Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli.
Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli.
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DOI：
10.3390/microorganisms9061195
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发表时间：
2021-05-31
期刊：
影响因子：
4.5
通讯作者：
Gimpel M
中科院分区：
文献类型：
--
作者：
Fan Q;Caserta G;Lorent C;Lenz O;Neubauer P;Gimpel M
氢化酶是一种丰富的金属酶，可催化分子H2可逆转化为质子和电子。在过去的二十年里，在对这些高度复杂的酶的理解方面取得了重要的成就。然而，大多数氢化酶的产量低，需要大量的努力和高成本的培养，限制了它们的研究。这些氢化酶在一个健壮的和遗传上可处理的表达宿主中的异源生产是一个有吸引力的策略，使这些酶更容易获得。在本研究中，我们选择了来自Ralstonia eutropha H16的耐氧h2传感调控[NiFe]-氢化酶（RH），因为它与其他[NiFe]-氢化酶相比结构相对简单，作为模型，在大肠杆菌中建立了异种氢化酶生产系统。在24个工作体积为3ml的深孔板上进行筛选实验，考察诱导剂浓度、表达温度、表达时间等相关培养参数。RH产率可以从14 mg/L增加到50 ~ 250 mg/L，通过从批处理切换到以EnPresso b为基础的饲料批处理，如摇瓶培养。这一产量超过了从同源宿主富营养菌中纯化的RH数100倍。此外，我们报告了RH单亚基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、无辅助因子的载脂蛋白形式分离出来的，但所提出的策略可能会有力地加速基础研究和应用研究的生物过程开发和结构研究。这些结果讨论了调节机制的背景下，控制组装的大型和小型氢化酶亚基。
Hydrogenases are abundant metalloenzymes that catalyze the reversible conversion of molecular H2 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 H2-sensing regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha H16 owing to its relatively simple architecture compared to other [NiFe]-hydrogenases as a model to develop a heterologous hydrogenase production system in Escherichia coli. 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 R. eutropha 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.
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