Recombinantly producing levodione reductase

Details Recombinantly producing levodione reductase Recombinantly producing levodione reductase

2002-12-23

2004-01-06

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Preparing oxygen-containing organic compound

C435S189000, C536S023200

Reexamination Certificate

active

06673583

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to DNA encoding levodione reductase, an expression vector containing the DNA, a recombinant vector containing the DNA, a microorganism into which the DNA has been introduced, and a method for producing (4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (hereinafter referred to as actinol) from (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (hereinafter referred to as levodione) using the microorganism. Actinol is a useful chiral building block of naturally occurring optically active compounds such as zeaxanthin.
BACKGROUND OF THE INVENTION
European Patent Application No. 98115564.1, filed on Aug. 19, 1998, discloses a process for the manufacture of actinol, which involves contacting levodione with a microorganism selected from the group consisting of microorganisms of the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter, which is capable of selective asymmetric reduction of levodione to actinol, and recovering the resulting actinol from the reaction mixture. In this process, one of the most effective strains was
Corynebacterium aquaticum
AKU611 (FERM BP-6448), which was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, in the name of F. Hoffmann-La Roche A G of Grenzacherstrasse 124, CH-4070 Basel, Switzerland on Aug. 4, 1998, under the Budapest Treaty.
European Patent Application No. 99102037.1, filed on Feb. 1, 1999, discloses an enzyme, levodione reductase, that acts on levodione to produce actinol, which was isolated from
Corynebacterium aquaticum
AKU611 (FERM BP-6448). This enzyme is characterized by the following physico-chemical properties: 1) The levodione reductase catalyzes the regio- and stereoselective reduction of levodione to actinol. 2) The relative molecular mass of the enzyme is estimated to be 142,000-155,000±10,000 Da, consisting of four homologous subunits having a molecular mass of 36,000±5,000 Da. 3) The optimum temperature is 15-20° C. at pH 7.0 and the optimum pH is 7.5. 4) The enzyme requires NAD
+
or NADH as a cofactor and is highly activated by monovalent cations, such as K
+
, Na
+
, Cs
+
, Rb
+
, and NH
4
+.
SUMMARY OF THE INVENTION
An object of the present invention is a DNA sequence encoding for an enzyme, levodione reductase, which is useful for the preparation of actinol, an important intermediate in the production of zeaxanthin.
The isolated DNA sequence may be more specifically characterized by the following:
(a) the nucleotide sequence codes for the enzyme having the amino acid sequence shown in SEQ ID No.: 1, or
(b) the nucleotide sequence codes for a variant of the enzyme selected from (i) an allelic variant, or (ii) an enzyme having one or more amino acid additions, insertions, deletions and/or substitutions, but still having the same type of enzymatic activity.
The isolated DNA sequence mentioned above may be derived from a gene of
Corynebacterium aquaticum
and selected from
(i) the nucleotide sequence shown in SEQ ID No.: 2;
(ii) a nucleotide sequence which, because of the degeneracy of the genetic code, encodes a levodione reductase having the same amino acid sequence as that encoded by SEQ ID No: 2, or
(iii) a nucleotide sequence which hybridizes to the complement of the nucleotide sequence from (i) or (ii) under standard hybridizing conditions.
Another object of the present invention is a recombinant DNA which codes for levodione reductase and which can be obtained by genetic recombination of the isolated DNA described above. Such a recombinant DNA may preferably be in the form of a vector. The recombinant DNA may contain regulatory regions, such as promoters and terminators, as well as an open reading frame of the gene described above.
A further object of the invention is a recombinant organism consisting of a host organism transformed with the recombinant DNA. A preferred form of the recombinant DNA is a vector. The host organism transformed with the recombinant DNA may be useful in improving the process of actinol production.
Another object of the present invention is a method for the biological production of actinol that includes introducing a recombinant DNA, as described above, into an appropriate host organism, and cultivating the resulting recombinant organism in the presence of levodione as a substrate.
Accordingly, the invention provides an isolated polynucleotide encoding a polypeptide having levodione reductase activity.
Another embodiment of the invention is a vector or a plasmid containing a polynucleotide sequence encoding a polypeptide having levodione reductase activity wherein the polypeptide has the properties as set forth above.
A further embodiment of the invention is a microorganism transformed or transfected with a polynucleotide sequence which encodes a polypeptide having levodione reductase activity wherein the polypeptide has the properties as set forth above.
Another embodiment of the invention is an isolated polypeptide having levodione reductase activity wherein the polypeptide has the properties as set forth above.
A further embodiment of the invention is a process for producing a polypeptide having levodione reductase activity. This process includes culturing a microorganism transformed or transfected with a polynucleotide encoding a polypeptide having levodione reductase activity in nutrient media and isolating the polypeptide produced by the microorganism.
The present invention also provides a process for producing actinol. This process includes contacting levodione with a polypeptide having levodione reductase activity.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, “levodione reductase” is a polypeptide that catalyzes, regio- and stereoselectively, the conversion of levodione to actinol in the presence of NADH. European Patent Application No. 99102037.1, filed on Feb. 1, 1999, discloses the physico-chemical properties of the levodione reductase. The levodione reductase of the invention can be prepared by cultivating an appropriate microorganism in an aqueous nutrient medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the levodione reductase from the cell-free extract. Many species have been found to catalyze this conversion, including the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter. A preferred strain for the enzyme is
Corynebacterium aquaticum
AKU611 (FERM BP-6448).
As used herein, an “allele” or “allelic variant” is an alternative form of a gene, which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutational changes that give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times, in a given sequence.
A “variant” of levodione reductase, as used herein, is an amino acid sequence that is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have “non-conservative” changes, e.g., replacement of glycine with tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. A “deletion”, as used herein, refers to a change in either the amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent. An “insertion” or “addition”, as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule. A “substitution”, as used herein, refers to the replacement of one or more amino acids or nucleot

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