Furuya Toshiki, Takahashi Shusuke, Ishii Yoshitaka, Kino Kuniki, Kirimura Kohtaro
Department of Applied Chemistry, School of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Biochem Biophys Res Commun. 2004 Jan 16;313(3):570-5. doi: 10.1016/j.bbrc.2003.11.157.
The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55 degrees C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.
嗜热二苯并噻吩（DBT）脱硫细菌枯草芽孢杆菌WU-S2B能够在高达50摄氏度的较宽温度范围内将DBT转化为2-羟基联苯并释放出无机硫。该转化过程由两种单加氧酶连续进行硫原子特异性氧化引发，黄素还原酶与这些黄素依赖性单加氧酶结合至关重要。表达来自枯草芽孢杆菌WU-S2B的DBT单加氧酶基因（bdsC）的重组大肠杆菌细胞在存在异源黄素还原酶的情况下也能将吲哚氧化为蓝色色素靛蓝。因此，为了从枯草芽孢杆菌WU-S2B中克隆编码黄素还原酶的基因，将该基因与bdsC在大肠杆菌中共表达产生靛蓝用作筛选方法。利用这种方法，从在营养琼脂平板上因产生靛蓝而形成蓝色菌落的重组菌株中获得了相应基因（frb），并证实该基因产物Frb具有黄素还原酶活性。frb推导的氨基酸序列由174个氨基酸残基组成，与解淀粉芽孢杆菌的硝基还原酶（YwrO）具有61%的同一性。此外，frb与来自枯草芽孢杆菌WU-S2B的DBT脱硫基因（bdsABC）共表达对于在20至55摄氏度的宽温度范围内具有高DBT脱硫能力至关重要。这种以靛蓝产生作为选择性指示的共表达筛选方法可能广泛适用于克隆编码黄素还原酶或黄素依赖性单加氧酶任一成分的新基因，这些成分能在双组分单加氧酶中与另一成分有效偶联。
