The unicellular cyanobacterium Synechocystis sp. PCC 6803 produces H2 during photosynthesis via a bidirectional Hox [NiFe]-hydrogenase. However, H2 production is short-lived in O2, an inherent byproduct of oxygenic photosynthesis. To overcome O2-sensitive H2 production, we are expressing a foreign, O2-tolerant hydrogenase in Synechocystis. Prior research in our laboratory uncovered an O2-tolerant [NiFe]-hydrogenase from the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS (CBS). Amino acid alignment suggests that this hexameric [NiFe]-hydrogenase (CooMKLXUH) belongs to Group 4 energy-conserving hydrogenases with a physiological function in H2 production from CO according to the equation CO + H2O --> H2 + CO2, which is verified by its expression and activity pattern. Additionally, six maturation genes, hyp(ABCDEF)1, were identified and shown to be expressed in a similar pattern to that of the hydrogenase, suggesting that these genes function in the assembly of the active site. Therefore, 4 of the 6 CBS hydrogenase genes, cooLXUH (CooM and K are membrane proteins and have been shown to be dispensable for in vitro activity), and the six maturation genes, hyp(ABCDEF)1, were transformed into a Synechocystis host lacking the native Hox hydrogenase. Initial studies revealed that the heterologously expressed CooLXUH can form a soluble complex in the Synechocystis host, albeit with no detectable activity. Work is ongoing to optimize CBS hydrogenase expression in Synechocystis and determine the minimal gene set required for heterologous expression and activity both in vivo ?and in vitro.

Genetic engineering in <i>Synechocystis</i> sp. PCC 6803 for solar hydrogen production

247th National Meeting (2014)

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Genetic engineering in Synechocystis sp. PCC 6803 for solar hydrogen production