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BackgroundHydrogen is considered a promising energy vector that can be produced from sustainable resources such as sunlight and water. In green algae, such as Chlamydomonas reinhardtii, photoproduction of hydrogen is catalyzed by the enzyme [FeFe]-hydrogenase (HydA). Although highly efficient, this process is transitory and thought to serve as a release valve for excess reducing power. Up to date, prolonged production of hydrogen was achieved by the deprivation of either nutrients or light, thus, hindering the full potential of photosynthetic hydrogen production. Previously we showed that the enzyme superoxide dismutase (SOD) can enhance HydA activity in vitro, specifically when tied together to a fusion protein.ResultsIn this work, we explored the in vivo hydrogen production phenotype of HydA–SOD fusion. We found a sustained hydrogen production, which is dependent on linear electron flow, although other pathways feed it as well. In addition, other characteristics such as slower growth and oxygen production were also observed in Hyd–SOD-expressing algae.ConclusionsThe Hyd–SOD fusion manages to outcompete the Calvin–Benson cycle, allowing sustained hydrogen production for up to 14 days in non-limiting conditions.

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The transition from ground to air in flying animals is often assisted by the legs pushing against the ground as the wings start to flap. Here, we show that when tiny whiteflies (Bemisia tabaci, body length ca. 1 mm) perform take-off jumps with closed wings, the abrupt push against the ground sends the insect into the air rotating forward in the sagittal ( pitch) plane. However, in the air, B. tabaci can recover from this rotation remarkably fast (less than 11 ms), even before spreading its wings and flapping. The timing of body rotation in air, a simplified biomechanical model and take-off in insects with removed wings all suggest that the wings, resting backwards alongside the body, stabilize motion through air to prevent somersaulting. The increased aerodynamic force at the posterior tip of the body results in a pitching moment that stops body rotation. Wing deployment increases the pitching moment further, returning the body to a suitable angle for flight. This inherent stabilizing mechanism is made possible by the wing shape and size, in which half of the wing area is located behind the posterior tip of the abdomen.

The integration of genes into the nuclear genome of Chlamydomonas reinhardtii is mediated by Non-Homologous-End-Joining, thus resulting in unpredicted insertion locations. This phenomenon defines 'the position-effect', which is used to explain the variation of expression levels between different clones transformed with the same DNA fragment. Likewise, nuclear transgenes often undergo epigenetic silencing that reduces their expression; hence, nuclear transformations require high-throughput screening methods to isolate clones that express the foreign gene at a desirable level. Here, we show that the number of integration sites of heterologous genes results in higher mRNA levels. By transforming both a synthetic ferredoxin-hydrogenase fusion enzyme and a Gaussia-Luciferase reporter protein, we were able to obtain 33 positive clones that exhibit a wide range of synthetic expression. We then performed a droplet-digital polymerasechain-reaction for these lines to measure their transgene DNA copy-number and mRNA levels. Surprisingly, most clones contain two integration sites of the synthetic gene (45.5%), whilst 33.3% contain one, 18.1% include three and 3.1% encompass four. Remarkably, we observed a positive correlation between the raw DNA copy-number values to the mRNA levels, suggesting a general effect of which transcription of transgenes is partially modulated by their number of copies in the genome. However, our data indicate that only clones harboring at least three copies of the target amplicon show a significant increment in mRNA levels of the reporter transgene. Lastly, we measured protein activity for each of the reporter genes to elucidate the effect of copynumber variation on heterologous expression.