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The regulation of 2,3-butanediol synthesis in Klebsiella pneumoniae as revealed by gene over-expressions and metabolic flux analysis

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Abstract

A variety of microorganism species are able naturally to produce 2,3-butanediol (2,3-BDO), although only a few of them are suitable for consideration as having potential for mass production purposes. Klebsiella pneumoniae (K. pneumoniae) is one such strain which has been widely studied and used industrially to produce 2,3-BDO. In the central carbon metabolism of K. pneumoniae, the 2,3-BDO synthesis pathway is dominated by three essential enzymes, namely acetolactate decarboxylase, acetolactate synthase, and butanediol dehydrogenase, which are encoded by the budA, budB, and budC genes, respectively. The mechanisms of the three enzymes have been characterized with regard to their function and roles in 2,3-BDO synthesis and cell growth (Blomqvist et al. in J Bacteriol 175(5):1392–1404, 1993), while a few studies have focused on the cooperative mechanisms of the three enzymes and their mutual interactions. Therefore, the K. pneumoniae KCTC2242::ΔwabG wild-type strain was utilized to reconstruct seven new mutants by single, double, and triple overexpression of the three enzymes key to this study. Subsequently, continuous cultures were performed to obtain steady-state metabolism in the organisms and experimental data were analyzed by metabolic flux analysis (MFA) to determine the regulation mechanisms. The MFA results showed that the seven overexpressed mutants all exhibited enhanced 2,3-BDO production, and the strain overexpressing the budBA gene produced the highest yield. While the enzyme encoded by the budA gene produced branched-chain amino acids which were favorable for cell growth, the budB gene enzyme rapidly enhanced the conversion of acetolactate to acetoin in an oxygen-dependent manner, and the budC gene enzyme catalyzed the reversible conversion of acetoin to 2,3-BDO and regulated the intracellular NAD+/NADH balance.

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Acknowledgments

This work was supported by the Industrial Strategic technology development program (10035147, Development of 2,3-butanediol and derivative production technology for C-Zero bio-platform industry) funded by the Ministry of Knowledge Economy (MKE), Republic of Korea.

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 121-742, Republic of Korea

    Mingshou Lu, Changhun Park, Soojin Lee, Borim Kim, Jungwook Kim & Jinwon Lee

  2. Department of Chemical and Biological Engineering, Korea University, Seoul, 136-713, Republic of Korea

    Min-Kyu Oh

  3. Clean Energy Research Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, 136-791, Republic of Korea

    Youngsoon Um

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  1. Mingshou Lu
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  2. Changhun Park
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  3. Soojin Lee
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Correspondence to Jinwon Lee.

Appendix: List of metabolic reactions

Appendix: List of metabolic reactions

  1. 1.

    Phosphotransferase system (PTS): glucose + pep → g6p + pyr

  2. 2.

    Phosphoglucose isomerase (PGI): g6p → f6p

  3. 3.

    Polysaccharide synthesis (PSS): g6p → Polysaccharide

  4. 4.

    Phosphafructokinase (PFK): f6p + [ATP] → fdp + [ADP]

  5. 5.

    Aldolases (ALDO): fdp → dhap + gap

  6. 6.

    Triosphosphate isomerase (TIS): dhap = gap

  7. 7.

    Glyceraldehyde-3-phosphate dehydrogenase (GAPHD): gap + [NAD] = pgp + [NADH] + [h]

  8. 8.

    Phosphoglycerate kinase (PGK): pgp + [ADP] + [Pi] = 3 pg + [ATP]

  9. 9.

    Phosphoglycerate mutase (PGLUMU): 3 pg = 2 pg

  10. 10.

    Enolase (ENO): 2 pg → pep

  11. 11.

    Pyruvate kinase (PK): pep + [ADP] + [h] = pyr + [ATP]

  12. 12.

    Glucose-6-phosphate dehydrogenase (G6PDH): g6p + [NADP] → 6 pg + [NADPH] + 2[h]

  13. 13.

    6-Phosphogluconate dehydrogenase (PGDH): 6 pg + [NADP] → ribu5p + [CO2] + [NADPH]

  14. 14.

    Ribulose phosphate 3-epimerase (RU5P): ribu5p = xyl5p

  15. 15.

    Ribose-5-phosphate isomerase (R5PI): ribu5p = rib5p

  16. 16.

    BIOMASS(ex): 0.101rib5p + 0.043e4p + 0.037g6p + 0.0556g6p + 0.0407pep + 0.333pyr + 0.17oxa + 0.12accoA + 1.73[ATP] + 2.120[NAD] + 1.89[NADPH] → Biomass + 1.73[ADP] + 2.12[NADH] + 1.89[NADP]

  17. 17.

    Transketolase A (TKA): rib5p + xyl5p = gap + sed7p

  18. 18.

    Transaldolase (TA): gap + sed7p = e4p + f6p

  19. 19.

    Transketolase B (TKB): e4p + xyl5p = f6p +gap

  20. 20.

    Pyruvate dehydrogenase (PDH): pyr + [NAD] + coA → accoA + [NADH] + [CO2]

  21. 21.

    Citrate synthase (CS): accoA + oxa + [H2O] → cit + coA

  22. 22.

    Aconitase (CAN): cit = icit

  23. 23.

    Isocitrate dehydrogenase (ICD): icit + [NAD] + [CO2] = akg + [NADH]

  24. 24.

    2-Ketoglutarate dehydrogenase (KDH): akg + [NAD] → succoA + [NADH] + [CO2]

  25. 25.

    Succinate thiokinase (SCAS): succoA + [ADP] + Pi = succinate + [ATP] + coA

  26. 26.

    Succ(ex): succinate → succinate(ex)

  27. 27.

    Succinate dehydrogenase (SDH): succinate + [fad] → fum + [fadh2]

  28. 28.

    Fumarase (FUM): fum + [H2O] = mal

  29. 29.

    Malate dehydrogenase (MDH): mal + [NAD] = oxa + [NADH] + [h]

  30. 30.

    PEP carboxykinase (PCK): oxa + [ATP] = pep + [ADP] + [CO2]

  31. 31.

    Lactate dehydrogenase (LDH): pyr + [NADH] → Lactate + [NAD]

  32. 32.

    Lactate → Lactate(ex)

  33. 33.

    Acetolactate synthase (ALS): 2 pyr → aclac

  34. 34.

    Acetolactate decarboxylase (ALDC): aclac → acet

  35. 35.

    Acetoin dehydrogenase (ACETDH): acet + [NADH] → 2,3-BDO + [NAD]

  36. 36.

    2,3-BDO → 2,3-BDOex

  37. 37.

    Aldehyde dehydrogenase (ACALDH): accoA + [NADH] → acal + [NAD] + coA

  38. 38.

    Alcohol dehydrogenase (ADH): acal + [NADH] + [h] → Ethanol + [NAD]

  39. 39.

    Ethanol → Ethanol(ex)

  40. 40.

    NADH dehydrogenase I: [NADH] + [Q] = [NAD] + [QH2] + 2 [h]

  41. 41.

    Cytochrome oxidase bo3: 2 [QH2] + [O2] = 2 [Q] + 4 [h]

  42. 42.

    Pyridine nucleotide transhydrogenase: [NADPH] + [NAD] = [NADP] + [NADH]

  43. 43.

    Succinate dehydrogenase complex: [fadh2] + [Q] = [QH2] + [fad]

  44. 44.

    Pyridine nucleotide transhydrogenase: [NADP] + [NADH] + 2 [h] = [NADPH] + [NAD]

  45. 45.

    ATP drain: [ATP] = [ADP] + [Pi]

  46. 46.

    CO2 → CO2(ex)

  47. 47.

    O2 = O2(ex)

  48. 48.

    Pi = Pi(ex)

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Lu, M., Park, C., Lee, S. et al. The regulation of 2,3-butanediol synthesis in Klebsiella pneumoniae as revealed by gene over-expressions and metabolic flux analysis. Bioprocess Biosyst Eng 37, 343–353 (2014). https://doi.org/10.1007/s00449-013-0999-y

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