Oxaloacetate hydrolase deficient fungal host cells

Details Oxaloacetate hydrolase deficient fungal host cells Oxaloacetate hydrolase deficient fungal host cells

2000-02-10

2003-04-08

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S320100, C435S252300, C435S325000, C435S252330, C536S023200

Reexamination Certificate

active

06544765

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to isolated nucleic acid sequences encoding polypeptides having oxaloacetate hydrolase activity. The present invention further relates to mutant host cells, in particular fungal mutant host cells such as cells of the genus Aspergillus, deficient in oxaloacetate hydrolase activity and thereby in oxalic acid production. The present invention also relates to the use of the mutant cells for producing desirable compounds, such as polypeptides, primary and secondary metabolites, and to methods of producing such compounds in the mutant cells of the invention. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides having oxaloacetate hydrolase activity.
2. Description of the Related Art
Filamentous fungi are widely used for the commercial production of a variety of compounds of interest, including homologous compounds, such as primary or secondary metabolites and polypeptides normally produced by the fungus in question or heterologous compounds, such as heterologous polypeptides encoded by foreign DNA which has been introduced into the fungus in question. Such products are produced by fermentation of the fungus in question and harvest of the desired product resulting from the fermentation.
The fungal species
A. niger
is widely used for the commercial production of desired compounds, eg citric acid and industrial enzymes. It is well-known that this species produces large amounts of oxalic acid. For a number of reasons the production of oxalic acid is undesirably when this species is used for commercial production of a compound of interest. For instance, the production of oxalic acid requires a lot of carbon and thus extra, expensive carbon sources must be added to the fermentation medium compared to what would be required only for the production of the desired compound; the presence of oxalic acid in the fermentation broth causes problems in the downstream processing involved in the recovery of the product of interest because oxalic acid forms a precipitate with calcium which interferes in the recovery; and oxalic acid is a toxic compound which means that its presence is considered a major problem in the production of food grade products from
A. niger.
Two possible routes for the pathway for biosynthesis of oxalic acid in
A. niger
have been suggested. The first route is oxaloacetate+water→Oxalic acid+acetate, the reaction being catalyzed by oxaloacetate hydrolase (Kubicek, C. P., G. Schreferl-Kunar, W. Wöhrer and M. Röhr (1988) Appl. Environ. Microbiol. 54, 633-637). The second route involves the glycoxylate pathway (Balmforth, A. J., A. Thomson (1984): Biochem. J. 218 113-118).
It has been attempted to control the formation of oxalic acid during commercial
A. niger
fermentations by conducting the fermentation at a low pH where only little oxalic acid is formed. However, fermentation at low pH may be undesirable since normally this pH is not optimal for the growth of
A. niger
and yield of a desired fermentation product.
A partially purified oxaloacetate hydrolase from
A. niger
has been described (Lenz et al., Partial purification and some properties of oxaloacetate from
Aspergillus niger,
1976, Eur.
J. Biochem. 65: 225-236), but the gene encoding this enzyme has not been described.
It is an object of the present invention to provide mutants of cells, such as filamentous fungal cells, in particular cells of
A. niger,
which is deficient in oxaloacetate hydrolase production and thereby oxalic acid production.
SUMMARY OF THE INVENTION
The present invention relates to isolated nucleic acid sequences encoding polypeptides having oxaloacetate hydrolase activity, selected from the group consisting of:
(a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 65% identity with the amino acid sequence of the oxaloacetate hydrolase of SEQ ID NO:2;
(b) a nucleic acid sequence having at least 65% homology with the coding part of the DNA sequence SEQ ID NO:1 (constituted by nucleotides 1157-1411, 1504-1651 and 1764-2383 of SEQ ID NO:1)
(c) a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1, (ii) the cDNA sequence of SEQ ID NO:1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
(d) an allelic variant of (a), (b), or (c); and
(e) a subsequence of (a), (b), (c), or (d), wherein the subsequence encodes a polypeptide fragment which has oxaloacetate hydrolase activity.
In a further important aspect the invention relates to a method for producing a mutant of a cell, which comprises disrupting or deleting the nucleic acid sequence of the invention or a control sequence thereof, which results in the mutant producing less of the oxaloacetate hydrolase and thus oxalic acid than the cell. The present invention also relates to a mutant produced by this method.
The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.


REFERENCES:
patent: 2089615 (1997-09-01), None
Ruijter et al., (1999) Microbiology 145:2569-2576.
Lenz et al., (1976) Eur. J. Biochem 65:225-236.
Kubicek et al., (1988) Applied and Environmental Microbiology 633-637.
Balmforth et al., (1984) Biochem. J. 218:113-118.
EMBL, Database Genbank, Acc. No. T82752.
Golubtsova et al., Mikrobiologiya, vol., XLVIII, Issue 6, (1979) (and a translation thereof).
Scherbakova et al., Microbiology, vol. 43, p. 432-436 (1974).
Kasatkina et al., Microbiology, vol. 94(3), p. 511, (1965).

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