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.
J Biosci Bioeng. 2012 Mar;113(3):360-6. doi: 10.1016/j.jbiosc.2011.10.017. Epub 2011 Nov 17.
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.
一种耐有机溶剂的细菌，红球菌 opacus B-4，经过代谢工程改造，可从原油中的二苯并噻吩 (DBT) 中去除硫。得到的重组菌株 ROD2-8 组成型表达红杆菌 IGTS8 基因 dszA、dszB 和 dszC，分别编码二苯并噻吩砜单加氧酶、2-(2'-羟苯基)苯磺酸盐脱磺酶和二苯并噻吩单加氧酶，该途径为 4S 途径，可避免硫酸盐终产物的转录抑制。与野生型菌株不同，ROD2-8 在含有 DBT 作为唯一硫源的无机盐培养基中生长。在水相条件下，ROD2-8 静止细胞将大于 85%的 DBT 转化为 2-羟基联苯 (2-HBP)，尽管以 DBT 作为唯一硫源预培养的 ROD2-8 细胞的消耗速率比在复杂培养基中培养的细胞高 3.3 倍。值得注意的是，在以正十六烷为有机溶剂的油-水两相反应混合物中，DBT 的消耗速率增加了 80%，并且静止细胞主要定位于乳化层中。两相反应混合物中脱硫活性随 DBT 浓度的增加而增加，并且 2-HBP 积累对其没有明显抑制。在两相条件下，细胞内 DBT 和 2-HBP 的浓度明显低于水相条件。我们的研究结果表明，在两相条件下增强的脱硫活性是由于减弱的反馈抑制以及由于 2-HBP 从细胞扩散和生物催化剂在乳化层中的积累而分别减少传质限制的综合作用所致。因此，耐溶剂和疏水性细菌红球菌 opacus B-4 似乎适合在含有最小水比的溶剂中的生物脱硫反应。
