Heterologous Hydrogenase Overproduction Systems for Biotechnology—An Overview

Abstract

Hydrogenases are complex metalloenzymes, showing tremendous potential as H2-converting redox catalysts for application in light-driven H2 production, enzymatic fuel cells and H2-driven cofactor regeneration. They catalyze the reversible oxidation of hydrogen into protons and electrons. The apo-enzymes are not active unless they are modified by a complicated post-translational maturation process that is responsible for the assembly and incorporation of the complex metal center. The catalytic center is usually easily inactivated by oxidation, and the separation and purification of the active protein is challenging. The understanding of the catalytic mechanisms progresses slowly, since the purification of the enzymes from their native hosts is often difficult, and in some case impossible. Over the past decades, only a limited number of studies report the homologous or heterologous production of high yields of hydrogenase. In this review, we emphasize recent discoveries that have greatly improved our understanding of microbial hydrogenases. We compare various heterologous hydrogenase production systems as well as in vitro hydrogenase maturation systems and discuss their perspectives for enhanced biohydrogen production. Additionally, activities of hydrogenases isolated from either recombinant organisms or in vivo/in vitro maturation approaches were systematically compared, and future perspectives for this research area are discussed.

Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli

“…Even though these proteins are only produced without [NiFe] cofactor, the availability of large quantities of recombinant precursors would allow to develop new artificial semisynthetic H 2 -producing catalysts in vitro, without the requirement of the canonical maturation machinery. This strategy has been proven successful in case of [FeFe]-and [Fe]-hydrogenases [22,63,64], where the incorporation of synthetic complexes contributed to the flourishing artificial enzymology.…”

“…The heterologous production of hydrogenases is a challenging task [22]. However, the relatively simple architecture of the RH compared to other [NiFe]-hydrogenases and its utilization as a model system for the investigation of the catalytic cycle of [NiFe]-hydrogenases [14,23,24], prompted us to develop new strategies for heterologous high-yield productions of RHstop in E. coli as a model for bioprocess development of heterologous [NiFe]-hydrogenase production.…”

“…However, the relatively simple architecture of the RH compared to other [NiFe]-hydrogenases and its utilization as a model system for the investigation of the catalytic cycle of [NiFe]-hydrogenases [14,23,24], prompted us to develop new strategies for heterologous high-yield productions of RHstop in E. coli as a model for bioprocess development of heterologous [NiFe]-hydrogenase production. The heterologous production of hydrogenases is a challenging task [22]. However, the relatively simple architecture of the RH compared to other [NiFe]-hydrogenases and its utilization as a model system for the investigation of the catalytic cycle of [NiFe]hydrogenases [14,23,24], prompted us to develop new strategies for heterologous highyield productions of RH stop in E. coli as a model for bioprocess development of heterologous [NiFe]-hydrogenase production.…”

High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli

“…Due to the complex maturation process, recombinant production of [NiFe]-hydrogenases is challenging, especially in heterologous systems, and makes scalability difficult (Fan et al, 2020). We have chosen the regulatory [NiFe]-hydrogenase (RH) from Cupriavidus necator (formerly Ralstonia eutropha) as a model for the development of a heterologous hydrogenase production system in E. coli.…”

Implementation of a high cell density fed-batch for heterologous production of active [NiFe]-hydrogenase in Escherichia coli bioreactor cultivations

“…However, at present, their biotechnological applications is still hardly feasible due to the low product yields and long process times by cultivation of the native producers [ 16 – 19 ]. Similarly, the heterologous production of these hydrogenases is challenging due to the complicated maturation process and the high specificity of the required maturation proteins [ 20 ]. To improve accessibility of hydrogenases, we recently developed a heterologous production system for [NiFe]-hydrogenases in the robust and genetically tractable production host Escherichia coli by the example of the O 2 -tolerant regulatory [NiFe]-hydrogenase from C. necator [ 21 ].…”