13C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids

Meng Zhang, Xiao-Wei Yu, Yan Xu, Paula Jouhten, Gurla V.T. Swapna, Ralf W. Glaser, John F. Hunt, Gaetano T. Montelione, Hannu Maaheimo, Thomas Szyperski

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Carbon metabolism of Crabtree-negative yeast Pichia pastoris was profiled using 13C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First, 13C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree-negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree-negative Pichia stipitis and Crabtree-positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly 2H, 13C, 15N-labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR-based structural biology. Enzymes: Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49), pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), l-serine hydroxymethyltransferase (EC 2.1.2.1), threonine aldolase (EC 4.1.2.5), threonine dehydratase (EC 4.3.1.19); transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2).

Original languageEnglish
Pages (from-to)3100-3113
Number of pages14
JournalFEBS Journal
Volume284
Issue number18
DOIs
Publication statusPublished - 2017
MoE publication typeA1 Journal article-refereed

Fingerprint

Branched Chain Amino Acids
Batch Cell Culture Techniques
Pichia
Yeast
Threonine Dehydratase
Glycine Hydroxymethyltransferase
Fumarate Hydratase
Metabolism
Pyruvate Carboxylase
Fluxes
Glucose
Pyruvate Kinase
Carbon
Biosynthesis
malate dehydrogenase (decarboxylating)
Ketol-Acid Reductoisomerase
Saccharomyces cerevisiae
Transaldolase
Magnetic Resonance Spectroscopy
Yeasts

Keywords

  • 13C NMR
  • Komagatella phaf?
  • Metabolic Flux
  • Metabolite transport
  • Pichia pastoris

Cite this

Zhang, Meng ; Yu, Xiao-Wei ; Xu, Yan ; Jouhten, Paula ; Swapna, Gurla V.T. ; Glaser, Ralf W. ; Hunt, John F. ; Montelione, Gaetano T. ; Maaheimo, Hannu ; Szyperski, Thomas. / 13C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids. In: FEBS Journal. 2017 ; Vol. 284, No. 18. pp. 3100-3113.
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abstract = "Carbon metabolism of Crabtree-negative yeast Pichia pastoris was profiled using 13C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First, 13C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree-negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree-negative Pichia stipitis and Crabtree-positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly 2H, 13C, 15N-labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR-based structural biology. Enzymes: Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49), pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), l-serine hydroxymethyltransferase (EC 2.1.2.1), threonine aldolase (EC 4.1.2.5), threonine dehydratase (EC 4.3.1.19); transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2).",
keywords = "13C NMR, Komagatella phaf?, Metabolic Flux, Metabolite transport, Pichia pastoris",
author = "Meng Zhang and Xiao-Wei Yu and Yan Xu and Paula Jouhten and Swapna, {Gurla V.T.} and Glaser, {Ralf W.} and Hunt, {John F.} and Montelione, {Gaetano T.} and Hannu Maaheimo and Thomas Szyperski",
year = "2017",
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language = "English",
volume = "284",
pages = "3100--3113",
journal = "FEBS Journal",
issn = "1742-464X",
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13C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids. / Zhang, Meng; Yu, Xiao-Wei; Xu, Yan; Jouhten, Paula; Swapna, Gurla V.T.; Glaser, Ralf W.; Hunt, John F.; Montelione, Gaetano T.; Maaheimo, Hannu; Szyperski, Thomas.

In: FEBS Journal, Vol. 284, No. 18, 2017, p. 3100-3113.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - 13C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids

AU - Zhang, Meng

AU - Yu, Xiao-Wei

AU - Xu, Yan

AU - Jouhten, Paula

AU - Swapna, Gurla V.T.

AU - Glaser, Ralf W.

AU - Hunt, John F.

AU - Montelione, Gaetano T.

AU - Maaheimo, Hannu

AU - Szyperski, Thomas

PY - 2017

Y1 - 2017

N2 - Carbon metabolism of Crabtree-negative yeast Pichia pastoris was profiled using 13C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First, 13C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree-negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree-negative Pichia stipitis and Crabtree-positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly 2H, 13C, 15N-labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR-based structural biology. Enzymes: Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49), pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), l-serine hydroxymethyltransferase (EC 2.1.2.1), threonine aldolase (EC 4.1.2.5), threonine dehydratase (EC 4.3.1.19); transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2).

AB - Carbon metabolism of Crabtree-negative yeast Pichia pastoris was profiled using 13C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First, 13C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree-negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree-negative Pichia stipitis and Crabtree-positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly 2H, 13C, 15N-labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR-based structural biology. Enzymes: Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49), pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), l-serine hydroxymethyltransferase (EC 2.1.2.1), threonine aldolase (EC 4.1.2.5), threonine dehydratase (EC 4.3.1.19); transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2).

KW - 13C NMR

KW - Komagatella phaf?

KW - Metabolic Flux

KW - Metabolite transport

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