Development of an Efficient Algal H2-production System

The direct photoevolution of H2 from water by green algae is a transient phenomenon, due to the rapid inactivation of the reversible hydrogenase (the enzyme catalyzing the reduction of protons to H2) by O2, a by-product of photosynthesis. Moreover, the expression of the algal H2 production activity requires prolonged anaerobic treatment of the cells in order to induce the enzyme. We are addressing the O2sensitivity problem of algal hydrogenase by means of both classical genetics and molecular biology approaches. The ultimate goal of our research is to generate a Chlamydomonas reinhardtii mutant that is sufficiently tolerant to O2 to produce H2 under aerobic conditions. The availability of such mutant will permit the development of a commercial photobiological H2-production system that is cost effective, renewable, scalable and non-polluting. The classical mutagenesis/selection approach that we have developed to obtain such a desirable mutant takes advantage of the reversible activity of the algal hydrogenase. We have designed two selective pressures that require mutagenized algal cells to survive by either metabolizing (photoreductive selective pressure) or evolving (H2-evolution selective pressure) H2 in the presence of O2 concentrations that inactivate the wild-type (WT) enzyme. Given the generally low specificity of the two selective pressures, the surviving organisms are subsequently subjected to a positive screen using a chemochromic sensor that detects H2 evolved by the algae. Clones that are found to exhibit high H2-evolution activity in the presence of O2 are characterized in more detail using biochemical assays. The strategy currently employed consists of re-mutagenizing, re-selecting and re-screening first generation mutants under higher selective stringency in order to accumulate single-point mutations, and thus, to further increase the O2 tolerance of the organism. Results for the past year include further optimization of the two selective pressures; the adoption of a new assay for characterizing the O2 tolerance of selected clones; the isolation of a clone, 104G5 with 14 times higher tolerance to O2 than the WT (obtained by the application of one round of photoreductive selective pressure); and the isolation of first and second generation mutants with, respectively, 3-4 and 10 times higher tolerance to O2 than the WT (by application of two rounds of the H2-evolution selective pressure). In order to enhance our probability of ultimately obtaining a commercially-viable organism, we have also been pursuing a molecular biology approach, which is synergistic with the classical genetic strategy described above. We intend to (a) clone the hydrogenase gene and use site-directed mutagenesis (based on information gathered from mutations generated by the classical approach that affect the O2 tolerance of the hydrogenase) to further increase the O2 tolerance of the enzyme and (b) identify other proteins whose presence may be require for optimal algal H2 evolution. Two techniques are currently being used to achieve these goals, mainly RT-PCR (which allows the amplification of a specific DNA sequence out of a population of isolated mRNA) and the construction of a subtractive library, which will contain mRNAs representing proteins that are expressed only upon anaerobic induction of the cells. This is on-going work, and current year advances consisted of the production of the subtracted DNA from induced minus uninduced cells corresponding to 1 and 4 h of anaerobic treatment. Preliminary results with the University of California, Berkeley on the development of a two-phase algal H2production system will not be presented here, but have suggested the scientific feasibility of employing indirect biophotolysis to develop a commercial algal H2-production system as a mid-term solution until an O2-tolerant organism becomes available.