Implementation of a high cell density fed-batch for heterologous production of active [NiFe]-hydrogenase in Escherichia coli bioreactor cultivations.
Implementation of a high cell density fed-batch for heterologous production of active [NiFe]-hydrogenase in Escherichia coli bioreactor cultivations.
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DOI：
10.1186/s12934-022-01919-w
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发表时间：
2022-09-19
影响因子：
6.4
通讯作者：
中科院分区：
文献类型：
--
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耐氧氢化酶在h2基技术中具有巨大的应用潜力。由于这些金属酶经历了一个复杂的成熟过程，需要一组专用的多种辅助蛋白，因此它们的异种生产具有挑战性，从而阻碍了对它们的基本理解和相关应用的发展。考虑到这些挑战，我们选择了Cupriavidus necator中相对简单的调控[NiFe]-氢化酶（RH）作为开发异源[NiFe]-氢化酶生产生物工艺的模型。我们最近已经报道了通过优化摇瓶培养条件，在大肠杆菌中高产产催化活性RH。在本研究中，我们通过合理设计高密度补料分批培养工艺，进一步提高RH产率，保证产品质量的一致性。总体而言，与C. necator的其他RH实验室生物反应器规模工艺相比，该生物反应器培养产生了130 mg L−1的催化活性RH，增加了100倍以上。此外，该工艺具有较高的重现性和较高的生产率。本研究为经济、高效地提供这种难以表达的复杂金属蛋白提供了良好的机会，以满足基础和应用研究的需求。在线版本包含补充材料，可在10.1186/s12934-022-01919-w获得。
O2-tolerant [NiFe]-hydrogenases offer tremendous potential for applications in H2-based technology. As these metalloenzymes undergo a complicated maturation process that requires a dedicated set of multiple accessory proteins, their heterologous production is challenging, thus hindering their fundamental understanding and the development of related applications. Taking these challenges into account, we selected the comparably simple regulatory [NiFe]-hydrogenase (RH) from Cupriavidus necator as a model for the development of bioprocesses for heterologous [NiFe]-hydrogenase production. We already reported recently on the high-yield production of catalytically active RH in Escherichia coli by optimizing the culture conditions in shake flasks. In this study, we further increase the RH yield and ensure consistent product quality by a rationally designed high cell density fed-batch cultivation process. Overall, the bioreactor cultivations resulted in ˃130 mg L−1 of catalytically active RH which is a more than 100-fold increase compared to other RH laboratory bioreactor scale processes with C. necator. Furthermore, the process shows high reproducibility of the previously selected optimized conditions and high productivity. This work provides a good opportunity to readily supply such difficult-to-express complex metalloproteins economically and at high concentrations to meet the demand in basic and applied studies. The online version contains supplementary material available at 10.1186/s12934-022-01919-w.
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影响因子：
4.5
作者：
Fan Q;Caserta G;Lorent C;Lenz O;Neubauer P;Gimpel M
通讯作者：
Gimpel M
影响因子：
15
作者：
Horch, Marius;Schoknecht, Janna;Zebger, Ingo
通讯作者：
Zebger, Ingo
影响因子：
5.2
作者：
通讯作者：
--
影响因子：
6.3
作者：
Ghosh D;Bisaillon A;Hallenbeck PC
通讯作者：
Hallenbeck PC
影响因子：
3.2
作者：
Kleihues, L;Lenz, O;Friedrich, B
通讯作者：
Friedrich, B