Kang Jie, Fang Huan, Dong Huina, Song Wenjun, Zhang Dawei
College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Sheng Wu Gong Cheng Xue Bao. 2017 Jan 25;33(1):55-67. doi: 10.13345/j.cjb.160255.
Biosynthesis of vitamin B₁₂ (VB₁₂) requires the methylation at positions C-2 and C-7 of the precursor uroporphyrinogen Ⅲ (urogen Ⅲ) to precorrin-2 by S-adenosyl-L-methionine uroporphyrinogen Ⅲ methyltransferase (SUMT), which is a potential bottleneck step. Most of SUMTs are inhibited by urogen Ⅲ and by-product S-adenosyl-L-homocysteine (SAH). In order to mine an SUMT that lacks such an inhibitory property to drive greater flux through the VB₁₂ biosynthetic pathway, we cloned two SUMT genes (RCcobA1, RCcobA2) from Rhodobacter capsulatus SB1003 and expressed them in Escherichia coli BL21 (DE3). Thereafter, the two enzymes were purified and their specific activity of 27.3 U/mg, 68.9 U/mg were determined respectively. The latter was 2.4 times higher than PDcobA (27.9 U/mg) from Pseudomonas denitrifican. Additionally, RCcobA2 could tolerate over 70 μmol/L urogen Ⅲ, which has never been reported before. Hence, RCcobA2 can be used as an efficient enzyme to regulate the VB₁₂ metabolic pathway and enhance VB₁₂ production in industrial strains.
维生素B₁₂（VB₁₂）的生物合成需要尿卟啉原Ⅲ（urogenⅢ）的C-2和C-7位通过S-腺苷-L-甲硫氨酸尿卟啉原Ⅲ甲基转移酶（SUMT）甲基化生成前咕啉-2，这是一个潜在的瓶颈步骤。大多数SUMT受到urogenⅢ和副产物S-腺苷-L-高半胱氨酸（SAH）的抑制。为了挖掘一种缺乏这种抑制特性的SUMT，以驱动更多通量通过VB₁₂生物合成途径，我们从荚膜红细菌SB1003中克隆了两个SUMT基因（RCcobA1、RCcobA2），并在大肠杆菌BL21（DE3）中表达。此后，纯化了这两种酶，其比活性分别测定为27.3 U/mg、68.9 U/mg。后者比反硝化假单胞菌的PDcobA（27.9 U/mg）高2.4倍。此外，RCcobA2能够耐受超过70μmol/L的urogenⅢ，这是以前从未报道过的。因此，RCcobA2可作为一种高效的酶来调节VB₁₂代谢途径，并提高工业菌株中VB₁₂的产量。
