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Title: Hydrogen from Water in a Novel Recombinant Cyanobacterial System

Photobiological processes are attractive routes to renewable H2 production. With the input of solar energy, photosynthetic microbes such as cyanobacteria and green algae carry out oxygenic photosynthesis, using sunlight energy to extract protons and high energy electrons from water. These protons and high energy electrons can be fed to a hydrogenase system yielding H2. However, most hydrogen-evolving hydrogenases are inhibited by O2, which is an inherent byproduct of oxygenic photosynthesis. The rate of H2 production is thus limited. Certain photosynthetic bacteria are reported to have an O2-tolerant evolving hydrogenase, yet these microbes do not split water, and require other more expensive feedstocks. To overcome these difficulties, the goal of this work has been to construct novel microbial hybrids by genetically transferring O2-tolerant hydrogenases from other bacteria into a class of photosynthetic bacteria called cyanobacteria. These hybrid organisms will use the photosynthetic machinery of the cyanobacterial hosts to perform the water-oxidation reaction with the input of solar energy, and couple the resulting protons and high energy electrons to the O2-tolerant bacterial hydrogenase, all within the same microbe (Fig. 1). The ultimate goal of this work has been to overcome the sensitivity of the hydrogenase enzyme to O2 and address one ofmore » the key technological hurdles to cost-effective photobiological H2 production which currently limits the production of hydrogen in algal systems. In pursuit of this goal, work on this project has successfully completed many subtasks leading to a greatly increased understanding of the complicated [NiFe]-hydrogenase enzymes. At the beginning of this project, [NiFe] hydrogenases had never been successfully moved across wide species barriers and had never been heterologously expressed in cyanobacteria. Furthermore, the idea that whole, functional genes could be extracted from complicated, mixed-sequence meta-genomes was not established. In the course of this work, we identified a new hydrogenase from environmental DNA sequence and successfully expressed it in a variety of hosts including cyanobacteria. This was one of the first examples of these complicated enzymes being moved across vastly different bacterial species and is the first example of a hydrogenase being “brought to life” from no other information than a DNA sequence from metagenomic data. The hydrogenase we identified had the molecular signature of other O2-tolerant hydrogenases, and we discovered that the resulting enzyme had exceptionally high oxygen- and thermo-tolerance. The new enzyme retained 80% of its activity after incubation at 80° C for 2 hours and retained 20% activity in 1% O2. We performed detailed analysis on the maturation genes required for construction of a functional enzyme of this class of hydrogenase, and found that seven additional maturation genes were required for minimal activity and a total of nine genes besides the hydrogenase were required for optimal maturation efficiency. Furthermore, we demonstrated that the maturation genes are functional on closely-related hydrogenase enzymes such as those from Alteromonas macleodii and Thiocapsa roseopersicina. Finally, we have extensively modified the hydrogenase to engineer new traits including higher H2 production and better interaction with electron donors. For example, combining two strategies increased hydrogenase activity in cyanobacteria by at least 20-fold over our initial expression level. The activity of this combined strain is almost twice that of the native hydrogenase activity in S. elongatus. This work validates the idea that these enzymes are broadly tolerant to modifications that may help integrate them into a successful photobiological H2 production system. While we did not achieve our ultimate goal of integrating the functional hydrogenase with the cyanobacterial photosynthetic apparatus, the work on this project has led to significant advances in the understanding of these complicated enzymes. This work will greatly benefit future projects focusing on photobiological H2 production. H2 is an excellent carbon-free storage molecule for solar energy. The vast diversity of hydrogenases is only now being appreciated as the number of bacterial genome sequences that are publically available has eclipsed the 10,000 mark and metagenomic DNA sequencing data from a wide variety of environments are now available. While this project focused on experiments with two hydrogenases, future work will have access to a much wider database of sequences to express and test in cyanobacteria. The results from this project justify and encourage future biotechnological approaches to realize direct H2 production from photobiological sources.« less
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  1. J. Craig Venter Institute
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Resource Type:
Technical Report
Research Org:
J. Craig Venter Institute
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Fuel Cell Technologies Office (EE-3V)
Country of Publication:
United States