Nicotinate dehydrogenase (NDHase) from Comamonas testosteroni JA1 catalyzes the C6 hydroxylation of 3-cyanopyridine with high regional selectivity, which is a very difficult and complex reaction for chemical synthesis. However, because NDHase is a membrane protein with three subunits (ndhS, ndhL and ndhM), it is difficult to express the enzyme in a functional form using common hosts such as Escherichia coli, Bacilus subtilis or Pichia pastoris. Furthermore, the enzyme requires special electron transfer chains in the membrane system for proper catalytic activity. Thus, we investigated the expression of NDHase in non-model bacterial strains, which are evolutionarily similar to C. testosteroni JA1, using several broad-host plasmids with different copy numbers as expression vectors. We successfully expressed NDHase in soluble from using the pVLT33 vector in C. testosteroni CNB-2, and found the activity of enzyme to be 40.6 U/L. To further improve the expression of NDHase in C. testosteroni CNB-2, we trialed a T7-like MmP1 system, composed of MmP1 RNA polymerase and an MmP1 promoter, which is used for transcriptional control in non-model bacteria. This increased protein expression and enzyme activity doubled to 90.5 U/L. A molecular chaperone was co-expressed using pBBR1 MCS-5 in the same host to improve the efficiency of folding and assembly of multi-subunit structures. The maximum activity was 115 U/L using the molecular chaperone GroES-EL, far surpassing the previously reported level, although expression was almost equivalent. These results indicate that a strategy involving the construction of a T7-like system and co-expression of a molecular chaperone offers an efficient approach for heterologous expression of enzymes that are difficult to express in functional forms using conventional hosts.

中文翻译：

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烟酸脱氢酶在睾丸毛单胞菌 CNB-2 中的高效异源表达，具有转录、折叠增强策略

来自睾丸毛单胞菌 JA1 的烟酸脱氢酶 (NDHase) 以高区域选择性催化 3-氰基吡啶的 C6 羟基化，这是化学合成中非常困难和复杂的反应。然而，由于 NDHase 是具有三个亚基（ndhS、ndhL 和 ndhM）的膜蛋白，因此很难使用常见宿主（如大肠杆菌、枯草芽孢杆菌或毕赤酵母）以功能形式表达该酶。此外，酶需要膜系统中特殊的电子转移链才能获得适当的催化活性。因此，我们使用几种具有不同拷贝数的广泛宿主质粒作为表达载体，研究了 NDHase 在非模型细菌菌株中的表达，这些菌株在进化上与睾丸睾酮 JA1 相似。我们在睾酮 C. testosteroni CNB-2 中使用 pVLT33 载体成功表达了可溶性 NDHase，发现酶的活性为 40.6 U/L。为了进一步提高 NDHase 在 C. testosteroni CNB-2 中的表达，我们试验了 T7 样 MmP1 系统，由 MmP1 RNA 聚合酶和 MmP1 启动子组成，用于非模型细菌中的转录控制。这种增加的蛋白质表达和酶活性翻了一番，达到 90.5 U/L。在同一宿主中使用 pBBR1 MCS-5 共表达分子伴侣，以提高多亚基结构的折叠和组装效率。使用分子伴侣 GroES-EL 的最大活性为 115 U/L，远远超过先前报告的水平，尽管表达几乎相同。