Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.United States. Dept. of EnergyUnited States. Advanced Research Projects Agency-Energ

The distribution and ecology of the fresh- and brackish-water medusae of the world.

Limnocnida and Craspedacusta are the two main genera of Cnidaria from continental waters which have a free-swimming medusa in their life cycle. Both originated in tropical-subtropical Asia, but Limnocnida is also found in Africa, with one species (L. tanganjicae) perhaps endemic to lake Tanganyika. Halmomises from Trinidad, and two genera (Mansariella, Keralica) from India have limited ranges. No freshwater medusae evolved in Europe and the Americas (aside from Trinidad), but Craspedacusta sowerbii, by virtue of its drought resistant stages, has managed to colonise all continents, except Antarctica, in the course of the twentieth century. In all, there are about 10–14 freshwater species of medusae. An additional 4–5 species of Moerisia, Australomedusa, and Craspedacusta are sequestered in continental salt-lakes, while a few genera occur in brackish-water seas and estuaries (e.g. Moerisia, Odessia, Ostroumovia). This reduced diversity is probably due to (1) a rarity of drought resistant stages, adjusted to upstream dispersal, in the life cycle, and (2) difficulties to adapt the osmoregulatory system to a hypotonic environment.The feeding ecology of the freshwater medusae is also examined. Like all Cnidaria, they are opportunistic predators. However, fish egg predation might be their major means of subsistence, other types of food being taken only occasionally, or when fish eggs and larvae are scarce. Their impact on the true zooplankton might therefore be limited to short pulses of planktivory. Whether they are themselves limited by predation remains to be studied; many fish in the invasive part of their range avoid them, but some macrocrustaceans readily consume them. It is hypothesized that this might explain their success in Lake Tanganyika, and their absence from lake Baical. Finally, the absence of endemic species from South America could relate to the great diversity bf small-sized predatory aquatic reptiles on this subcontinent