Towards In Vitro synthesis of oxygen-tolerant CpI hydrogenase variants for photosynthetic hydrogen production

Hydrogen is an attractive alternative to non-renewable energy sources like fossil fuels due to its carbon-free nature and high energy density. However, most of hydrogen production uses feedstock chemicals derived from fossil fuels, which leads to carbon dioxide emissions and thus contributes to the climate crisis. Photosynthetic hydrogen technology can provide an alternative, sustainable and entirely carbon-free energy cycle. However, oxygen sensitivity of the hydrogenase enzymes that catalyze hydrogen production is one of the most significant barriers against photosynthetic hydrogen production, as oxygen is a by-product of photosynthesis. Therefore, the hydrogenase enzyme to be used in the production of photosynthetic hydrogen must tolerate gaseous oxygen. In this thesis, four locations known to be important for oxygen tolerance in the Clostridium pasteurianum (CpI) hydrogenase were replaced with the amber (TAG) stop codon for non-canonical amino acid (ncAA) incorporation. CpI was chosen as it is one of the most efficient and fast [Fe-Fe] hydrogenases characterized in terms of hydrogen production. Significant progress was made toward ncAA insertion using cell free protein synthesis (CFPS) to the selected spots on the enzyme. The genes of six M. jannaschii-derived aminoacyl-tRNA synthetases (aaRSs) required for cysteine and tyrosine analog incorporation were first cloned into the high- copy pY71 vector. These aaRSs were then produced in vivo and purified with IMAC as they have genetically fused N-terminal 6xHis-tags. The orthogonal tRNACUA template necessary for ncAA insertion was also synthesized with PCR and purified. In the next part of the thesis, experiments were performed to synthesize M. barkeri- derived aminoacyl tRNA synthetase, HRS, necessary for histidine analog incorporation. Different conditions were tested in vivo and in vitro (CFPS) synthesis of the enzyme. However, HRS formed insoluble aggregates and could not be purified. Lastly, the M. barkeri-derived orthogonal tRNA template was produced by PCR assembly and purified. In the last part, wild-type CpI was anaerobically produced and matured in vivo, followed by purification using StrepTactin affinity chromatography. The components required for the two biochemical assays to measure the hydrogen production activity of CpI, namely NADPH- and DTH-driven assays, were synthesized. With the activity assays, the anaerobically produced and purified CpI enzyme was shown to be in the active state. Attempts were made to produce CpI anaerobically in CFPS as well, however this was unsuccessful. But everything necessary for CFPS production of both wild-type and mutant CpI was prepared.