Nowadays, the use of formate dehydrogenase (FDH, EC 1.17.1.9) is well established as a means of NADH regeneration from NAD+ via the coupled conversion of formate into carbon dioxide. Recent studies have been reported that specifically Chaetomium thermophilum FDH (CtFDH) is the most efficient FDH catalyzing this reaction in reverse (i.e. using CO2 as a substrate to produce formate, and thereby regenerating NAD+). However, to date the production of active CtFDH at high protein expression levels has received relatively little attention. In this study, we have tested the effect of batch and high cell density fermentation (HCDF) strategies in a small stirred fermenter, as well as the effect of supplementing the medium with casamino acids, on the expressed level of secreted CtFDH using P. pastoris. We have established that the amount of expressed CtFDH was indeed enhanced via a HCDF strategy and that extracellular protease activity was eliminated via the addition of casamino acids into the fermentation medium. On this basis, secreted CtFDH in an active form can be easily separated from the fermentation and can be used for subsequent biotechnological applications.

中文翻译：

---

活性嗜热毛壳菌 FDH 在毕赤酵母中的高水平异源表达

如今，甲酸脱氢酶 (FDH, EC 1.17.1.9) 的使用已成为一种通过甲酸耦合转化为二氧化碳从 NAD+ 再生 NADH 的手段。最近的研究报告称，具体而言，嗜热毛壳菌 FDH (CtFDH) 是最有效的 FDH 催化反向反应（即使用 CO2 作为底物产生甲酸盐，从而再生 NAD+）。然而，迄今为止，以高蛋白表达水平生产活性 CtFDH 的关注相对较少。在这项研究中，我们测试了在小型搅拌发酵罐中分批和高细胞密度发酵 (HCDF) 策略的影响，以及用酪蛋白氨基酸补充培养基对使用毕赤酵母表达的分泌 CtFDH 水平的影响. 我们已经确定表达的 CtFDH 的量确实通过 HCDF 策略增强，并且通过向发酵培养基中添加酪蛋白氨基酸消除了细胞外蛋白酶活性。在此基础上，活性形式的分泌型 CtFDH 可以很容易地从发酵中分离出来，并可用于后续的生物技术应用。