Koji Takeda

Production of α,ω-Alkanediols Using Escherichia coli Expressing a Cytochrome P450 from Acinetobacter sp. OC4

Our biotransformation using Escherichia coli expressing a cytochrome P450 (CYP) belonging to the CYP153A family from Acinetobacter sp. OC4 produced a great amount of 1-octanol (2,250 mg per liter) from n-octane after 24 h of incubation. This level of production is equivalent to the maximum level previously achieved in biotransformation experiments of alkanes. In addition, the initial production rate of 1-octanol was maintained throughout the entire incubation period. These results indicate that we have achieved the functional and stable expression of a CYP in E. coli for the first time. Further, our biotransformation system showed α,ω-diterminal oxidation activity of n-alkanes, and a large amount of 1,8-octanediol (722 mg per liter) was produced from 1-octanol after 24 h of incubation. This is the first report on the bioproduction of α,ω-alkanediols from n-alkanes or 1-alkanols.

Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Vitamin D(3) (VD(3)) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD(3) into its physiologically active forms, namely, 25(OH)VD(3) or 1alpha,25(OH)(2)VD(3). In this study, we isolated and characterized Vdh (vitamin D(3) hydroxylase), which hydroxylates VD(3) from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V(max) values for VD(3) 25-hydroxylation and 25(OH)VD(3) 1alpha-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD(3). We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD(3) 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD(3) into 25(OH)VD(3) was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD(3) by a single cytochrome P450.

Biotransformation of Various Alkanes Using the Escherichia coli Expressing an Alkane Hydroxylase System from Gordonia sp. TF6

Biotransformation using alkane-oxidizing bacteria or their alkane hydroxylase (AH) systems have been little studied at the molecular level. We have cloned and sequenced genes from Gordonia sp. TF6 encoding an AH system, alkB2 (alkane 1-monooxygenase), rubA3 (rubredoxin), rubA4 (rubredoxin), and rubB (rubredoxin reductase). When expressed in Escherichia coli, these genes allowed the construction of biotransformation systems for various alkanes. Normal alkanes with 5 to 13 carbons were good substrates for this biotransformation, and oxidized to their corresponding 1-alkanols. Surprisingly, cycloalkanes with 5 to 8 carbons were oxidized to their corresponding cycloalkanols as well. This is the first study to achieve biotransformation of alkanes using the E. coli expressing the minimum component genes of the AH system. Our biotransformation system has facilitated assays and analysis leading to improvement of AH systems, and has indicated a cycloalkane oxidation pathway in microorganisms for the first time.

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.

Isolation and identification of 2α,25-dihydroxyvitamin D3, a new metabolite from Pseudonocardia autotrophica 100U-19 cells incubated with Vitamin D3

Pseudonocardia autotrophica converted Vitamin D(3) to 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3). The hydroxylation of Vitamin D(3) with P. autotrophica was enhanced by the addition of cyclodextrin. In this microbial hydroxylation, a new Vitamin D(3) metabolite was observed in the reaction mixture of P. autotrophica and Vitamin D(3), and was isolated in a pure form by several steps of chromatography. The structure of the new metabolite was determined to be 2alpha,25-dihydroxyvitamin D(3) by UV, NMR and mass spectroscopic analyses. Biological evaluation of the new metabolite was conducted by means of several experiments.