Heterologe Produktion und Reifung einer löslichen sauerstofftoleranten [NiFe]-Hydrogenase aus Cupriavidus necator in Escherichia coli

The aim of this thesis was the production and maturation of an O2-tolerant [NiFe]-hydrogenase, the SH from Cupriavidus necator (Cn), in Escherichia coli (Ec). For this purpose, an available cloning system (StarGate®, IBA) was initially subjected to an upgrade in order meet the requirements for a multigene and high-yield Escherichia coli expression platform. The first development consisted of the design of new fusion vectors, which place each gene under control of individual T7-promoters and –terminators in the course of gene assembly and enable the fusion of each gene product with an N- or C-terminal StrepII-tag. Using the novel fusion system, a total number of 21 hydrogenase-associated Cn genes, including five SH-structural genes and two independent sets of auxiliary genes, were systematically assembled yielding functional multigene units (modules). Co-expression of up to 10 genes assembled as SH- or maturation-related modules from one plasmid was successfully demonstrated. In order to combine SH- and accessory modules in one cell, a set of compatible plasmids was designed. Thereby, generation of expression strains harboring different combinations of vectors and modules was enabled. SH-production as well as maturation studies were thus made possible in a systematic and integrative way. SH-production studies using recombinant Ec-strains indicated that the activity of maturation-related proteins was not only dependent on gene doses and the rate of expression, but also on specific cellular environments transiently present during growth. Therefore, a cultivation strategy based on a previously presented lactose-autoinduction system was applied for SH-production. In the course of optimization studies, aerobic long-term cultivations with sequential modification of both physical growth parameters and the medium composition were carried out. The strategy was subsequently applied to recombinant SH-production strains and consistently yielded maximal SH-activities between 36–40 hours after inoculation. The highest specific activity in cell-free extracts amounted to 7.2 U•mg-1, which is in range of the values achieved with an optimized SH-production process in Cn. Hence, combination of the expression platform presented herein with the developed cultivation strategy represents the first high-yield production system for a recombinant [NiFe]-hydrogenase to date. StrepII-tagged SH-variants were homogeneously isolated from recombinant Ec strains. The specific activity of purified recombinant SH reached 227 U•mg-1, which represents the highest activity for the enzyme reported to date. In case of these ultrapure preparations, yields were reduced but still in the mg•(Liter culture)-1 range. The oligomeric enzyme structures and catalytic properties obtained for the recombinant variants nicely matched the data available for the wildtype-SH isolated from Cn cells. Development of the combinatorial expression platform further enabled innovative strategies to investigate the maturation process of [NiFe]-hydrogenases. This was demonstrated for the SH as a model enzyme in this study. Individual maturation-related proteins as well as complex intermediates of two independent Cn maturation sets were recombinantly produced and isolated by StrepTactin affinity chromatography. These results indicated that the in-vivo-stability of the maturation factors was largely dependent on the formation of oligomeric conformations as well as stoichiometric complexes with other parts of the maturation machinery. Purification of the SH-specific endopeptidase HoxW revealed the existence of a redox-active [4Fe-4S]-cluster with hitherto unknown properties. This is the first documented isolation of a HoxW-analog and the first indication for an electron-transferring co-factor in a maturation-related specific endopeptidase to date. In-vivo-deletion and substitution studies with analogous Hyp-proteins from different Cn and Ec maturation-related sets provided further insights into specificities and thus, complementation capabilities. An in-vitro-maturation system was developed, which allowed the dissection of the maturation process in a cell-free environment. Essential components and limiting factors of the SH maturation process were determined using this system. Initial pull-down experiments with in vitro matured SH suggested that the cell-free system might be used as an efficient technique to identify previously unknown maturation complex-intermediates and to study the general maturation process in detail.