Engineering protein reagents and pathways for in vivo and in vitro hydrogen production [electronic resource]
Keywords: Chemical Engineering

In this dissertation we report our efforts to engineer protein reagents and pathways for two biohydrogen production technologies: (1) direct solar biohydrogen and (2) biomass to hydrogen production. Hydrogen is an important industrial chemical and feedstock and has great potential for use as a clean fuel. However, the CO2 released from the production of hydrogen accounts for over 1% of global annual CO2 emissions. Future utilization of hydrogen as a transportation fuel using current technologies would increase CO2 emissions. For this reason, we are interested in developing novel hydrogen production biotechnologies that utilize renewable resources such as sunlight or biomass. In the direct solar biohydrogen application, oxygen produced during photosynthesis inactivates the hydrogenase which stops hydrogen production. In order to engineer a hydrogenase with improved oxygen tolerance we first improved the yield of active hydrogenase produced from linear DNA template by anaerobic cell-free protein synthesis (CFPS). Second, we developed a more effective and reproducible methodology for challenging mutated hydrogenase libraries by exposure to oxygen and measuring their oxygen tolerance. We created and tested hydrogenase mutants designed to reduce oxygen diffusion to the active site. No impact in oxygen tolerance was observed. We went on to investigate technologies for producing hydrogen from biomass. Previously published conversion technologies have suffered from low yields and low volumetric productivities. In vivo efforts to develop biological routes for hydrogen production from biomass-derived sugars have been limited by the low yields associated with fermentative hydrogen production (2-4 mole hydrogen per mole glucose). While in vitro approaches have enabled higher yields (approaching the theoretical maximum of 12 mole hydrogen per mole glucose) through utilization of the pentose phosphate pathway (PPP), this required expensive purified protein reagents. Both approaches suffer from impractical volumetric productivities (< 1 mmole H2 L-1 hr-1). We describe the development of an in vitro synthetic enzyme pathway for the production of hydrogen from glucose and other biomass constituent sugars that has the potential to overcome these two limitations. We utilized the [FeFe] hydrogenase from Clostridium pasteurianum (CpI) which is much faster than the [NiFe] hydrogenases used in previously reported biomass to hydrogen studies. Ferredoxin and ferredoxin-NADPH-reductase (FNR) were utilized to transfer electrons from NADPH (the output of the PPP) to the CpI hydrogenase. In its final application, this pathway would be composed of unpurified proteins in a crude cell extract, avoiding the need for using expensive purified proteins. We present data obtained by testing a variety of FNRs and ferredoxins from different microorganisms for hydrogen production with the synthetic enzyme pathway. Temperature, stirring, and the concentration of the FNR and ferredoxin had a strong effect on the rate of hydrogen production from NADPH. From the enzymes we produced, purified, and tested for hydrogen production we identified the FNR from Anabaena variabilis (AnFNR) and the 2[4Fe4S] ferredoxin from C. pasteurianum (CpFd) as allowing the fastest transfer of electrons from NADPH to CpI. The full pathway, consisting of a (1) crude cell extract containing the PPP enzymes, (2) AnFNR, (3) CpFd and (4) CpI allowed a rate of hydrogen production from glucose of 4.7 mmole H2 L-1 hr-1. While this is a large improvement over previously reported rates it is still 30-fold slower, on an energy productivity basis, than that of US bioethanol processes. These results required very high concentrations of the FNR enzyme, and the resulting FNR turnover number was two orders of magnitude lower than expected. We therefore focused subsequent research toward improving the turnover rate of the FNR in order to increase the rate of hydrogen production through the pathway. Accordingly, we report the production and testing of a series of FNR-CpI fusion proteins with varying linker lengths designed to improve the function of the FNR. We produced these fusion proteins by cell-free protein synthesis and tested the unpurified proteins for hydrogen production from NADPH. We observed a more than 100-fold improvement in the turnover of the FNR enzymes relative to our previous results. This significant improvement in FNR performance should enable significantly higher volumetric hydrogen productivities, which in turn will facilitate further development of an economic process for the production of hydrogen from biomass.