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Quantitative metabolic flux analysis revealed uneconomical utilization of ATP and NADPH in Acremonium chrysogenum fed with soybean oil

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Abstract

A metabolic network was constructed for the Acremonium chrysogenum cultivation fed with soybean oil. Metabolic flux analysis indicated that the shift from exponential growth to rapid cephalosporin C (CPC) formation was accompanied by 1.63- and 5-fold carbon flux enlargement in TCA cycle and glyoxylate by-pass, respectively. The flux via pentose phosphate pathway branch was little affected during the rapid CPC formation period; the contributory explanation was that 35.6% of NADPH was consumed in the dissimilation of fatty acids. Estimation of NADPH, ATP generation, and consumption demonstrated that, with soybean oil as carbon source in rapid CPC formation phase, the NADPH consumed in fatty acid catabolism was fourfold greater than that used in the CPC biosynthesis-relevant part; simultaneously, more than 90% energy spent was not directly related to the CPC formation. Therefore, the improvement of CPC production yield through optimization of the NADPH, ATP generation, and consumption was put forward.

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Abbreviations

G-6-P:

Glucose-6-phosphate

F-6-P:

Fructose-6-phosphate

GAP:

Glyceraldehyde-3-phosphate

3-PG:

3-Phosphoglycerate

Ru-5-P:

Ribulose-5-phosphate

PEP:

Phosphoenolpyruvate

PYR:

Pyruvate

OAA:

Oxaloacetic acid

ISOCIT:

Isocitrate

α-KG:

α-Ketoglutarate

α-AAA:

α-Amino adipinic acid

CPC:

Cephalosporin C

Metex :

Extracellular methionine

ATP:

Adenosine triphosphate

NADH:

Reduced diphosphopyridine nucleotide

FADH2 :

Reduced flavin adenine dinucleotide

NADPH:

Reduced nicotinamide adenine dinucleotide phosphate

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Acknowledgments

This work was financially supported by a grant from the National High Technology Research and Development Program of China (863 Program), No. 2006AA020302, the Major State Basic Research Development Program of China (973 Program), No. 2007CB714303, National Key Technology Research and Development Program, No. 2008BAI63B01, 2007BAI26B02, and Shanghai Leading Academic Discipline Project, No. B505.

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, P.O. Box 329, Shanghai, 200237, People’s Republic of China

    Jianhua Li, Yiming Yang, Ju Chu, Mingzhi Huang, Liang Li, Yonghong Wang, Yingping Zhuang & Siliang Zhang

  2. Shanxi Weiqida Pharmaceutical Co. Ltd, Shanxi, 037000, People’s Republic of China

    Xiaochun Zhang

Authors
  1. Jianhua Li
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  2. Yiming Yang
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  3. Ju Chu
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  4. Mingzhi Huang
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  5. Liang Li
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  6. Xiaochun Zhang
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  7. Yonghong Wang
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  8. Yingping Zhuang
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  9. Siliang Zhang
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Corresponding authors

Correspondence to Ju Chu or Mingzhi Huang.

Appendix: Cellular biochemical reactions included in metabolic model

Appendix: Cellular biochemical reactions included in metabolic model

  1. (1)

    Uptake reactions

  1. 1.

    glucose + ATP → G-6-P

  2. 2.

    soybean oil → glycerol + fatty acids

  3. 3.

    palmitic acid + ATP + 8CoASH → 8acetyl-CoA + 7FADH2 + 7NADH + AMP + 2Pi

  4. 4.

    stearic acid + ATP + 9CoASH → 9acetyl-CoA + 8FADH2 + 8NADH + AMP + 2Pi

  5. 5.

    arachidic acid + ATP + 10CoASH → 10acetyl-CoA + 9FADH2 + 9NADH + AMP + 2Pi

  6. 6.

    oleic acid + ATP + 9CoASH → 9acetyl-CoA + 7FADH2 + 8NADH + AMP + 2Pi

  7. 7.

    linoleic acid + ATP + 9CoASH + NADPH → 9acetyl-CoA + 6FADH2 + 8NADH + AMP + 2Pi

  8. 8.

    linolenic acid + ATP + 9CoASH + NADPH → 9acetyl-CoA + 5FADH2 + 8NADH + AMP + 2Pi

  9. 9.

    glycerol + ATP → GAP + NADH + ADP + Pi

  1. (2)

    Embden–Meyerhof–Parns pathway

  1. 10.

    G-6-P → F-6-P

  2. 11.

    F-6-P + ATP → 2GAP + ADP + Pi

  3. 12.

    GAP → 3-PG + ATP + NADH

  4. 13.

    3-PG → PEP

  5. 14.

    PEP → PYR + ATP

  1. (2X)

    Gluconeogenesis

  1. 10X.

    F-6-P → G-6-p

  2. 11X.

    2GAP + H2O → F-6-P + Pi

  3. 12X.

    3-PG + ATP + NADH → GAP

  4. 13X.

    PEP → 3-PG

  5. 14.

    PEP → PYR + ATP

  6. 15X.

    OAA → PEP + CO2

  1. (3)

    TCA cycle

  1. 15.

    PYR + CoASH → acetyl-CoA + CO2 + NADH

  2. 16.

    OAA + acetyl-CoA → ISOCIT + CoASH

  3. 17.

    ISOCIT → α-KG + CO2 + NADH

  4. 18.

    α-KG + CoASH → succinyl-CoA + NADH + CO2

  5. 19.

    succinyl-CoA → succinate + ATP + CoASH

  6. 20.

    Succinate → malate + FADH2

  7. 21.

    Malate → OAA + NADH

  1. (4)

    Glyoxylate bypass

  1. 22.

    ISOCIT → succinate + glyoxylate

  2. 23.

    glyoxylate + acetyl-CoA → malate + CoASH

  1. (5)

    Pentose phosphate pathway

  1. 24.

    G-6-P + H2O+2NADP+ → Ru-5-P + 2NADPH + CO2 + 2H+

  2. 25.

    3Ru-5-P→ 2F-6-P + GAP

  1. (6)

    Miscellaneous reactions and oxidative phosphorylation

  1. 26.

    lactate → PYR + FADH2

  2. 27.

    α-ketobutyrate + ATP + HSCoA → succinyl-CoA + NADH + ADP + Pi

  3. 28.

    NADH+ 0.5O2→ 2.5ATP

  4. 29.

    FADH2+ 0.5O2 → 1.5ATP

  1. (7)

    Biosynthesis of CPC

  1. 30.

    2PYR + glutamate + NADPH → Valine + α-KG+CO2

  2. 31.

    glutamate + acetyl-CoA → α-AAA + NADH + CO2 +HSCoA

  3. 32.

    methionine + ATP → homocysteine + Adensine + PPi+Pi

  4. 33.

    homocysteine + serine → cysteine +α-ketobutyrate+NH4 +

  5. 34.

    serine + acetyl-CoA + SO4 2− + 4ATP + 4NADPH → cysteine + 4ADP + 4Pi + acetate

  6. 35.

    α-AAA + cysteine + valine + 6ATP + 3O2 + 2α-KG + acetyl-CoA → CPC + 2succinate + 2CO2

  1. (8)

    Biosynthesis of amino acid and macromoleculars

  1. 36.

    G-6-P → Biomass

  2. 37.

    Ru-5-P → Biomass

  3. 38.

    F-6-P → Biomass

  4. 39.

    serine → Biomass

  5. 40.

    PEP → Biomass

  6. 41.

    PYR → Biomass

  7. 42.

    acetyl-CoA → Biomass

  8. 43.

    OAA → Biomass

  9. 44.

    α-KG → Biomass

  10. 45.

    α-AAA + glutamate + 2ATP + 2NADPH → lysine + α-KG + NADH + 2ADP + 2Pi

  11. 46.

    lysine → Biomass

  12. 47.

    methionine → Biomass

  13. 48.

    valine → Biomass

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Li, J., Yang, Y., Chu, J. et al. Quantitative metabolic flux analysis revealed uneconomical utilization of ATP and NADPH in Acremonium chrysogenum fed with soybean oil. Bioprocess Biosyst Eng 33, 1119–1129 (2010). https://doi.org/10.1007/s00449-010-0439-1

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  • DOI: https://doi.org/10.1007/s00449-010-0439-1

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