation and subsequent bottle fermentations. Bacterial growth is also usually accompanied by the production of carbon dioxide which may result in “fizzy” wine.
Yeast strains which can degrade L-malic acid have also been used in wine fermentations. Fermentations using the fission yeast
S. pombe
which completely degrades malate to ethanol through a malo-ethanolic fermentation have been attempted. Thornton (U.S. Pat. No. 4,830,968) describes a method involving inoculating grape juice with a strain of
Saccharomyces malidevorans
which is capable of some degradation of L-malic acid under wine making conditions. However, these yeast strains (i.e.
Schizosaccharomyces pombe
and
Saccharomyces malidevorans
) are not desirable in wine making since off-flavours are produced. High density cell suspensions of several yeasts, including
S. cerevisiae
have also been used to try to increase the rate at which L-malate is degraded during fermentation (Gao, 1995).
Attempts have been made to hybridize wine yeasts with malate-metabolizing yeast strains. Protoplast fusion (Carrau et al., 1982; Svoboda, 1980, U.S. Pat. No. 5,330,774 to Carrau et al.), transformation (Lautensach and Subden, 1984; Williams et al., 1984), and other means (Fernandez, 1967; Goto et al., 1978; Kuczynski and Radler, 1982) have not been successful.
Metabolic engineering of
S. cerevisiae
strains to carry out alcoholic fermentation and malolactic or malo-ethanolic fermentation simultaneously has been explored. The malolactic gene (mleS) from
Lactobacillus delbrueckii
(Williams et al., 1984) and
Lactococcus lactis
(Ansanay et al., 1993, Denayrolles et al., 1994) have been cloned, characterized and several attempts have been made to introduce and express this gene in
S. cerevisiae
. However, recombinant strains of
S. cerevisiae
expressing the mleS gene were unable to degrade malate effectively to L-lactate (Williams et al., 1984; Ansanay et al., 1993, Denayrolles et al., 1995).
SUMMARY OF THE INVENTION
The present inventors have identified a gene in
S. pombe
, designated mae1 or malate permease gene, which encodes a dicarboxylic acid permease (referred to herein as “malate permease” or “Mae1”). This is the first molecular characterization of a dicarboxylic acid permease in a eukaryotic cell. The
S.pombe
mae1 gene encodes a single mRNA of 1.5 kb. The gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease gene of
C. utilis
(Cassio and Leas, 1993) and
H. anomala
(Corte-Real and Leao, 1990). The mae1 gene was mapped to 2842 bp 5′ to the MFm1 gene on Chromosome I.
Transport assays revealed that the mae1 gene encodes a malate permease involved in the transport of L-malate, succinate, and malonate. The
S. pombe
malate permease has 435 amino acid residues with a molecular weight of approximately 49 kDa.
Mae1 from
S. pombe
contains a number of well-characterized regions including two protein kinase C phosphorylation sites, a PEST region, a leucine zipper region, two hydrophillic linker regions, and ten membrane-spanning helices. In particular, a well conserved PEST region (amino acids 421-434 in
FIG. 3
, SEQ ID NO:2) is found at the C-terminal end, consisting of proline (P), glutamic acid (E), serine (S), threonine (T) and to a lesser extent aspartic acid. A leucine zipper motif (amino acids 214 to 235 in
FIG. 3
, SEQ ID NO:2), consisting of four leucine residues spaced by 6 amino acids, is located between membrane-spanning domains six and seven. Protein kinase C phosphorylation sites were found at positions 28: phvplSqrlkh and at position 94: ikypsTikdsw. Mae1 from
S.pombe
also contains three potential N-linked glycosylation sites located at amino acids 193, 277 and 336 (
FIG. 3
, SEQ ID NO:2).
The present inventors have introduced an efficient pathway for malate degradation in
S. cerevisiae
by cloning and expressing the
S. pombe
malate permease (mae1) and malic enzyme (mae2) genes in this yeast. Recombinant strains efficiently degraded 8 g/l of malate within 7 days. A recombinant strain of
S. cerevisiae
containing both the
S. pombe
mae 1 and
L. lactis
mleS genes was also shown to efficiently and rapidly degrade L-malate to L-lactate in grape must in a significantly short period of time. The present inventors have shown the efficacy of these recombinant strains (mae1, mae2, and mae1mleS) for maloethanolic fermentation, and malolactic fermentation, respectively.
The present invention therefore provides an isolated nucleic acid molecule comprising a sequence which encodes a polypeptide which mediates the uptake of L-malate, succinate, and malonate. The nucleic acid molecule may comprise the malate per
Grobler Jandre
Krizus Aldis
Osothsilp-Deeknamakul Chauanpit
Pretorius Isak S.
Subden Ronald E.
Bereskin & Parr
Gravelle Micheline
Nashed Nashaat T.
University of Guelph
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