Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris

Aina Solà, Hannu Maaheimo, Katri Ylönen, Pau Ferrer (Corresponding Author), Thomas Szyperski (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

72 Citations (Scopus)

Abstract

Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional 13C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h−1, 0.16 h−1) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h−1 on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h−1 (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h−1. Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h−1. This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.
Original languageEnglish
Pages (from-to)2462 - 2470
Number of pages9
JournalEuropean Journal of Biochemistry
Volume271
Issue number12
DOIs
Publication statusPublished - 2004
MoE publication typeA1 Journal article-refereed

Fingerprint

Pichia
Biosynthesis
Glycerol
Oxaloacetic Acid
Dilution
Fluxes
Amino Acids
Glucose
Chemostats
Saccharomyces cerevisiae
Carbon
Metabolism
Mitochondrial Membranes
Pyruvic Acid
Yeast
Labeling
Membranes
Enzymes

Keywords

  • 13C NMR
  • central metabolism
  • flux profiling
  • metabolic engineering
  • Pichia pastoris

Cite this

Solà, Aina ; Maaheimo, Hannu ; Ylönen, Katri ; Ferrer, Pau ; Szyperski, Thomas. / Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris. In: European Journal of Biochemistry. 2004 ; Vol. 271, No. 12. pp. 2462 - 2470.
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abstract = "Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional 13C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h−1, 0.16 h−1) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h−1 on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h−1 (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h−1. Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33{\%} to 48{\%} when increasing the dilution rate for glycerol supply, while 38{\%} is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h−1. This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.",
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Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris. / Solà, Aina; Maaheimo, Hannu; Ylönen, Katri; Ferrer, Pau (Corresponding Author); Szyperski, Thomas (Corresponding Author).

In: European Journal of Biochemistry, Vol. 271, No. 12, 2004, p. 2462 - 2470.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris

AU - Solà, Aina

AU - Maaheimo, Hannu

AU - Ylönen, Katri

AU - Ferrer, Pau

AU - Szyperski, Thomas

PY - 2004

Y1 - 2004

N2 - Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional 13C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h−1, 0.16 h−1) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h−1 on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h−1 (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h−1. Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h−1. This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.

AB - Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional 13C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h−1, 0.16 h−1) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h−1 on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h−1 (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h−1. Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h−1. This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.

KW - 13C NMR

KW - central metabolism

KW - flux profiling

KW - metabolic engineering

KW - Pichia pastoris

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DO - 10.1111/j.1432-1033.2004.04176.x

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JO - FEBS Journal

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SN - 1742-464X

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