Limits...
Experimental evidence and isotopomer analysis of mixotrophic glucose metabolism in the marine diatom Phaeodactylum tricornutum.

Zheng Y, Quinn AH, Sriram G - Microb. Cell Fact. (2013)

Bottom Line: 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).We validated the latter result for mixotrophic growth on glycerol by performing a 2-(13)C glycerol isotope labeling experiment.We further analyzed the metabolic mechanisms underlying the mixotrophic metabolism of glucose and found isotopic evidence for unusual pathways active in Pt.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, MD 20742, USA. gsriram@umd.edu.

ABSTRACT

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-(13)C glucose and naturally abundant (~99% (12)C) 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-(13)C 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-(13)C 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.

Show MeSH

Related in: MedlinePlus

Unique carbon-carbon bond re-arrangements explain abnormal isotope abundances from the 100% 1-13C Glc ILE. (a) Five different Metabolic Models (V-IX) were constructed to explain the ILE data; each column represents a different model. Pathways included in a model are denoted by a “Y”, color-coded according to the color of the pathway in Figure 3. (b) Vertical bars represent the SSR of each model and horizontal lines represent the acceptable SSR (65) corresponding to the number of redundant isotopomer measurements (45) in each model. (c) The carbon rearrangements of SGAT and SHMT demonstrate how 3-13C triose phosphates (derived from 1-13C glucose through glycolysis) result in 2-13C serine and glycine. Serine is conventionally known to be synthesized directly from 3-phosphoglycerate without carbon rearrangements via PSAT shown with the dashed arrow. (d) The photorespiratory action of RuBisCO yields glyoxylate from pentose phosphate, whereas the PPK pathway yields acetate that is converted to glyoxylate via the glyoxylate shunt. Each pathway yields 2-13C glycine and serine from 1-13C pentose phosphate arising from the reductive PPP. (e) Anaplerotic fixation of a mixture of intracellular 12CO2 and 13CO2 results in 1-13C pyruvate through reversible reactions in the TCA cycle. Succinate is a symmetric molecule; therefore C-1 and C-4 are equivalent. Oxaloacetate is 6% 13C-enriched at the C-4 position and 13% enriched at C-1. In comparison, pyruvate is 18% enriched at C-1, indicating that anaplerotic fixation cannot fully account for the labeling on pyruvate. (f) The oxidative PPP yields 13CO2 and U-12C ribulose 5-phosphate from 1-13C glucose-6-phosphate. Photosynthetic fixation of CO2 via RuBisCO then results in 1-13C pyruvate. (g) The predominant isotopomers of serine and glycine that were simulated in the poorly fit Model VII and the well fit Model IX are compared against the experimental abundance of each isotopomer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842785&req=5

Figure 5: Unique carbon-carbon bond re-arrangements explain abnormal isotope abundances from the 100% 1-13C Glc ILE. (a) Five different Metabolic Models (V-IX) were constructed to explain the ILE data; each column represents a different model. Pathways included in a model are denoted by a “Y”, color-coded according to the color of the pathway in Figure 3. (b) Vertical bars represent the SSR of each model and horizontal lines represent the acceptable SSR (65) corresponding to the number of redundant isotopomer measurements (45) in each model. (c) The carbon rearrangements of SGAT and SHMT demonstrate how 3-13C triose phosphates (derived from 1-13C glucose through glycolysis) result in 2-13C serine and glycine. Serine is conventionally known to be synthesized directly from 3-phosphoglycerate without carbon rearrangements via PSAT shown with the dashed arrow. (d) The photorespiratory action of RuBisCO yields glyoxylate from pentose phosphate, whereas the PPK pathway yields acetate that is converted to glyoxylate via the glyoxylate shunt. Each pathway yields 2-13C glycine and serine from 1-13C pentose phosphate arising from the reductive PPP. (e) Anaplerotic fixation of a mixture of intracellular 12CO2 and 13CO2 results in 1-13C pyruvate through reversible reactions in the TCA cycle. Succinate is a symmetric molecule; therefore C-1 and C-4 are equivalent. Oxaloacetate is 6% 13C-enriched at the C-4 position and 13% enriched at C-1. In comparison, pyruvate is 18% enriched at C-1, indicating that anaplerotic fixation cannot fully account for the labeling on pyruvate. (f) The oxidative PPP yields 13CO2 and U-12C ribulose 5-phosphate from 1-13C glucose-6-phosphate. Photosynthetic fixation of CO2 via RuBisCO then results in 1-13C pyruvate. (g) The predominant isotopomers of serine and glycine that were simulated in the poorly fit Model VII and the well fit Model IX are compared against the experimental abundance of each isotopomer.

Mentions: We limited our models to simulate the confirmed cytosolic and mitochondrial amino acids from a 100% 1-13C glucose ILE, because amongst the glucose labels used, 1-13C glucose has a higher information yield than U-13C glucose for a network consisting of glycolysis and related pathways [27]. Additionally, we wanted to eliminate errors from amino acids with identical precursors but different MIDs due to differing compartmentalization. The entire metabolic network is shown in Figure 3, with each constituent pathway distinguished by color. Models I-IX contain different combinations of pathways, as specified in Figures 4a and 5a. A simple model, Model I, encompassed only the EMP and PPP pathways. This model simulated the MIDs of serine, glycine and alanine, comprising 22 redundant mass isotopomers. Model I yielded an SSR of 87 (Figure 4b), which is much higher than the statistically acceptable SSR of 37 corresponding to the set of 22 mass isotopomers. The two alanine fragments contributed a majority (58%) of this SSR (Additional file 3: Figure S3), specifically because this model was unable to mimic the high measured abundance of alanine{123}. The inability of the Model I to account for the isotopomer data is evident from the carbon atom rearrangements in Figure 4c (dark blue squares denote 13C atoms). Processing of 1-13C glucose by the EMP pathway or the PPP results in alanine{123}. In contrast, the ED pathway cleaves the first three carbons of glucose directly to pyruvate yielding the alanine{123} isotopomer that we experimentally observed. Therefore, we extended Model I by incorporating the ED pathway, resulting in Model II. This extension reduced the SSR from 87 in Model I to 42 in Model II (Figure 4b). The improvement of Model II over Model I is evident from a comparison of measured and simulated isotopomers of alanine (Figure 4d).


Experimental evidence and isotopomer analysis of mixotrophic glucose metabolism in the marine diatom Phaeodactylum tricornutum.

Zheng Y, Quinn AH, Sriram G - Microb. Cell Fact. (2013)

Unique carbon-carbon bond re-arrangements explain abnormal isotope abundances from the 100% 1-13C Glc ILE. (a) Five different Metabolic Models (V-IX) were constructed to explain the ILE data; each column represents a different model. Pathways included in a model are denoted by a “Y”, color-coded according to the color of the pathway in Figure 3. (b) Vertical bars represent the SSR of each model and horizontal lines represent the acceptable SSR (65) corresponding to the number of redundant isotopomer measurements (45) in each model. (c) The carbon rearrangements of SGAT and SHMT demonstrate how 3-13C triose phosphates (derived from 1-13C glucose through glycolysis) result in 2-13C serine and glycine. Serine is conventionally known to be synthesized directly from 3-phosphoglycerate without carbon rearrangements via PSAT shown with the dashed arrow. (d) The photorespiratory action of RuBisCO yields glyoxylate from pentose phosphate, whereas the PPK pathway yields acetate that is converted to glyoxylate via the glyoxylate shunt. Each pathway yields 2-13C glycine and serine from 1-13C pentose phosphate arising from the reductive PPP. (e) Anaplerotic fixation of a mixture of intracellular 12CO2 and 13CO2 results in 1-13C pyruvate through reversible reactions in the TCA cycle. Succinate is a symmetric molecule; therefore C-1 and C-4 are equivalent. Oxaloacetate is 6% 13C-enriched at the C-4 position and 13% enriched at C-1. In comparison, pyruvate is 18% enriched at C-1, indicating that anaplerotic fixation cannot fully account for the labeling on pyruvate. (f) The oxidative PPP yields 13CO2 and U-12C ribulose 5-phosphate from 1-13C glucose-6-phosphate. Photosynthetic fixation of CO2 via RuBisCO then results in 1-13C pyruvate. (g) The predominant isotopomers of serine and glycine that were simulated in the poorly fit Model VII and the well fit Model IX are compared against the experimental abundance of each isotopomer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842785&req=5

Figure 5: Unique carbon-carbon bond re-arrangements explain abnormal isotope abundances from the 100% 1-13C Glc ILE. (a) Five different Metabolic Models (V-IX) were constructed to explain the ILE data; each column represents a different model. Pathways included in a model are denoted by a “Y”, color-coded according to the color of the pathway in Figure 3. (b) Vertical bars represent the SSR of each model and horizontal lines represent the acceptable SSR (65) corresponding to the number of redundant isotopomer measurements (45) in each model. (c) The carbon rearrangements of SGAT and SHMT demonstrate how 3-13C triose phosphates (derived from 1-13C glucose through glycolysis) result in 2-13C serine and glycine. Serine is conventionally known to be synthesized directly from 3-phosphoglycerate without carbon rearrangements via PSAT shown with the dashed arrow. (d) The photorespiratory action of RuBisCO yields glyoxylate from pentose phosphate, whereas the PPK pathway yields acetate that is converted to glyoxylate via the glyoxylate shunt. Each pathway yields 2-13C glycine and serine from 1-13C pentose phosphate arising from the reductive PPP. (e) Anaplerotic fixation of a mixture of intracellular 12CO2 and 13CO2 results in 1-13C pyruvate through reversible reactions in the TCA cycle. Succinate is a symmetric molecule; therefore C-1 and C-4 are equivalent. Oxaloacetate is 6% 13C-enriched at the C-4 position and 13% enriched at C-1. In comparison, pyruvate is 18% enriched at C-1, indicating that anaplerotic fixation cannot fully account for the labeling on pyruvate. (f) The oxidative PPP yields 13CO2 and U-12C ribulose 5-phosphate from 1-13C glucose-6-phosphate. Photosynthetic fixation of CO2 via RuBisCO then results in 1-13C pyruvate. (g) The predominant isotopomers of serine and glycine that were simulated in the poorly fit Model VII and the well fit Model IX are compared against the experimental abundance of each isotopomer.
Mentions: We limited our models to simulate the confirmed cytosolic and mitochondrial amino acids from a 100% 1-13C glucose ILE, because amongst the glucose labels used, 1-13C glucose has a higher information yield than U-13C glucose for a network consisting of glycolysis and related pathways [27]. Additionally, we wanted to eliminate errors from amino acids with identical precursors but different MIDs due to differing compartmentalization. The entire metabolic network is shown in Figure 3, with each constituent pathway distinguished by color. Models I-IX contain different combinations of pathways, as specified in Figures 4a and 5a. A simple model, Model I, encompassed only the EMP and PPP pathways. This model simulated the MIDs of serine, glycine and alanine, comprising 22 redundant mass isotopomers. Model I yielded an SSR of 87 (Figure 4b), which is much higher than the statistically acceptable SSR of 37 corresponding to the set of 22 mass isotopomers. The two alanine fragments contributed a majority (58%) of this SSR (Additional file 3: Figure S3), specifically because this model was unable to mimic the high measured abundance of alanine{123}. The inability of the Model I to account for the isotopomer data is evident from the carbon atom rearrangements in Figure 4c (dark blue squares denote 13C atoms). Processing of 1-13C glucose by the EMP pathway or the PPP results in alanine{123}. In contrast, the ED pathway cleaves the first three carbons of glucose directly to pyruvate yielding the alanine{123} isotopomer that we experimentally observed. Therefore, we extended Model I by incorporating the ED pathway, resulting in Model II. This extension reduced the SSR from 87 in Model I to 42 in Model II (Figure 4b). The improvement of Model II over Model I is evident from a comparison of measured and simulated isotopomers of alanine (Figure 4d).

Bottom Line: 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).We validated the latter result for mixotrophic growth on glycerol by performing a 2-(13)C glycerol isotope labeling experiment.We further analyzed the metabolic mechanisms underlying the mixotrophic metabolism of glucose and found isotopic evidence for unusual pathways active in Pt.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, MD 20742, USA. gsriram@umd.edu.

ABSTRACT

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-(13)C glucose and naturally abundant (~99% (12)C) 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-(13)C 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-(13)C 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.

Show MeSH
Related in: MedlinePlus