Browsing by Author "Zheng, Yuting"
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Item 13C and 15N Metabolic Flux Analysis on the Marine Diatom Phaeodactylum tricornutum to Investigate Efficient Unicellular Carbon and Nitrogen Assimilation Mechanisms(2013) Zheng, Yuting; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Photosynthesis is indispensable in carbon cycling and obtaining renewable carbon. Operated by cyanobacteria, algae and plants, this process provides reduced carbon and molecular oxygen, consumes atmospheric CO2 and harnesses solar energy. Photosynthesis is also central to the production of biofuels. Diatoms, a class of marine algae, contribute 20% to 40% of global photosynthetic productivity despite surviving in CO2-depleted and nitrogen-limited environments. This makes diatoms ideal models to study efficient photosynthetic, specifically carbon concentrating mechanisms (CCM). It has been long debated that whether the unicellular marine diatom Phaeodactylum tricornutum operates a CCM, and whether the CCM is biophysical or biochemical (C4) in nature, with existing (circumstantial) experimental evidence divided amongst the two possibilities. Through isotope labeling experiments (ILE) and metabolic flux analysis (MFA), we provide for the first time significant, direct evidence for a biochemical CCM and the potential combined operation of a biochemical and a biophysical CCM. Additionally, we shed light on how genes regulating this complex process respond to critical environmental variables. Furthermore, we report the use of isotope-assisted metabolic flux analysis to study organic carbon (especially glucose) assimilation in P. tricornutum. Our steady state ILEs reveal glucose assimilation under light and potentially which genes may be responsible for glucose metabolism. We then studied nitrogen (mainly urea) assimilation through instationary 15N and 13C labeling experiments, to find indications of an unusual pathway of urea assimilation. Gene expression trends under various environmental conditions suggest the possible participation of the urea cycle in assimilating nitrogen in P. tricornutum, and how this metabolically differs from nitrate and ammonium assimilation. We anticipate that this work will not only improve understanding of unicellular C4 CCMs, but provide insights to explain the ecological success of diatoms in adapting to challenging environments.Item Experimental evidence and isotopomer analysis of mixotrophic glucose metabolism in the marine diatom Phaeodactylum tricornutum(Springer Nature, 2013-11-14) Zheng, Yuting; Quinn, Andrew H; Sriram, GaneshHeterotrophic fermentation using simple sugars such as glucose is an established and cost-effective method for synthesizing bioproducts from bacteria, yeast and algae. Organisms incapable of metabolizing glucose have limited applications as cell factories, often despite many other advantageous characteristics. Therefore, there is a clear need to investigate glucose metabolism in potential cell factories. One such organism, with a unique metabolic network and a propensity to synthesize highly reduced compounds as a large fraction of its biomass, is the marine diatom Phaeodactylum tricornutum (Pt). Although Pt has been engineered to metabolize glucose, conflicting lines of evidence leave it unresolved whether Pt can natively consume glucose. Isotope labeling experiments in which Pt was mixotrophically grown under light on 100% U-13C glucose and naturally abundant (~99% 12C) dissolved inorganic carbon resulted in proteinogenic amino acids with an average 13C-enrichment of 88%, thus providing convincing evidence of glucose uptake and metabolism. The dissolved inorganic carbon was largely incorporated through anaplerotic rather than photosynthetic fixation. Furthermore, an isotope labeling experiment utilizing 1-13C glucose and subsequent metabolic pathway analysis indicated that (i) the alternative Entner-Doudoroff and Phosphoketolase glycolytic pathways are active during glucose metabolism, and (ii) during mixotrophic growth, serine and glycine are largely synthesized from glyoxylate through photorespiratory reactions rather than from 3-phosphoglycerate. We validated the latter result for mixotrophic growth on glycerol by performing a 2-13C glycerol isotope labeling experiment. Additionally, gene expression assays showed that known, native glucose transporters in Pt are largely insensitive to glucose or light, whereas the gene encoding cytosolic fructose bisphosphate aldolase 3, an important glycolytic enzyme, is overexpressed in light but insensitive to glucose. We have shown that Pt can use glucose as a primary carbon source when grown in light, but cannot use glucose to sustain growth in the dark. We further analyzed the metabolic mechanisms underlying the mixotrophic metabolism of glucose and found isotopic evidence for unusual pathways active in Pt. These insights expand the envelope of Pt cultivation methods using organic substrates. We anticipate that they will guide further engineering of Pt towards sustainable production of fuels, pharmaceuticals, and platform chemicals.