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Numerous fast growing and highly competitive exotic crops are being selected for production of renewable bioenergy. Tolerance of poor growing conditions with minimal inputs are ideal characteristics for bioenergy feedstocks, but have attracted concern for their potential to become invasive. Miscanthus × giganteus is one of the most promising bioenergy crops in the US, but grower adoption is hindered by high establishment costs due to sterility. Newly developed fertile tetraploid M. × giganteus may streamline cultivation while reducing establishment costs. However, fertile seed dramatically increases the potential propagule pressure, and thus probability of off-site plant establishment. To empirically evaluate the invasive potential of fertile M. × giganteus in the Southeastern US, we compared fitness and spread potential relative to ten grass species comprising 19 accessions under both high and low levels of competition and disturbance. We chose species known to be invasive in the US (positive controls: Arundo donax, naturalized M. sinensis, M. sacchariflorus, Phalaris arundinacea, Sorghum halepense) and non-invasive (negative controls; Andropogon gerardii, ornamental M. sinensis, Panicum virgatum, Sorghum bicolor, Saccharum spp.). This novel design allows us to make relative comparisons of risk among species with varying invasiveness. After three years of establishment and growth in Blacksburg, Virginia, neither aboveground disturbance nor interspecific weed competition influenced fitness for fertile M. × giganteus or our positive and negative control groups. Fertile M. × giganteus produced 346% and 283% greater aboveground biomass than our positive and negative species, respectively. However, fertile M. × giganteus produced 74% fewer inflorescences m-2 than our positive controls and 7% and 51% fewer spikelets inflorescence-1 than the positive and negative control species. After 18 months of growth, we observed the vegetative and seedling spread of three of our positive control species (S. halepense, P. arundinacea, and M. sacchariflorus) outside the cultivated plot into receiving areas of both high and low competition. After 24 months of growth, numerous species were observed outside the cultivated plot including fertile M. × giganteus and 50% of negative control species. Notably, in three years sterile M. × giganteus ‘Illinois’ and Arundo donax never moved from the cultivated plot. The addition of fertile seed appears to increase the potential for offsite movement, but within the geographic confines of our empirical evaluation, fertile M. × giganteus seedlings are more similar to native P. virgatum and were not nearly as fast growing or as competitive as our positive control S. halepense. The use of numerous species providing relative comparisons allow us to draw important conclusions which may help prepare for widespread commercialization, while providing novel methodology for ecological risk assessment of new species.
Background: The branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol.
Results: Isobutanol production could be improved by re-locating the valine biosynthesis enzymes Ilv2, Ilv5 and Ilv3 from the mitochondrial matrix into the cytosol. To prevent the import of the three enzymes into yeast mitochondria, N-terminally shortened Ilv2, Ilv5 and Ilv3 versions were constructed lacking their mitochondrial targeting sequences. SDS-PAGE and immunofluorescence analyses confirmed expression and re-localization of the truncated enzymes. Growth tests or enzyme assays confirmed enzymatic activities. Isobutanol production was only increased in the absence of valine and the simultaneous blockage of the mitochondrial valine synthesis pathway. Isobutanol production could be even more enhanced after adapting the codon usage of the truncated valine biosynthesis genes to the codon usage of highly expressed glycolytic genes. Finally, a suitable ketoisovalerate decarboxylase, Aro10, and alcohol dehydrogenase, Adh2, were selected and overexpressed. The highest isobutanol titer was 0.63 g/L at a yield of nearly 15 mg per g glucose.
Conclusion: A cytosolic isobutanol production pathway was successfully established in yeast by re-localization and optimization of mitochondrial valine synthesis enzymes together with overexpression of Aro10 decarboxylase and Adh2 alcohol dehydrogenase. Driving forces were generated by blocking competition with the mitochondrial valine pathway and by omitting valine from the fermentation medium. Additional deletion of pyruvate decarboxylase genes and engineering of co-factor imbalances should lead to even higher isobutanol production.
Background: The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.
Results: The previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L−1 in a 72-h fermentation. The additional accumulation of 90 mg L−1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L−1. However, in growth tests concentrations even lower than 50.0 mg L−1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.
Conclusions: By providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.