Project description:These research areas concentrate on stress induced proteases in recombinant Escherichia coli, glycosylation heterogeneity due to bioprocess conditions produced in mammalian cells, and metabolic engineering of E. coli. The hypothesis of this project is that recombinant protein glycosylation is inefficient under normal bioreactor conditions since the additional glycosylation reactions necessary for the recombinant protein exceed the metabolic capacity of the cells. Normal bioreactor conditions have been optimized for cell growth, and sometimes for protein productivity. Only recently has it been accepted that optimal glycosylation may not occur under optimal growth or protein productivity conditions. Specific Aim: Determine the relationship between bioreactor conditions and glycosylation gene expression in NS0 cells. EXPERIMENT: Mouse NS0 myeloma cells were grown in culture, stressed with 5 mM NaCl, 10 mM proline plus 5 mM ammonia, or 5 mM ammonia, along with an unstressed control group. The growth of the cultures were followed until the late exponential phase (90 hours), at which time two 50 mLs of cells were harvested and RNA extracted. Samples were prepared in triplicate, for a total of 12 samples. The RNA was amplified and labeled by Microarray Core (E) and hybridized to the GLYCOv3 microarrays.
Project description:The industrially important fungus Aspergillus niger feeds naturally on decomposing plant material, for which it is equipped with a range of enzyme systems. A significant proportion of plant material are lipids that might be available either as for energy storage or as membrane building blocks. With 63 potential lipase-encoding genes in its genome, A. niger has the tools to degrade these extracellular lipids. In contrast to polysaccharide-degrading enzyme networks not much is known about the signalling and regulatory processes that control lipase expression and activity in fungi both under laboratory and natural occurring conditions. A pulse of 1 mM of various oils was applied to four bioreactor-grown A. niger cultures to examine (i) whether A. niger responds at the level of gene transcription, (ii) at what time point this effect is detected most accurately, and (iii) whether differences between the response towards oils are observed. The triglyceride olive oil induces genes encoding peroxins and enzymes of fatty acid metabolism. A complex oil mixture extracted from wheat gluten, which is enriched for digalactosyl-diglycerides, induces genes encoding peroxins as well as enzymes of fatty acid metabolism, but with different expression profile when compared to olive oil. Pure digalactosyldiglyceride, a proxy for plant membrane lipids, does not trigger a transcriptional response. Keywords: time course; induction experiment
Project description:These research areas concentrate on stress induced proteases in recombinant Escherichia coli, glycosylation heterogeneity due to bioprocess conditions produced in mammalian cells, and metabolic engineering of E. coli. The hypothesis of this project is that recombinant protein glycosylation is inefficient under normal bioreactor conditions since the additional glycosylation reactions necessary for the recombinant protein exceed the metabolic capacity of the cells. Normal bioreactor conditions have been optimized for cell growth, and sometimes for protein productivity. Only recently has it been accepted that optimal glycosylation may not occur under optimal growth or protein productivity conditions. Specific Aim: Determine the relationship between bioreactor conditions and glycosylation gene expression in NS0 cells.
Project description:The industrially important fungus Aspergillus niger feeds naturally on decomposing plant material, for which it is equipped with a range of enzyme systems. A significant proportion of plant material are lipids that might be available either as for energy storage or as membrane building blocks. With 63 potential lipase-encoding genes in its genome, A. niger has the tools to degrade these extracellular lipids. In contrast to polysaccharide-degrading enzyme networks not much is known about the signalling and regulatory processes that control lipase expression and activity in fungi both under laboratory and natural occurring conditions. A pulse of 1 mM of various oils was applied to four bioreactor-grown A. niger cultures to examine (i) whether A. niger responds at the level of gene transcription, (ii) at what time point this effect is detected most accurately, and (iii) whether differences between the response towards oils are observed. The triglyceride olive oil induces genes encoding peroxins and enzymes of fatty acid metabolism. A complex oil mixture extracted from wheat gluten, which is enriched for digalactosyl-diglycerides, induces genes encoding peroxins as well as enzymes of fatty acid metabolism, but with different expression profile when compared to olive oil. Pure digalactosyldiglyceride, a proxy for plant membrane lipids, does not trigger a transcriptional response. Keywords: time course; induction experiment In one week, 4 fermentor cultures were run in 2.2-liter batch fermentors in which A. niger was grown on 100 mM sorbitol. At 14 hours after oxygen supply had switched from headspace to sparger-inlet each fermentor was induced with 22 mL medium which contained 100 mM (final concentration in fermentor: 1 mM) of either olive oil, a complex oil mixture extracted from wheat gluten, pure wheat digalactosyldiglycerides, or was induced with a solution of minimal medium containing only 0.2% (in fermentor, final concentration 0.002%) triton X-100 which served as emulsifier agent. For each fermentor vessel, a sample of 10 mL was taken prior to induction (T=0), or 30 minutes, 1 hour, or 2 hours after induction. Every sample was hybridized onto a single microarray, yielding in total 16 DNA microarrays.
Project description:Microalgal lipid, a feasible substrate for biofuel, is typically accumulated during the stationary growth phase. Generating strains which trigger lipogenesis from the exponential growth phase will enhance lipid productivity, reduce cost of biofuel production. We characterized a lipid-rich microalgal mutant, Dunaliella tertiolecta, which exhibited a six-fold enhancement of neutral lipids production in the exponential growth phase with marginal compromise on growth (4%). Using transcriptomics and metabolomics, regulatory mechanisms of the mutant were uncovered.
Project description:Volatile fatty acids found in effluents of the dark fermentation of biowastes can be used for mixotrophic growth of microalgae, improving productivity and reducing the cost of the feedstock. Microalgae can use the acetate in the effluents very well, but butyrate is poorly assimilated and can inhibit growth above 1 gC.L-1. The non-photosynthetic chlorophyte alga Polytomella sp. SAG 198.80 was found to be able to assimilate butyrate fast. To decipher the metabolic pathways implicated in butyrate assimilation, a large-scale differential proteomics study was developed comparing Polytomella sp. cells grown on acetate and butyrate at 1 gC.L-1.
Project description:Solventogenic Clostridium species ferment carbohydrates to acetone, butanol and ethanol which are well-known next-generation biofuels. However, repeated subculture of or continuous fermentation by Clostridium often decreases and eventually terminates the solvent production and spore formation, which is a process called strain degeneration. Supplementation of CaCO3 to fermentation medium could partially recover metabolism of degenerated strain by more than 50% increase of cell growth and solvent production. The transcriptome profile of Clostridium beijerinckii NCIMB 8052 (DG-8052) and its response to CaCO3 treatment were analysed by microarray. Since fermentation by C. beijerinckii NCIMB 8052 is a biphasic process, gene expressions of two fermentations were compared at each stage, i.e. 12h and 24h fermentation time representing acidogenic phase and solventogenic phase, respectively. This study examined expression of 5168 genes capturing 98.6% of the C. beijerinckii NCIMB 8052 genome. With the addition of CaCO3, DG-8052 had 565 and 916 genes significantly up-regulated at acidogenic phase and solventogenic phase, respectively. According to the enrichment analysis of pathway and Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, these genes were significantly overrepresented in cellular functions such as Amino acid transport and metabolism, organic acid biosynthetic process, bacteria chemotaxis and defense mechanisms. On the other hand, there were 704 and 1044 genes significantly down-regulated at acidogenic phase and solventogenic phase, respectively. These repressed genes were mainly enriched in functions such as ion transmembrane transport, ATP synthesis, oxidative phosphorylation.