Expression of engineered carbonyl reductase from Ogataea minuta in Rhodococcus opacus and its application to whole-cell bioconversion in anhydrous solvents

• Published in: Journal of bioscience and bioengineering 2019-02, Vol.127 (2), p.145-149
• Main Authors: Honda, Kohsuke, Ono, Tomohiro, Okano, Kenji, Miyake, Ryoma, Dekishima, Yasumasa, Kawabata, Hiroshi
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - isolation & purification; Alcohol Oxidoreductases - metabolism; Alcohols - metabolism; Biocatalysis; Carbonyl reductase; Catalysis; Gene Expression Regulation, Enzymologic; Organic solvent; Oxidation-Reduction; Protein Engineering; Rhodococcus - genetics; Rhodococcus - metabolism; Rhodococcus opacus; Solvents - chemistry; Thermal stability; Thermoanaerobacter - metabolism; Water - chemistry; Whole-cell biocatalysis; Yeasts - enzymology; Yeasts - genetics
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2018.07.011

The carbonyl reductase from the methylotrophic yeast Ogataea minuta can catalyze the regio- and enantio-selective reduction of prochiral ketones to chiral alcohols, and is available for industrial manufacturing of statin drugs. We previously conducted a directed evolution experiment of the enzyme, and obtained a mutant (OCR_V166A) with improved tolerance to organic solvents. This expanded the applicability of the enzyme to the bioconversion of water-insoluble compounds (Honda et al., J. Biosci. Bioeng., 123, 673–678, 2017). In the present study, we expressed OCR_V166A in Rhodococcus opacus cells, which have a highly lipophilic surface structure and are dispersible in anhydrous organic solvents, and developed a whole-cell biocatalyst which can function in an organic-solvent-based reaction medium. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeADH) was employed as an NADPH-regenerating enzyme and co-expressed with OCR_V166A in R. opacus. The whole-cell bioconversion of 2,2,2-trifluoroacetophenone to α-(trifluoromethyl)benzyl alcohol was performed in organic solvents, including isopropanol, isobutanol, and cyclohexanol, which served both as reaction media and as substrates for TeADH. The type of organic solvents markedly affected not only the product titer but also the enantio-purity of the product. When isobutanol was used as the reaction medium, the whole-cell biocatalyst showed higher stability than the isolated enzyme. Consequently, a high concentration (1 M) of the substrate was converted to the product with an overall conversion yield of 81% (mol/mol) in 24 h.