High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli.
High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli.
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DOI：
10.3389/fmicb.2022.894375
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发表时间：
2022
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
--
作者：
氢化酶是生物技术相关的金属酶，其催化分子氢可逆转化为质子和电子。来自钩虫贪铜菌（以前的真养罗尔斯通氏菌）的耐O2 [NiFe]-氢化酶特别令人感兴趣，因为它们即使在分子氧存在下也能保持催化作用。然而，为了满足生物技术应用和科学研究的需求，需要异源生产策略来克服其天然宿主中的低产量。我们以前使用的调节氢化酶（RH）从C。necator作为在E.杆菌虽然获得了高的蛋白质产量，纯化的酶是无活性的，由于缺乏催化中心，其中包含一个无机的镍-铁辅因子。在本研究中，我们显着改进的生产工艺，以获得催化活性RH。优化了O2含量、金属有效性、生产温度和时间以及RH特异性成熟酶基因的共表达等重要因素。在好氧培养过程中，RH成功成熟。大肠杆菌通过共同生产的七个氢化酶特异性成熟酶和镍渗透酶，这是证实了纯化的酶的活性测量和光谱调查。改进后的生产条件使催化活性RH的产率达到80 mg L-1左右，E. coli中的表达量与天然宿主C. necator [<0.1 U（L d）-1]。我们的策略对E.大肠杆菌K-12和B菌株在重组生产复杂的金属酶，并提供了一个蓝图，生产催化活性的[NiFe]-氢化酶的生物技术相关的数量。
Hydrogenases are biotechnologically relevant metalloenzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. The O2-tolerant [NiFe]-hydrogenases from Cupriavidus necator (formerly Ralstonia eutropha) are of particular interest as they maintain catalysis even in the presence of molecular oxygen. However, to meet the demands of biotechnological applications and scientific research, a heterologous production strategy is required to overcome the low production yields in their native host. We have previously used the regulatory hydrogenase (RH) from C. necator as a model for the development of such a heterologous hydrogenase production process in E. coli. Although high protein yields were obtained, the purified enzyme was inactive due to the lack of the catalytic center, which contains an inorganic nickel-iron cofactor. In the present study, we significantly improved the production process to obtain catalytically active RH. We optimized important factors such as O2 content, metal availability, production temperature and time as well as the co-expression of RH-specific maturase genes. The RH was successfully matured during aerobic cultivation of E. coli by co-production of seven hydrogenase-specific maturases and a nickel permease, which was confirmed by activity measurements and spectroscopic investigations of the purified enzyme. The improved production conditions resulted in a high yield of about 80 mg L–1 of catalytically active RH and an up to 160-fold space-time yield in E. coli compared to that in the native host C. necator [<0.1 U (L d) –1]. Our strategy has important implications for the use of E. coli K-12 and B strains in the recombinant production of complex metalloenzymes, and provides a blueprint for the production of catalytically active [NiFe]-hydrogenases in biotechnologically relevant quantities.
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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
DOI：
10.1073/pnas.76.9.4530
发表时间：
1979-01-01
影响因子：
11.1
作者：
CASADABAN, MJ;COHEN, SN
通讯作者：
COHEN, SN
影响因子：
5.6
作者：
Blokesch, M;Albracht, SPJ;Böck, A
通讯作者：
Böck, A
影响因子：
3.4
作者：
Hartmann, Sven;Frielingsdorf, Stefan;Lenz, Oliver
通讯作者：
Lenz, Oliver
影响因子：
4.8
作者：
Buhrke, T;Lenz, O;Friedrich, B
通讯作者：
Friedrich, B