Fan Qin, Caserta Giorgio, Lorent Christian, Lenz Oliver, Neubauer Peter, Gimpel Matthias
Institute of Biotechnology, Technische Universität Berlin, Chair of Bioprocess Engineering, Ackerstraße 76, D-13355 Berlin, Germany.
Department of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Microorganisms. 2021 May 31;9(6):1195. doi: 10.3390/microorganisms9061195.
Hydrogenases are abundant metalloenzymes that catalyze the reversible conversion of molecular H into protons and electrons. Important achievements have been made over the past two decades in the understanding of these highly complex enzymes. However, most hydrogenases have low production yields requiring many efforts and high costs for cultivation limiting their investigation. Heterologous production of these hydrogenases in a robust and genetically tractable expression host is an attractive strategy to make these enzymes more accessible. In the present study, we chose the oxygen-tolerant H-sensing regulatory [NiFe]-hydrogenase (RH) from H16 owing to its relatively simple architecture compared to other [NiFe]-hydrogenases as a model to develop a heterologous hydrogenase production system in . Using screening experiments in 24 deep-well plates with 3 mL working volume, we investigated relevant cultivation parameters, including inducer concentration, expression temperature, and expression time. The RH yield could be increased from 14 mg/L up to >250 mg/L by switching from a batch to an EnPresso B-based fed-batch like cultivation in shake flasks. This yield exceeds the amount of RH purified from the homologous host by several 100-fold. Additionally, we report the successful overproduction of the RH single subunits HoxB and HoxC, suitable for biochemical and spectroscopic investigations. Even though both RH and HoxC proteins were isolated in an inactive, cofactor free apo-form, the proposed strategy may powerfully accelerate bioprocess development and structural studies for both basic research and applied studies. These results are discussed in the context of the regulation mechanisms governing the assembly of large and small hydrogenase subunits.
氢化酶是一类丰富的金属酶，可催化分子氢可逆转化为质子和电子。在过去二十年里，人们对这些高度复杂的酶的认识取得了重要进展。然而，大多数氢化酶的产量较低，培养需要付出诸多努力且成本高昂，这限制了对它们的研究。在一个强大且易于进行基因操作的表达宿主中进行这些氢化酶的异源生产，是一种让这些酶更易获取的有吸引力的策略。在本研究中，我们选择了来自嗜热栖热放线菌（Thermus thermophilus）H16的耐氧H感应调节[NiFe]氢化酶（RH），因为与其他[NiFe]氢化酶相比，其结构相对简单，以此作为模型在嗜热栖热放线菌中开发异源氢化酶生产系统。通过在工作体积为3 mL的24孔深孔板中进行筛选实验，我们研究了相关培养参数，包括诱导剂浓度、表达温度和表达时间。通过从摇瓶中的分批培养转换为基于EnPresso B的补料分批培养，RH产量可从14 mg/L提高到>250 mg/L。这个产量比从同源宿主中纯化得到的RH量高出数百倍。此外，我们报告了RH单个亚基HoxB和HoxC的成功过量生产，适用于生化和光谱研究。尽管RH和HoxC蛋白都是以无活性、不含辅因子的脱辅基形式分离得到的，但所提出的策略可能会有力地加速基础研究和应用研究中的生物工艺开发和结构研究。这些结果将在控制大小氢化酶亚基组装的调控机制背景下进行讨论。
