Chemical & Biomolecular Engineering
Permanent URI for this communityhttp://hdl.handle.net/1903/2219
Formerly known as the Department of Chemical Engineering.
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Item Metabolic Flux Analysis for Metabolic Engineering of Marine Organisms(2018) Quinn, Andrew Higgins; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We explored the metabolic pathways in two industrially relevant marine microorganisms to understand their core metabolic capabilities. It is necessary to track how an organism distributes organic building blocks throughout its metabolic pathways so that we can devise strategies to alter its metabolism and reroute substantial metabolic flux towards target compound(s). Though we cannot measure intracellular metabolic fluxes directly, we can retro-biosynthetically calculate them by supplying substrates labeled with non-radioactive isotopes to an organism. We then measure the resulting isotope labeling patterns of metabolites and calculate the fluxes that produced them. We addressed three goals with our research, (i) resolving questions surrounding organic carbon metabolism in the diatom Phaeodactylum tricornutum (P. tricornutum), (ii) identifying reactions in a putative photosynthetic carbon concentrating mechanism in P. tricornutum and (iii) mapping central carbon metabolism of the cellulolytic aerobe Saccharophagus degradans (S. degradans). Towards goal (i) we show that P. tricornutum predominantly consumes glucose, as opposed to atmospheric CO2, under mixotrophic conditions using the Entner-Doudoroff (ED) glycolytic pathway instead of the more common Embden-Meyerhof-Parnas pathway (EMP). We utilized metabolic flux analysis (MFA) to discover that acetate is metabolized for energy production instead of for biomass formation during mixotrophic growth on CO2 and acetate. Finally, we developed a method for measuring isotopic labeling in polyunsaturated fatty acids via gas chromatography-mass spectrometry (GC-MS), and demonstrated its utility in resolving outstanding questions about glucose metabolism by P. tricornutum. Towards goal (ii) we utilized isotope labeling and gene silencing in combination to identify pyruvate carboxylase as a key enzyme in a C4 carbon concentrating mechanism in P. tricornutum, while also ruling out phosphoenolpyruvate carboxylase as a key enzyme in the pathway. Towards goal (iii) we present 13C-MFA of aerobic consumption of glucose, xylose, and cellobiose by S. degradans. This is the first reported MFA of cellobiose metabolism and one of only a handful analyzing xylose metabolism in an aerobic microorganism.Item OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION FOR CELL-PENETRATING PEPTIDE FUSIONS TO PROTEIN CARGO(2017) Adhikari, Sayanee; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recombinant production of cell-penetrating peptides (CPPs) as fusions to protein “cargo” leads to low yields for some CPP-cargo fusions; thus, ways to enhance the recombinant expression of peptide-cargo fusions need to be identified. We optimized expression conditions for fusions of five CPPs (NPFSD, pVEC, SynB, histatin-5 and MPG) to the cargo proteins biotin carboxyl carrier protein (BCCP), maltose binding protein (MBP) and green fluorescent protein GFP. Glutathione-S-transferase was incorporated as a fusion partner to improve expression. In general, expression at 37 oC for 6 h and 10 h led 2 to the highest levels of expression for the different CPP-cargo constructs. The fusion of histatin-5 to GFP was purified, and its translocation into the fungal pathogen Candida albicans was studied. The purified protein translocated into the nearly 3% of C. albicans cells. These results provide the foundation for future studies to improve translocation of varied CPP-cargo fusions into C. albicans cells.Item The Effect of Zinc in Carbon Concentrating Mechanisms in Phaeodactylum tricornutum(2015) Zhou, Nairui; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Photosynthesis is crucial for life but is a slow process because the CO2 concentration near the principal carbon-assimilation enzyme RuBisCO is extremely low. Very few plants and algae perform a carbon-concentrating mechanism (CCM) to overcome the insufficiency, which are classified into biophysical and biochemical (C4) mechanism. The enzyme CA catalyzes the reversible dehydration of HCO3- to CO2 in biophysical CCMs and its active site contains a Zn2+. In this study, we hypothesized that Zn2+ availability can impact CCMs and therefore investigated the effect of Zn2+ availability on photosynthetic metabolism in a unicellular marine diatom Phaeodactylum tricornutum. P. tricornutum has a sequenced genome and can conduct both biophysical and C4 CCMs. We observed that Zn2+ has a significant effect on cell growth rate but no significant interference on intracellular metabolism, suggesting no essential compensation of C4 CCMs for biophysical CCMs even at low CA activity anticipated at low Zn2+ concentration.