Approaches to developing biological H(2)-photoproducing organisms and processes.

The development of efficient biological systems for the direct photoproduction of H(2) gas from water faces several challenges, the more serious of which is the sensitivity of the H(2)-evolving enzymes (hydrogenases) to O(2), an obligatory by-product of photosynthesis. This high sensitivity is common to both FeFe and NiFe hydrogenases, and is caused by O(2) binding to their respective metallocatalytic sites. This overview describes approaches to (i) molecular engineering of algal FeFe-hydrogenase to prevent O(2) access to its catalytic site; (ii) transform a cyanobacterium with an O(2)-tolerant bacterial NiFe hydrogenase or (c) partially inactivate algal O(2)-evolution activity to create physiologically anaerobiosis and induce hydrogenase expression.

Identification of genes required for hydrogenase activity in Chlamydomonas reinhardtii.

The eukaryotic green alga, Chlamydomonas reinhardtii, produces H(2) under anaerobic conditions, in a reaction catalysed by an [FeFe]-hydrogenase. To identify genes that influence H(2) production in C. reinhardtii, a library of 6000 colonies on agar plates was screened with sensitive chemochromic H(2)-sensor films for clones defective in H(2) production. Two mutants of particular interest were fully characterized. One mutant, hydEF-1, is unable to assemble an active [FeFe]-hydrogenase. This is the first reported C. reinhardtii mutant that is not capable of producing any H(2). The second mutant, sta7-10, is not able to accumulate insoluble starch and has significantly lowered H(2)-photoproduction rates in comparison with the wild-type. In hydEF-1, anaerobiosis induces transcription of the two reported C. reinhardtii hydrogenase genes, HydA1 and HydA2, indicating a normal transcriptional response to anaerobiosis. In contrast, the transcription of both hydrogenase genes in sta7-10 is significantly attenuated.

Response of hya expression to external pH in Escherichia coli.

The hya operon of Escherichia coli is composed of the genes which synthesize uptake hydrogenase isoenzyme 1 (Hyd1). Although hya expression and Hyd1 synthesis occur only under anaerobic conditions, Hyd1 is not essential for growth. In this study we used a hya'-'lacZ fusion to characterize parameters of anaerobic growth that maximize hya expression in an attempt to further elucidate Hyd1 function. We found that the expression pattern of hya followed a decline of external pH. In buffered media where the pH value was set, the onset of hya expression initiated earlier in growth and reached a greater peak level in acidic than in alkaline medium. When cultures expressing hya were shifted from acidic to alkaline conditions, hya expression was arrested; shifting from alkaline to acidic conditions stimulated hya expression. Maximal expression of hya under all growth conditions required the sigma factor RpoS and transcriptional regulators AppY and ArcA. In the absence of RpoS or AppY, the response of hya expression onset to external pH was evident and maximal hya levels remained greater in acidic than in alkaline medium. However, the absence of ArcA led to a diminished response of expression onset to external pH and the loss of elevated expression at an acidic external pH. The fermentation end product formate slightly altered hya expression levels but was not required for hya to respond to external pH. In contrast to hya expression, the onset of hyb operon expression, encoding uptake hydrogenase isoenzyme 2, was constitutive with respect to external pH. However, external pH did affect hyb expression levels, which, in contrast to hya, were maximal in alkaline rather than acidic medium.