title: Isolation and characterization of a thermotolerant ene reductase from Geobacillus sp. 30 and its heterologous expression in Rhodococcus opacus.
abstract: Rhodococcus opacus B-4 cells are adhesive to and even dispersible in water-immiscible hydrocarbons owing to their highly lipophilic nature. In this study, we focused on the high operational stability of thermophilic enzymes and applied them to a biocatalytic conversion in an organic reaction medium using R. opacus B-4 as a lipophilic capsule of enzymes to deliver them into the organic medium. A novel thermo- and organic-solvent-tolerant ene reductase, which can catalyze the enantioselective reduction of ketoisophorone to (6R)-levodione, was isolated from Geobacillus sp. 30, and the gene encoding the enzyme was heterologously expressed in R. opacus B-4. Another thermophilic enzyme which catalyzes NAD(+)-dependent dehydrogenation of cyclohexanol was identified from the gene-expression library of Thermus thermophilus and the gene was coexpressed in R. opacus B-4 for cofactor regeneration. While the recombinant cells were not viable in the mixture due to high reaction temperature, 634 mM of (6R)-levodione could be produced with an enantiopurity of 89.2 % ee by directly mixing the wet cells of the recombinant R. opacus with a mixture of ketoisophorone and cyclohexanol at 50 °C. The conversion rate observed with the heat-killed recombinant cells was considerably higher than that obtained with a cell-free enzyme solution, demonstrating that the accessibility between the substrates and enzymes could be improved by employing R. opacus cells as a lipophilic enzyme capsule. These results imply that a combination of thermophilic enzymes and lipophilic cells can be a promising approach for the biocatalytic production of water-insoluble chemicals.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, 565-0871, Osaka, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Tsuji
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Honda
authors.forename: Mayumi
authors.initials: M
authors.lastname: Wada
authors.forename: Kenji
authors.initials: K
authors.lastname: Okano
authors.forename: Hisao
authors.initials: H
authors.lastname: Ohtake
mesh_terms: D003001:Cloning, Molecular; D003511:Cyclohexanols / Q000378:metabolism; D003512:Cyclohexanones / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012240:Rhodococcus / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics
chemical_list: D003511:Cyclohexanols; D003512:Cyclohexanones; D004269:DNA, Bacterial; C494596:levodione; C476259:ketoisophorone; D010088:Oxidoreductases