Sasoh Mikio, Masai Eiji, Ishibashi Satoko, Hara Hirofumi, Kamimura Naofumi, Miyauchi Keisuke, Fukuda Masao
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
Appl Environ Microbiol. 2006 Mar;72(3):1825-32. doi: 10.1128/AEM.72.3.1825-1832.2006.
We isolated Comamonas sp. strain E6, which utilizes terephthalate (TPA) as the sole carbon and energy source via the protocatechuate (PCA) 4,5-cleavage pathway. Two almost identical TPA degradation gene clusters, tphRICIA2IA3IBIA1I and tphRIICIIA2IIA3IIBIIA1II, were isolated from this strain. Based on amino acid sequence similarity, the genes tphR, tphC, tphA2, tphA3, tphB, and tphA1 were predicted to code, respectively, for an IclR-type transcriptional regulator, a periplasmic TPA binding receptor, the large subunit of the oxygenase component of TPA 1,2-dioxygenase (TPADO), the small subunit of the oxygenase component of TPADO, a 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) dehydrogenase, and a reductase component of TPADO. The growth of E6 on TPA was not affected by disruption of either tphA2I or tphA2II singly; however, the tphA2I tphA2II double mutant no longer grew on TPA, suggesting that both TPADO genes are involved in TPA degradation. Introduction of a plasmid carrying tphRIICIIA2IIA3IIBIIA1II conferred the TPA utilization phenotype on Comamonas testosteroni IAM 1152, which is able to grow on PCA but not on TPA. Disruption of either tphRII or tphCII on this plasmid resulted in the loss of the growth of IAM 1152 on TPA, suggesting that these genes are essential to convert TPA to PCA in E6. The genes tphA1II, tphA2II, tphA3II, and tphBII were expressed in Escherichia coli, and the resultant cell extracts containing TphA1II, TphA2II, and TphA3II converted TPA in the presence of NADPH into a product which was transformed to PCA by TphBII. On the basis of these results, TPADO was strongly suggested to be a two-component dioxygenase which consists of the terminal oxygenase component (TphA2 and TphA3) and the reductase (TphA1), and tphB codes for the DCD dehydrogenase.
我们分离出了食酸菌属菌株E6，该菌株通过原儿茶酸（PCA）4,5-裂解途径将对苯二甲酸（TPA）作为唯一碳源和能源利用。从该菌株中分离出了两个几乎相同的TPA降解基因簇，即tphRICIA2IA3IBIA1I和tphRIICIIA2IIA3IIBIIA1II。基于氨基酸序列相似性，预测基因tphR、tphC、tphA2、tphA3、tphB和tphA1分别编码一种IclR型转录调节因子、一种周质TPA结合受体、TPA 1,2-双加氧酶（TPADO）加氧酶组分的大亚基、TPADO加氧酶组分的小亚基、一种1,2-二羟基-3,5-环己二烯-1,4-二羧酸（DCD）脱氢酶以及TPADO的一种还原酶组分。单独破坏tphA2I或tphA2II对E6在TPA上的生长没有影响；然而，tphA2I tphA2II双突变体不再能在TPA上生长，这表明两个TPADO基因都参与TPA降解。携带tphRIICIIA2IIAIIBIIA1II的质粒导入能在PCA上生长但不能在TPA上生长的睾丸酮食酸菌IAM 1152后，赋予了其利用TPA的表型。该质粒上tphRII或tphCII的破坏导致IAM 1152在TPA上无法生长，这表明这些基因对于E6中TPA转化为PCA至关重要。tphA1II、tphA2II、tphA3II和tphBII基因在大肠杆菌中表达，所得含有TphA1II、TphA2II和TphA3II的细胞提取物在NADPH存在下将TPA转化为一种产物，该产物由TphBII转化为PCA。基于这些结果，强烈提示TPADO是一种双组分双加氧酶，由末端加氧酶组分（TphA2和TphA3）和还原酶（TphA1）组成，且tphB编码DCD脱氢酶。
