School of Energy and Chemical Engineering, UNIST, UNIST-gil 50, Ulsan 44919, Republic of Korea.
School of Chemical and Biomolecular Engineering, Pusan National University, Busan 609-735, Republic of Korea.
FEMS Microbiol Lett. 2018 Nov 1;365(21). doi: 10.1093/femsle/fny211.
Coenzyme B12 is an essential cofactor for many enzymes such as glycerol dehydratase, methionine synthase and methylmalonyl-CoA mutase. Herein, we revisited the B12 biosynthetic gene clusters (I and II) in Pseudomonas denitrificans, a well-known industrial producer of the coenzyme B12, to understand the regulation of gene expression and improve the production of coenzyme B12. There were eight operons, seven in cluster I and one in cluster II, and four operons were regulated by B12-responsive riboswitches with a switch-off concentration at ∼5 nM coenzyme B12. DNA sequences of the four riboswitches were partially removed, individually or in combination, to destroy the structures of riboswitches, but no improvement was observed. However, when the whole length of riboswitches in cluster I were completely removed and promoters regulated by the riboswitches were replaced with strong constitutive ones, B12 biosynthesis was improved by up to 2-fold. Interestingly, modification of the promoter region for cluster II, where many (>10) late genes of B12 biosynthesis belong, always resulted in a significant, greater than 6-fold reduction in B12 biosynthesis.
辅酶 B12 是许多酶的必需辅助因子，如甘油脱水酶、蛋氨酸合成酶和甲基丙二酰辅酶 A 变位酶。在此，我们重新研究了众所周知的辅酶 B12 工业生产菌——脱氮假单胞菌中的 B12 生物合成基因簇（I 和 II），以了解基因表达的调控并提高辅酶 B12 的产量。有八个操纵子，七个在簇 I 中，一个在簇 II 中，其中四个操纵子由 B12 响应的核糖开关调控，其开关浓度约为 5 nM 辅酶 B12。四个核糖开关的 DNA 序列被部分删除，单独或组合使用，以破坏核糖开关的结构，但没有观察到任何改善。然而，当完全去除簇 I 中的所有核糖开关并将受核糖开关调控的启动子替换为强组成型启动子时，B12 的生物合成提高了 2 倍。有趣的是，对 B12 生物合成的许多（>10）晚期基因所在的簇 II 的启动子区域进行修饰，总是导致 B12 生物合成显著降低，大于 6 倍。
