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Citrate Stimulates Oligosaccharide Synthesis in Metabolically Engineered Agrobacterium sp.

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Abstract

Agrobacterium sp. ATCC 31749 was previously shown to be an advantageous host for oligosaccharide production. Unexpectedly, the addition of citrate to the oligosaccharide synthesis reaction resulted in up to a sixfold improvement in the production N-aceytl-lactosamine, a disaccharide. The possible mechanisms for this citrate-induced stimulation of oligosaccharide production were investigated, including the consumption of citrate as a carbon and energy source, enhanced metal ion solubility from citrate chelation, and the ability of citrate to act as a buffer. The main mechanisms for the effect of citrate on oligosaccharide production were determined to be carbon and energy provision from citrate consumption and pH maintenance. ATCC 31749 was shown to co-metabolize citrate along with sucrose, a preferred carbon source, indicating the lack of a catabolite repression system in this Agrobacterium. Metabolic flux analysis suggested an increase in flux through TCA cycle for the citrate-containing reaction, which may provide additional energy supply to support enhanced oligosaccharide production. The citrate stimulation of oligosaccharide synthesis was shown to be unique to the Agrobacterium strain, as a similarly engineered Escherichia coli strain did not show significant improvement in oligosaccharide production with citrate addition. This work provides insight into the metabolism of Agrobacterium sp. ATCC 31749 and highlights important factors in whole-cell oligosaccharide synthesis.

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Abbreviations

ACoA:

Acetyl coenzyme A

ADP:

Adenosine diphosphate

aKG:

Alpha-ketoglutarate

ATP:

Adenosine triphosphate

CIT:

Citrate

Crd:

Curdlan

ED:

Entner–Doudoroff

F6P:

Fructose-6-phosphate

FAD:

Flavin adenine dinucleotide (oxidized)

FADH2 :

Flavin adenine dinucleotide (reduced)

Fru:

Fructose

G1P:

Glucose-1-phosphate

G6P:

Glucose-6-phosphate

GAP:

Glyceraldehyde-3-phosphate

Glc:

Glucose

Glycerol-3-P:

Glycerol-3-phosphate

ICT:

Isocitrate

LacNAc:

N-Aceytl-lactosamine

MAL:

Malate

NAD:

Nicotinamide adenine dinucleotide (oxidized)

NADH:

Nicotinamide adenine dinucleotide (reduced)

OAA:

Oxaloacetate

P3:

Product 3 (galactose-β1,4-mannose)

PPP:

Pentose phosphate pathway

PTS:

Phosphotransferase system

PYR:

Pyruvate

SUCC:

Succinate

SUCC-CoA:

Succinyl coenzyme A

TCA:

Tricarboxylic acid

UDP-Gal:

Uridine diphosphate galactose

UDP-Glc:

Uridine diphosphate glucose

UTP:

Uridine triphosphate

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Acknowledgments

This work was funded by National Science Foundation (BES 0455193) and the American Cancer Society. A. Ruffing acknowledges a NSF graduate fellowship.

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Authors and Affiliations

  1. School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA, 30332-0100, USA

    Anne M. Ruffing & Rachel Ruizhen Chen

Authors
  1. Anne M. Ruffing
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  2. Rachel Ruizhen Chen
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Corresponding author

Correspondence to Rachel Ruizhen Chen.

Appendix

Appendix

  1. (1)

    Sucrose → Glc + Fru

  2. (2)

    Glc → Glc (extracellular)

  3. (3)

    Fru → Fru (extracellular)

  4. (4)

    Glc + ATP → G6P + ADP

  5. (5)

    Fru + ATP → F6P + ADP

  6. (6)

    F6P → G6P

  7. (7)

    G6P → G1P

  8. (8)

    G1P + ATP → UDP-Glc + ADP

  9. (9)

    UDP-Glc → Crd

  10. (10)

    UDP-Glc → UDP-Gal

  11. (11)

    UDP-Gal + Glc → Lactose

  12. (12)

    UDP-Gal + GlcNAc → LacNAc

  13. (13)

    UDP-Gal + F6P → P3

  14. (14)

    G6P → 6PG

  15. (15)

    6PG → GAP + PYR

  16. (16)

    Glycerol-3-P + NAD → GAP + NADH

  17. (17)

    Glycerol + ATP → Glycerol-3-P + ADP

  18. (18)

    GAP + 2 ADP + NAD → PYR + 2 ATP + NADH

  19. (19)

    PYR + NAD → ACoA + NADH

  20. (20)

    ACoA + ADP → Acetate + ATP

  21. (21)

    ACoA + OAA → CIT

  22. (22)

    CIT (extracellular) → CIT

  23. (23)

    CIT → ICT

  24. (24)

    ICT + NAD → aKG + NADH

  25. (25)

    aKG + NAD → SUCC-CoA + NADH

  26. (26)

    SUCC-CoA + ADP → SUCC + ATP

  27. (27)

    SUCC + FAD → MAL + FADH2

  28. (28)

    MAL + NAD → OAA + NADH

  29. (29)

    ICT + ACoA → SUCC + MAL

  30. (30)

    NADH + 3 ADP → NAD + 3 ATP

  31. (31)

    FADH2 + 2 ADP → FAD + 2 ATP

  32. (32)

    ATP → ADP

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Ruffing, A.M., Chen, R.R. Citrate Stimulates Oligosaccharide Synthesis in Metabolically Engineered Agrobacterium sp.. Appl Biochem Biotechnol 164, 851–866 (2011). https://doi.org/10.1007/s12010-011-9179-1

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  • DOI: https://doi.org/10.1007/s12010-011-9179-1

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