A number of metalloenzymes harbor unique cofactors, which are incorporated into the apo-enzymes via protein-assisted maturation. In the case of [NiFe]-hydrogenases, minimally seven maturation factors (HypABCDEF and a specific endopeptidase) are involved, making these enzymes an excellent example for studying metallocenter assembly in general. Here, we describe an innovative toolbox to study maturation involving multiple putative gene products. The two core elements of the system are a modular, combinatorial cloning system and a cell-free maturation system, which is based on recombinant Escherichia coli extracts and/or purified maturases. Taking maturation of the soluble, oxygen-tolerant [NiFe]-hydrogenase (SH) from Cupriavidus necator as an example, the capacities of the toolbox are illustrated. In total 18 genes from C. necator were analyzed, including four SH-structural genes, the SH-dedicated hyp-genes and a second set of hyp-genes putatively involved in maturation of the Actinobacterium-like hydrogenase (AH). The two hyp-sets were either expressed in their entirety from single vectors or split into functional modules, which enabled flexible approaches to investigate limitations, specificities and the capabilities of individual constituents to functionally substitute each other. Affinity-tagged Hyp-Proteins were used in pull-down experiments to demonstrate direct interactions between dedicated or non-related constituents. The dedicated Hyp-set from C. necator exhibited the highest maturation efficiency in vitro. Constituents of non-related maturation machineries were found to interact with and to accomplish partial activation of SH. In contrast to homologues of the Hyp-family, omission of the SH-specific endopeptidase HoxW completely abolished in vitro maturation. We detected stoichiometric imbalances inside the recombinant production system, which point to limitations by the cyanylation complex HypEF and the premature subunit HoxH. Purification of HoxW revealed strong indications for the presence of a putative [4Fe-4S]-cluster, which is unique among this class of maturases. Results are discussed in the context of [NiFe]-hydrogenase maturation, and in light of the capacity of the novel toolbox. Biotechnol. Bioeng. 2015;112: 2360-2372. (c) 2015 Wiley Periodicals, Inc.