Structure elucidation of natural products including the absolute configuration is a complex task that involves different analytical methods like mass spectrometry, NMR spectroscopy, and chemical derivation, which are usually performed after the isolation of the compound of interest. Here, a combination of stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transaminase mutants followed by detailed MS analysis enabled the structure elucidation of novel cyclopeptides named GameXPeptides including their absolute configuration in crude extracts without their actual isolation.

BackgroundThe 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.ResultsIsobutanol 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.ConclusionA 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.

Fatty acid-derived ether lipids are present not only in most vertebrates but also in some bacteria. Here we describe what is to our knowledge the first gene cluster involved in the biosynthesis of such lipids in myxobacteria that encodes the multifunctional enzyme ElbD, which shows similarity to polyketide synthases. Initial characterization of elbD mutants in Myxococcus xanthus and Stigmatella aurantiaca showed the importance of these ether lipids for fruiting body formation and sporulation.

dAcetogenic bacteria can grow by the oxidation of various substrates coupled to the reduction of CO 2 in the Wood-Ljungdahl pathway. Here, we show that growth of the acetogen Acetobacterium woodii on 1,2-propanediol (1,2-PD) as the sole carbon and energy source is independent of acetogenesis. Enzymatic measurements and metabolite analysis revealed that 1,2-PD is dehydrated to propionaldehyde, which is further oxidized to propionyl coenzyme A (propionyl-CoA) with concomitant reduction of NAD. NADH is reoxidized by reducing propionaldehyde to propanol. The potential gene cluster coding for the responsible enzymes includes genes coding for shell proteins of bacterial microcompartments. Electron microscopy revealed the presence of microcompartments as well as storage granules in cells grown on 1,2-PD. Gene clusters coding for the 1,2-PD pathway can be found in other acetogens as well, but the distribution shows no relation to the phylogeny of the organisms.A cetogenic bacteria are a diverse group of anaerobic bacteria able to reduce two molecules of CO 2 to acetate by the WoodLjungdahl pathway (WLP) (1-4). Electrons may derive from molecular hydrogen (autotrophic growth) or from organic donors (heterotrophic growth), such as hexoses, pentoses, formate, lactate, alcohols, and methyl group donors (1). This not only provides the cell with organic material for biomass formation, but the pathway is also coupled to energy conservation for ATP supply (2, 5). The energy-conserving reactions remained an enigma for a long time, but recent discoveries in the model acetogen Acetobacterium woodii provided insights into the energy metabolism of this group of anaerobic bacteria (6, 7). In A. woodii, the reactions for the oxidation of the substrate can in general be regarded as isolated modules separate from the reactions of the WLP for the reoxidation of the electron carriers by the reduction of CO 2 . One has to emphasize that all enzymes of the WLP are soluble and located in the cytoplasm (6). With molecular hydrogen as the electron donor, only one enzyme is necessary for its oxidation, providing the reducing equivalents as reduced ferredoxin (Fd) and NADH in a 1:1 stoichiometry (8). Oxidation of organic substrates, such as hexoses, also yields reduced ferredoxin and NADH that are reoxidized in the WLP. The WLP in A. woodii does not use both electron carriers in equal amounts; therefore, a membranebound Fd:NAD oxidoreductase (presumably the Rnf complex) provides NADH from reduced ferredoxin, thereby translocating sodium ions across the cytoplasmic membrane that are used for subsequent ATP synthesis by a membrane-bound, sodium iondependent ATP synthase (7, 9, 10). The reaction is reversible, and the enzyme can drive Fd reduction at the expense of the electrochemical sodium ion potential.Besides CO 2 , acetogenic bacteria can use different alternative electron acceptors, e.g., nitrate (Moorella thermoacetica [11]) or phenylacrylates (A. woodii [12]). These acceptors have a more positive redox potential than the CO 2 -acetate pair,...