Doukyu N, Toyoda K, Aono R
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan.
Appl Microbiol Biotechnol. 2003 Feb;60(6):720-5. doi: 10.1007/s00253-002-1187-1. Epub 2002 Dec 19.
Acinetobacter sp. strain ST-550 produces indigo from indole in the presence of a large volume of diphenylmethane and a high level of indole. Particular proteins increased remarkably in strain ST-550 grown in the two-phase culture system for indigo production. One of the proteins showed a N-terminal amino acid sequence that was identical to that of the largest subunit of phenol hydroxylase (MopN) from A. calcoaceticus NCBI8250. The indigo-producing activity was strongly induced when ST-550 was grown with phenol as a sole carbon source. Genes coding for the multicomponent phenol hydroxylase were cloned, based on the homology with mopKLMOP from A. calcoaceticus NCBI8250. Escherichia coli carrying the genes produced indigo from indole. E. coli JA300 and its cyclohexane-resistant mutant, OST3410, carrying the hydroxylase genes and the NADH regeneration system were grown in the two-phase culture system for indigo production. The OST3410 recombinant produced 52 microg indigo ml(-1) of medium in the presence of diphenylmethane. This productivity was 4.3-fold higher than that of the JA300 recombinant.
不动杆菌属菌株ST-550在大量二苯甲烷和高浓度吲哚存在的情况下，能从吲哚产生靛蓝。在用于靛蓝生产的两相培养系统中生长的ST-550菌株中，特定蛋白质显著增加。其中一种蛋白质的N端氨基酸序列与乙酸钙不动杆菌NCBI8250的苯酚羟化酶最大亚基（MopN）的序列相同。当ST-550以苯酚作为唯一碳源生长时，靛蓝生产活性被强烈诱导。基于与乙酸钙不动杆菌NCBI8250的mopKLMOP的同源性，克隆了编码多组分苯酚羟化酶的基因。携带这些基因的大肠杆菌能从吲哚产生靛蓝。携带羟化酶基因和NADH再生系统的大肠杆菌JA300及其环己烷抗性突变体OST3410在用于靛蓝生产的两相培养系统中生长。在二苯甲烷存在的情况下，OST3410重组体在培养基中产生了52微克靛蓝/毫升。该生产力比JA300重组体高4.3倍。
