Joji Sasaki

Transformation of vitamin D3 to 1α,25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains

To enzymatically synthesize active metabolites of vitamin D3, we screened about 500 bacterial strains and 450 fungal strains, of which 12 strains were able to convert vitamin D3 to 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] via 25-hyroxyvitamin D3 [25(OH)D3]. The conversion activity was only detected in strains belonging to the genus Amycolata among all the organisms tested. A preparative-scale conversion of vitamin D3 to 25(OH)D3 and 1α,25(OH)2D3 in a 200-1 tank fermentor using A. autotrophica FERM BP-1573 was accomplished, yielding 8.3 mg 25(OH)D3/l culture and 0.17 mg 1α,25(OH)2D3/l culture. A related compound, vitamin D2, could be also converted to 25-hydroxyvitamin D2 and 1α,25-dihydroxyvitamin D2 using the same strain. The cytochrome P-450 of FERM BP-1573 was detected by reduced CO difference spectra in whole-cell suspensions. Vitamin D3 in the culture induced cytochrome P-450 and the conversion activity simultaneously, suggesting that the hydroxylation at C-25 of vitamin D3 and at C-1 of 25(OH)D3 originates from cytochrome P-450.

Application of cyclodextrin to microbial transformation of vitamin D3 to 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3

Abstract Amycolata autotrophica converts vitamin D3(VD3) to 1α,25-dihydroxyvitamin D3(1α,25(OH)2VD3) via 25-hydroxyvitamin D3(25(OH)VD3) by hydroxylation of VD3 at C-25 and C-1. In this microbial hydroxylation, it was found that cyclodextrin (CD) had the ability to enhance the hydroxylation of VD3. Addition of partially-methylated-β-cyclodextrin (PMCD) increased the productivity of 25(OH)VD3 about seven-fold compared to that without CD. Combined use of PMCD and γ-CD increased the production of 1α,25(OH)2VD3 in a tank fermentor about sixteen-fold compared to that without CD.

Cloning and nucleotide sequence of a bacterial cytochrome P-450VD25 gene encoding vitamin D-3 25-hydroxylase

The gene encoding an enzyme that catalyzes the hydroxylation at position 25 of vitamin D-3 was cloned from an actinomycete strain, Amycolata autotrophica, by use of a host-vector system of Streptomyces lividans. The amino acid sequence deduced from the nucleotide sequence revealed that this enzyme, tentatively named P-450VD25, contains several regions of strong similarity with amino acid sequences of cytochromes P-450 from a variety of organisms, primarily in the regions of an oxygen-binding site and a heme ligand pocket. Especially, P-450VD25 shows end-to-end similarity in amino acid sequence to P-450dNIR of Fusarium oxysporum and P-450SU2 of Streptomyces griseolus. The recombinant S. lividans strain containing the P-450VD25 gene on a multicopy plasmid converted vitamin D-3 in the medium into 25-hydroxyvitamin D-3 at a maximum yield of 10%.

Microbial Oxidation of KE-298 Metabolites by Rhizopus sp. and Rhodococcus sp. Strains

The metabolites of the antirheumatic agent KE-298 in humans, (-)-(2R)-M-4 [(-)-(2R)-4-(4-hydroxymethylphenyl)-2-methylthiomethyl-4-oxobutanoic acid], (-)-(2R)-M-5 [diastereomers of (-)-(2R)-4-(4-hydroxymethyl-phenyl)-2-methylsulfinyl-methyl-4-oxobutanoic acid], (-)-(2R)-M-6 [(-)-(2R)-4-(4-carboxyphenyl)-2-methylthio-methyl-4-oxobutanoic acid], and (-)-(2R)-M-7 [di- astereomers of (-)-(2R)-4-(4-carboxyphenyl)-2-methyl-sulfinylmethyl-4-oxobutanoic acid] were synthesized based on microbial transformation. The substrate KE-748 (racemic form of (-)-(2R)- and (+)-(2S)-4-(4-methyl-phenyl)-2-methylthiomethyl-4-oxobutanoic acid: 7.5 g) was converted to (-)-(2R)-M-4 (1.84 g) using Rhizopus sp. TF0040 in a 50-l jar fermentor. Specific cytochrome P-450 inhibitors, SKF-525-A and metyrapone strongly inhibited the hydroxylation reaction. It was suggested that cytochrome P-450 is responsible for the microbial reaction. Furthermore, (-)-(2R)-M-4 (200 mg) was transformed to (-)-(2R)-M-6 (144 mg) by co-oxidation with n-hexadecane as a carbon source using Rhodococcus sp. TA0250 in a 1.4-l jar fermentor. Starting from (-)-(2R)-M-4 and (-)-(2R)-M-6 obtained as above, (-)-(2R)-M-5 and (-)-(2R)-M-7, respectively were chemically synthesized by m-chloroperoxybenzoic acid oxidation.