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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.

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Principal pathways for the mixotrophic metabolism of glucose and CO2 to amino acids in Pt. Central carbon metabolic pathways convert glucose and/or CO2 (fixed photosynthetically or anaplerotically) to the 15 amino acids (metabolites shown as open circles) experimentally detected by GC-MS in hydrolysates of Pt cell pellets. In most organisms, glycolysis proceeds via the EMP pathway. However, two alternate glycolytic pathways of bacterial origin were found in this organism’s annotated genome. Of these, the phosphoketolase (PKP) enzyme converts phosphorylated pentose and/or hexose sugars to glyceraldehyde 3-phosphate/erythrose 4-phosphate and acetylphosphate, which is then converted to either acetate via acetate kinase, or acetyl-CoA via phosphate acetyltransferase. Both phosphorylated pentose and hexose sugars are shown as substrates for the PPK pathway because the enzyme specificity in Pt is unknown. The second alternative pathway (ED) uses two enzymes to convert 6-phospho-D-gluconate to pyruvate and glyceraldehyde 3-phosphate. Differences in the carbon atom rearrangements of the EMP, PPK and ED pathways become evident in the MIDs of glycolytic amino acids.
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Figure 1: Principal pathways for the mixotrophic metabolism of glucose and CO2 to amino acids in Pt. Central carbon metabolic pathways convert glucose and/or CO2 (fixed photosynthetically or anaplerotically) to the 15 amino acids (metabolites shown as open circles) experimentally detected by GC-MS in hydrolysates of Pt cell pellets. In most organisms, glycolysis proceeds via the EMP pathway. However, two alternate glycolytic pathways of bacterial origin were found in this organism’s annotated genome. Of these, the phosphoketolase (PKP) enzyme converts phosphorylated pentose and/or hexose sugars to glyceraldehyde 3-phosphate/erythrose 4-phosphate and acetylphosphate, which is then converted to either acetate via acetate kinase, or acetyl-CoA via phosphate acetyltransferase. Both phosphorylated pentose and hexose sugars are shown as substrates for the PPK pathway because the enzyme specificity in Pt is unknown. The second alternative pathway (ED) uses two enzymes to convert 6-phospho-D-gluconate to pyruvate and glyceraldehyde 3-phosphate. Differences in the carbon atom rearrangements of the EMP, PPK and ED pathways become evident in the MIDs of glycolytic amino acids.

Mentions: To test our hypothesis that Pt metabolizes glucose, we grew Pt cultures for 21 d on media supplemented with 1.917 ± 0.012 g L-1 of U-13C glucose under both light and dark, and examined if the supplied 13C label appeared in biomass components of Pt. In accordance with previously reported results [13-15], cultures kept in the dark exhibited no growth (Additional file 1: Figure S1); therefore, only cultures grown under light were analyzed for 13C-enrichment and glucose consumption. Toward this, we harvested biomass at the end of the 21-d steady state ILE (Additional file 2: Figure S2), acid-hydrolyzed the biomass to degrade cellular protein to amino acids and measured the 13C enrichments of the amino acids by mass spectrometry (MS). We also analyzed the media and found that the final glucose concentration was 0.791 ± 0.008 g L-1, indicating that 59% of the glucose was consumed. Furthermore, on finding evidence of mixotrophic glucose metabolism, we employed MPA to identify the metabolic pathways through which glucose is metabolized. Figure 1 depicts a diagram of potential metabolic pathways including the EMP, the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle, anaplerosis, glyoxylate shunt and RuBisCO-mediated photosynthesis. The 15 proteinogenic amino acids measured by us are synthesized from precursor metabolites belonging to these pathways; therefore, the labeling patterns in these precursors can be retrobiosynthetically evaluated from those of the amino acids [24]. In our analyses, we also considered the alternative glycolysis pathways (PPK using both hexose and pentose substrates; ED) (Figure 1, right).


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)

Principal pathways for the mixotrophic metabolism of glucose and CO2 to amino acids in Pt. Central carbon metabolic pathways convert glucose and/or CO2 (fixed photosynthetically or anaplerotically) to the 15 amino acids (metabolites shown as open circles) experimentally detected by GC-MS in hydrolysates of Pt cell pellets. In most organisms, glycolysis proceeds via the EMP pathway. However, two alternate glycolytic pathways of bacterial origin were found in this organism’s annotated genome. Of these, the phosphoketolase (PKP) enzyme converts phosphorylated pentose and/or hexose sugars to glyceraldehyde 3-phosphate/erythrose 4-phosphate and acetylphosphate, which is then converted to either acetate via acetate kinase, or acetyl-CoA via phosphate acetyltransferase. Both phosphorylated pentose and hexose sugars are shown as substrates for the PPK pathway because the enzyme specificity in Pt is unknown. The second alternative pathway (ED) uses two enzymes to convert 6-phospho-D-gluconate to pyruvate and glyceraldehyde 3-phosphate. Differences in the carbon atom rearrangements of the EMP, PPK and ED pathways become evident in the MIDs of glycolytic amino acids.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 1: Principal pathways for the mixotrophic metabolism of glucose and CO2 to amino acids in Pt. Central carbon metabolic pathways convert glucose and/or CO2 (fixed photosynthetically or anaplerotically) to the 15 amino acids (metabolites shown as open circles) experimentally detected by GC-MS in hydrolysates of Pt cell pellets. In most organisms, glycolysis proceeds via the EMP pathway. However, two alternate glycolytic pathways of bacterial origin were found in this organism’s annotated genome. Of these, the phosphoketolase (PKP) enzyme converts phosphorylated pentose and/or hexose sugars to glyceraldehyde 3-phosphate/erythrose 4-phosphate and acetylphosphate, which is then converted to either acetate via acetate kinase, or acetyl-CoA via phosphate acetyltransferase. Both phosphorylated pentose and hexose sugars are shown as substrates for the PPK pathway because the enzyme specificity in Pt is unknown. The second alternative pathway (ED) uses two enzymes to convert 6-phospho-D-gluconate to pyruvate and glyceraldehyde 3-phosphate. Differences in the carbon atom rearrangements of the EMP, PPK and ED pathways become evident in the MIDs of glycolytic amino acids.
Mentions: To test our hypothesis that Pt metabolizes glucose, we grew Pt cultures for 21 d on media supplemented with 1.917 ± 0.012 g L-1 of U-13C glucose under both light and dark, and examined if the supplied 13C label appeared in biomass components of Pt. In accordance with previously reported results [13-15], cultures kept in the dark exhibited no growth (Additional file 1: Figure S1); therefore, only cultures grown under light were analyzed for 13C-enrichment and glucose consumption. Toward this, we harvested biomass at the end of the 21-d steady state ILE (Additional file 2: Figure S2), acid-hydrolyzed the biomass to degrade cellular protein to amino acids and measured the 13C enrichments of the amino acids by mass spectrometry (MS). We also analyzed the media and found that the final glucose concentration was 0.791 ± 0.008 g L-1, indicating that 59% of the glucose was consumed. Furthermore, on finding evidence of mixotrophic glucose metabolism, we employed MPA to identify the metabolic pathways through which glucose is metabolized. Figure 1 depicts a diagram of potential metabolic pathways including the EMP, the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle, anaplerosis, glyoxylate shunt and RuBisCO-mediated photosynthesis. The 15 proteinogenic amino acids measured by us are synthesized from precursor metabolites belonging to these pathways; therefore, the labeling patterns in these precursors can be retrobiosynthetically evaluated from those of the amino acids [24]. In our analyses, we also considered the alternative glycolysis pathways (PPK using both hexose and pentose substrates; ED) (Figure 1, right).

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