Metabolic engineering of hydrophobic Rhodococcus opacus for biodesulfurization in oil–water biphasic reaction mixtures Hideo Kawaguchi, Hajime Kobayashi, and Kozo Sato ⁎ Frontier Research Center for Energy and Resources, School of Engineering, The University of Tokyo, Eng. Bldg. No. 4, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received 25 August 2011; accepted 20 October 2011 Available online 17 November 2011 An organic solvent-tolerant bacterium, Rhodococcus opacus B-4, was metabolically engineered to remove sulfur from dibenzothiophene (DBT), a component of crude oil. The resulting recombinant strain ROD2-8 constitutively expressed the Rhodococcus erythropolis IGTS8 genes dszA, dszB, and dszC, encoding dibenzothiophene sulfone monooxygenase, 2-(2′- hydroxyphenyl) benzenesulfinate desulfinase, and dibenzothiophene monooxygenase, respectively, of the 4S pathway to avoid transcriptional inhibition by the sulfate end-product. Unlike the wild-type strain, ROD2-8 grew in mineral salts medium containing DBT as the sole sulfur source. Under aqueous conditions, ROD2-8 resting cells converted greater than 85% of DBT to 2-hydroxybiphenyl (2-HBP), although the consumption rate by ROD2-8 cells precultured on DBT as the sole sulfur source was 3.3-fold higher than that of cells cultured in complex medium. Notably, DBT consumption rates increased by 80% in oil–water biphasic reaction mixtures with n-hexadecane as the organic solvent, and resting cells were predominantly localized in the emulsion layer. Desulfurization activity in biphasic reaction mixtures increased with increasing concentrations of DBT and was not markedly inhibited by 2-HBP accumulation. Intracellular concentrations of DBT and 2-HBP were significantly lower under biphasic conditions than aqueous conditions. Our findings suggest that the enhanced desulfurization activity under biphasic conditions results from the combined effects of attenuated feedback inhibition and reduced mass transfer limitations due to 2-HBP diffusion from cells and accumulation of both substrate and biocatalyst in the emulsion layer, respectively. Therefore, the solvent-tolerant and hydrophobic bacterium R. opacus B-4 appears suitable for biodesulfurization reactions in solvents containing a minimum ratio of water. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Biodesulfurization; Dibenzothiophene; Hydrophobic bacterium; Rhodococcus opacus; Biphasic reaction mixture] Unconventional oils, including heavy oils and oil sands, are promising candidates to meet increasing worldwide energy demands. However, these oils often contain high concentrations of sulfuric compounds, ranging from 500 to 80,000 ppm (1). Recently, many countries have applied tighter restrictions on the allowable sulfur content in transportation fuels, such as diesel. For example, in 2006, the United States Environmental Protection Agency adopted new sulfur control regulations to reduce the sulfur content of on-road diesel fuel to a maximum of 15 ppm (US EPA: Ultra-low sulfur diesel fuel program. http://www.epa.gov/region1/dieselcollaborative/pdf/ ULSD-Implementation-Nov06.pdf, accessed 16 December 2010). To meet these standards, petroleum refiners are required to produce ultra-low sulfur diesel (ULSD) from heavier crudes containing high sulfur contents. Because the production of ULSD is expensive, alternative approaches are needed to reduce sulfur concentrations in unconventional oils with lower energetic consumption. Hydrodesulfurization (HDS) is a catalytic process that is commer- cially utilized for upgrading heavy oil. During this process, sulfur atoms in organosulfur compounds are reduced to H 2 S under conditions of high temperature and hydrogen pressure (290–455°C and 1.0–20.7 MPa, respectively) (2). To reach the low concentrations of sulfur required for ULDS (b 15 ppm), even higher temperatures and pressures are required, which requires substantially increased external energy. However, middle-distillate fractions, including diesel, contain significant amounts of refractory organosulfur compounds, such as dibenzothiophene (DBT) and its alkyl substituted derivatives, which are resistant to HDS treatment (3). In addition, unselective hydrogenation during this process produces substantial amounts of carbon dioxide (CO 2 ) and unavailable organic compounds as by-products, reducing the calorific value of fuels. To address these limitations, advanced technological and environmentally conscious alternatives to HDS that can improve oil availability and reduce costs, such as bioprocessing, are needed. Several microorganisms capable of selectively removing sulfur from DBT and its derivatives under ambient conditions have been identified and are considered suitable for energy-saving biodesulfur- ization (BDS) processes as alternatives to HDS (4). For example, the aerobic bacterium Rhodococcus erythropolis IGTS8 removes sulfur from DBT without degrading its carbon skeleton in four enzymatic steps, termed the 4S pathway (Kilbane, J.J., II: Mutant microorganisms useful for cleavage of organic C\S bonds. U.S. patent 5104801, 1992). In this pathway, DBT is sequentially oxidized to DTB sulfoxide (DBTO) Journal of Bioscience and Bioengineering VOL. 113 No. 3, 360 – 366, 2012 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +81 3 5841 7041; fax: +81 3 3818 7492. E-mail address: sato@frcer.t.u-tokyo.ac.jp (K. Sato). 1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2011.10.017