Improved production of the NiFe-hydrogenase from Pyrococcus furiosus by increased expression of maturation genes

The NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) from the hyperthermophile Pyrococcus furiosus, which grows optimally near 100 degrees C, is extremely thermostable and has many in vitro applications, including cofactor generation and hydrogen production. In particular, SHI is used in a cell-free synthetic pathway that contains more than a dozen other enzymes and produces three times more hydrogen (12 H-2/glucose) from sugars compared to cellular fermentations (4 H-2/glucose). We previously reported homologous over-expression and rapid purification of an affinity-tagged (9x-His) version of SHI, which is a heterotetrameric enzyme. However, about 30% of the enzyme that was purified contained an inactive trimeric form of SHI lacking the catalytic [NiFe]-containing subunit. Herein, we constructed a strain of P. furiosus that contained a second set of the eight genes involved in the maturation of the catalytic subunit and insertion of the [NiFe]-site, along with a second set of the four genes encoding the SHI structural subunits. This resulted in a 40% higher yield of the purified affinity-tagged enzyme and the content of the inactive trimeric form decreased to 5% of the total protein. These results bode well for the future production of active SHI for both basic and applied purposes.