Journal
MICROBIAL CELL FACTORIES
Volume 12, Issue -, Pages -Publisher
BMC
DOI: 10.1186/1475-2859-12-109
Keywords
Phaeodactylum tricornutum; Glucose; Mixotrophy; Isotope labeling; Metabolic pathway analysis; Entner-Doudoroff
Categories
Funding
- National Science Foundation [CBET-1134115]
- Hulka Energy Research fellowship from the University of Maryland Energy Research Center
- Directorate For Engineering
- Div Of Chem, Bioeng, Env, & Transp Sys [1134115] Funding Source: National Science Foundation
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Background: Heterotrophic 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. Results: Isotope labeling experiments in which Pt was mixotrophically grown under light on 100% U-C-13 glucose and naturally abundant (similar to 99% C-12) dissolved inorganic carbon resulted in proteinogenic amino acids with an average C-13-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-C-13 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-C-13 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. Conclusion: 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.
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