Presentation Information
[P04-474]Metabolome analysis identifies pyruvate accumulation as a key control step for ethanol production in Komagataella phaffii
○Genki Sato1, Nobuyuki Okahashi1, Matsuda Fumio1 (1. Osaka Univ. (Japan))
Keywords:
Komagataella phaffii,Metabolome analysis,Proteome analysis,Ethanol production,Metabolic shift
[Purpose]
Some yeast species exhibit a metabolic shift from non-ethanol-producing respiratory growth to ethanol-producing fermentation during glucose batch cultivation. The Crabtree-negative yeast Komagataella phaffii (Pichia pastoris) is known to rapidly activate ethanol production in response to a decline in dissolved oxygen (DO). However, the precise metabolic regulatory mechanisms responsible for the shift remain unclear. This study aims to investigate metabolic regulatory mechanisms using metabolomics to artificially control metabolic shifts.
[Method]
K. phaffii CBS7435 was cultivated at a flask scale in 50 mL of BMD medium (1.34% YNB w/o amino acids, 2% glucose, 4 mg/L biotin, and 10 mM potassium phosphate buffer, pH 6.0) at 30 °C and 120 rpm. Extracellular metabolites were quantified by HPLC, and DO was monitored using an optical sensor (PreSens). For metabolomics, intracellular metabolites were extracted using the chloroform-methanol-water method and quantified via LC-MS/MS and GC-MS (Shimadzu).
[Results and discussion]
During batch cultivation, K. phaffii initially exhibited non-ethanol-producing growth. A rapid shift to ethanol production was observed at approximately 13 hours in the mid-log phase, when DO levels fell below 50%. This shift was accompanied by a significant increase in the specific glucose consumption rate, which rose by 24% by 16 hours, confirming a swift acceleration of glycolytic flux under oxygen-limited conditions[1].
A metabolome analysis of 121 intracellular metabolites revealed 2.8- and 3.0-folds accumulations of pyruvate and fructose1,6-bisphosphate (FBP) during the metabolic shift, respectively. Thermodynamic analysis using the ΔΔG method[2] revealed that the ΔΔG of pyruvate kinase (PYK) was positive (6.0 kJ/mol) during the metabolic shift. Furthermore, proteomic analysis showed that PYK (Cdc19p) expression increased by 180%. Given that FBP is an allosteric activator of PYK[3], these results suggest that PYK activation was driven by both expression levels and post-translational regulation, which contributes to the enhanced glycolytic flux. Conversely, ΔΔG for pyruvate carboxylase (PYC) was negative (-7.9 kJ/mol), suggesting inactivation of the anaplerotic pathway for the TCA cycle.
Based on these findings, we conducted control of the metabolic shift using the PYC inhibitor, phenylacetate[4]. The addition of 2.5 mM phenylacetate successfully accelerated the onset of the metabolic shift by 4 hours. These results indicate that decreased PYC activity contributes to the metabolic shift through the accumulation of pyruvate.
[References]
[1] Baumann, K., Carnicer, M., Dragosits, M., et al., BMC Syst. Biol. (2010).
[2] Matsuda, F., Kamiyama, A., Yamasaki, K., et al., Anal. Chem. (2025).
[3] Schormann, N., Hayden, K. L., Lee, P., et al., Protein Sci. (2019).
[4] Vijay Kumar, N., & Rangarajan, P. N., Microbiology (2011).
Some yeast species exhibit a metabolic shift from non-ethanol-producing respiratory growth to ethanol-producing fermentation during glucose batch cultivation. The Crabtree-negative yeast Komagataella phaffii (Pichia pastoris) is known to rapidly activate ethanol production in response to a decline in dissolved oxygen (DO). However, the precise metabolic regulatory mechanisms responsible for the shift remain unclear. This study aims to investigate metabolic regulatory mechanisms using metabolomics to artificially control metabolic shifts.
[Method]
K. phaffii CBS7435 was cultivated at a flask scale in 50 mL of BMD medium (1.34% YNB w/o amino acids, 2% glucose, 4 mg/L biotin, and 10 mM potassium phosphate buffer, pH 6.0) at 30 °C and 120 rpm. Extracellular metabolites were quantified by HPLC, and DO was monitored using an optical sensor (PreSens). For metabolomics, intracellular metabolites were extracted using the chloroform-methanol-water method and quantified via LC-MS/MS and GC-MS (Shimadzu).
[Results and discussion]
During batch cultivation, K. phaffii initially exhibited non-ethanol-producing growth. A rapid shift to ethanol production was observed at approximately 13 hours in the mid-log phase, when DO levels fell below 50%. This shift was accompanied by a significant increase in the specific glucose consumption rate, which rose by 24% by 16 hours, confirming a swift acceleration of glycolytic flux under oxygen-limited conditions[1].
A metabolome analysis of 121 intracellular metabolites revealed 2.8- and 3.0-folds accumulations of pyruvate and fructose1,6-bisphosphate (FBP) during the metabolic shift, respectively. Thermodynamic analysis using the ΔΔG method[2] revealed that the ΔΔG of pyruvate kinase (PYK) was positive (6.0 kJ/mol) during the metabolic shift. Furthermore, proteomic analysis showed that PYK (Cdc19p) expression increased by 180%. Given that FBP is an allosteric activator of PYK[3], these results suggest that PYK activation was driven by both expression levels and post-translational regulation, which contributes to the enhanced glycolytic flux. Conversely, ΔΔG for pyruvate carboxylase (PYC) was negative (-7.9 kJ/mol), suggesting inactivation of the anaplerotic pathway for the TCA cycle.
Based on these findings, we conducted control of the metabolic shift using the PYC inhibitor, phenylacetate[4]. The addition of 2.5 mM phenylacetate successfully accelerated the onset of the metabolic shift by 4 hours. These results indicate that decreased PYC activity contributes to the metabolic shift through the accumulation of pyruvate.
[References]
[1] Baumann, K., Carnicer, M., Dragosits, M., et al., BMC Syst. Biol. (2010).
[2] Matsuda, F., Kamiyama, A., Yamasaki, K., et al., Anal. Chem. (2025).
[3] Schormann, N., Hayden, K. L., Lee, P., et al., Protein Sci. (2019).
[4] Vijay Kumar, N., & Rangarajan, P. N., Microbiology (2011).
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