Recombinantly producing levodione reductase

#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.

#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 NH4+.

#The N-terminal amino acid sequence of purified Corynebacterium aquaticum levodione reductase was determined by automated Edman degradation using a model 476A pulsed liquid protein sequencer (PE Biosystems). The sequence is similar to the NH2-terminal amino acid sequences of other short-chain dehydrogenase/reductase (SDR) family enzymes, such as the biphenyl-2,3-dihydro-2,3-diol dehydrogenase of Pseudomonas sp. strain KKS102 (Biochem. Biophys. Res. Commun. 2.02, 850-856, 1994). Moreover, this NH2-terminal amino acid sequence contains G-X-X-X-G-X-G, which is a highly conserved motif in the NH2-terminal regions of SDR family proteins. The amino acid sequence obtained from the N-terminal of levodione reductase is shown in Table 1.

#TABLE 1 N-terminal amino acid sequence (SEQ ID NO:3) TATSSPTTRFTDRVVLITGGGSGLGRATAVRLAAEGAKLSSVD

#The levodione reductase protein was digested with lysyl endopeptidase, and the resulting digest was separated by the Smart system (Amersham Pharmacia Biotech). The K-1, K-2, and K-3 peptides were isolated, and the amino acid sequences of these peptides were analyzed with a protein sequencer. The K-1, K-2, and K-3 sequences are shown in Table 2. When these sequences were compared with the sequences in three protein sequence databases (PIR, PRF, and SWISS-PROT) using the sequence similarity search programs Blast and Fasta, the K-1 and K-2 sequences were found to be significantly similar to partial amino acid sequences of a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase of S. paucimobilis (J. Bacteriol. 176, 3117-3125, 1994) and a 3-oxoacyl-[acyl-carrier protein] reductase of Haemophilus influenzae (Whole-genome random sequencing and assembly of Haemophilus influenzae Rd, Science 269, 495-512, (1995)), respectively, both of which belong to the SDR family. However, K-3 did not exhibit significant similarity to any other SDR family proteins.