Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2000-05-14
2002-10-22
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S440000, C536S023200
Reexamination Certificate
active
06468773
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to mutant 1,3-propanediol dehydrogenase having an altered Km for 1,3-propanediol. The present invention provides the nucleic acid and amino acid sequence of the mutant form of 1,3-propanediol dehydrogenase. The present invention also provides expression vectors and host cells comprising mutant 1,3-propanediol dehydrogenase.
BACKGROUND OF THE INVENTION
1,3-Propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds. The production of 1,3-propanediol has been disclosed in U.S. Pat. No. 5,686,276 issued Nov. 11, 1997 and WO 98/21341. One representative pathway for the production of 1,3-propanediol from glucose can be accomplished by the following series of steps. Glucose is converted in a series of steps by enzymes of the glycolytic pathway to dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, or reduction of DHAP to glycerol 3-phosphate (G3P) followed by hydrolysis. The hydrolysis step can be catalyzed by any number of cellular phosphatases which are known to be specific or non-specific with respect to their substrates or the activity can be introduced into the host by recombination. The reduction step can be catalyzed by a NAD
+
(or NADP
+
) linked host enzyme or the activity can be introduced into the host by recombination. It is notable that the dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6) which catalyzes the reversible reaction of Equation 3.
Glycerol→3-HP+H
2
O (Equation 1)
3-HP+NADH+H
+
→1,3-Propanediol+NAD
+
(Equation 2)
Glycerol+NAD
+
→DHA+NADH+H
+
(Equation 3)
Glycerol is converted to 1,3-propanediol via the intermediate 3-hydroxypropionaldehye (3-HP) as has been described in detail above. The intermediate 3-HP is produced from glycerol (Equation 1) by a dehydratase enzyme which can be encoded by the host or can introduced into the host by recombination. This dehydratase can be glycerol dehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28), or any other enzyme able to catalyze this transformation. Glycerol dehydratase is encoded by the dha regulon. 1,3-Propanediol is produced from 3-HP (Equation 2) by a NAD
+
- (or NADP
+
) linked host enzyme or the activity can introduced into the host by recombination. This final reaction in the production of 1,3-propanediol can be catalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or other alcohol dehydrogenases.
In
Klebsiella pneumoniae
and
Citrobacter freundii
, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon. The dha regulons from Citrobacter and Klebsiella have been expressed in
Escherichia coli
and have been shown to convert glycerol to 1,3-propanediol.
Nucleic acid and amino acid sequences for a 1,3-propanediol dehydrogenase that have been disclosed in the art, including Klebsiella pneumoniae GenBank accession #U30903 (Williard, 1994, “Investigation of the Klebsiella pneumoniae 1,3-propanediol pathway: Characterization and expression of glycerol dehydratase and 1,3-propanediol oxidoreductase” Thesis Chemical Engineering, University of Wisconsin-Madison);
Citrobacter freundii
GenBank accession #U09771 (Daniel, R. et al., 1995, Purification of 1,3-propanediol dehydrogenase from
Citrobacter freundii
: cloning, sequencing, and overexpression of the corresponding gene in
Escherichia coli. J. Bacteriol
. 177:2151-2156); and
Clostridium pasteurianum
GenBank accession #AF006034 (Luers, F. et al., 1997, Glycerol conversion to 1,3-propanediol by
Clostridium pasteurianum
: cloning and expression of the gene encoding 1,3-propanediol dehydrogenase.
FEMS Microbiol. Lett
. 154:337-345).
SUMMARY OF THE INVENTION
The present invention relates to the discovery of a mutant form of 1,3-propanediol dehydrogenase (PDD) isolated from a derivative of
E.blattae
capable of growth in the presence of at least 105 g/l 1,3-propanediol, levels normally toxic to wild-type
E.blattae
. The present invention is therefore based in part upon the discovery that the mutant form of PDD is associated with
E.blattae's
resistance to normally toxic levels of 1,3-propanediol. The present invention is also based in part upon the finding that this mutant PDD has an altered Km for 1,3-propanediol and NAD.
Accordingly, the present invention provides a mutant PDD having a Km for 1,3-propanediol that is increased over the wild-type PDD Km for 1,3-propanediol. In one embodiment, the Km of the mutant PDD is about 3 times the Km of wild-type PDD for 1,3-propanediol. In another embodiment, the mutant PDD has a Km of about 80 mM for 1,3-propanediol. In a further embodiment, the mutant PDD is obtainable from
E.blattae
ATCC accession number PTA-92.
In yet another embodiment of the present invention, the mutant PDD comprises a mutation corresponding to residue His105 to Leu in
E.blatte
PDD as shown in FIG.
3
. In an additional embodiment, the mutant PDD comprises the amino acid shown in SEQ ID NO:2 and is encoded by nucleic acid having the sequence as shown in SEQ ID NO:1.
The present invention also provides expression vectors and host cells comprising the isolated nucleic acid having the sequence as shown in SEQ ID NO:1. In one embodiment, the host cell includes Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces and Pseudomonas.
In an additional aspect, the present invention relates to methods for producing 1,3-propanediol comprising the use of a microorganism comprising mutant PDD.
REFERENCES:
Boronat et al., “Experimental Evolution of Propanediol Oxidoreductase inEscherichia Coli,”Biochimica et Biophysica Acta, 672 (1981) pp. 98-107.
Daniel et al., “Purification of 1,3-Propanediol Dehydrogenase fromCitrabacter freundiiand Cloning, Sequencing, and Overexpression of the Corresponding Gene inEscherichia coli,”Journal of Bacteriology, Apr. 1995, pp. 2151-2156.
Obradors et al., “Site-directed mutagenesis studies of the metal-binding center of the iron-dependent propanediol oxidoreductase fromEscherichia coli,”Eur. J. Biochem., 258, 207-213 (1998).
Reimann et al., “1,3-Propanediol formation with product-Tolerant mutants ofClostridium butyricumDSM 5431 in continuous culture: productivity, carbon and electron flow,”Journal of Applied Microbiology1998, 84, pp. 1125-1130.
Copy of the PCT Search Report.
Donald Trimbur E.
Gregory Whited M.
Selifonova Olga V.
Achutamurthy Ponnathapu
Genencor International Inc.
Ito Richard T.
Pak Y
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