Applied Microbiology and Biotechnology

, Volume 102, Issue 8, pp 3723–3737 | Cite as

Molecular and functional characterization of two pyruvate decarboxylase genes, PDC1 and PDC5, in the thermotolerant yeast Kluyveromyces marxianus

  • Jin Ho Choo
  • Changpyo Han
  • Dong Wook Lee
  • Gyu Hun Sim
  • Hye Yun Moon
  • Jae-Young Kim
  • Ji-Yoon Song
  • Hyun Ah KangEmail author
Applied genetics and molecular biotechnology


Pyruvate decarboxylase (Pdc) is a cytosolic enzyme located at the branch point between fermentative and respiratory sugar catabolism. Here, we identified and functionally characterized KmPDC1 and KmPDC5 encoding two homologs of Pdc in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555. Despite the conservation of important Pdc domains, a few amino acid sequences essential for enzymatic activity are not conserved in KmPdc5p. Deletion of KmPDC1 alone eliminated most of Pdc activity, but the growth of the Kmpdc1Δ strain on glucose was comparable to that of the wild type (WT) strain under aerobic conditions. In contrast to the WT, Kmpdc1Δ could not grow on glucose under oxygen-limited conditions. The KmPDC5 deletion did not generate any apparent change in Pdc activity or growth patterns under several tested conditions. Whereas the expression of KmPDC1 was enhanced by glucose, the basic expression levels of KmPDC5 were very low, without a detectable difference between glucose and nonfermentable carbon sources. Moreover, KmPDC5 overexpression was unable to complement the growth defect of Kmpdc1Δ in the presence of antimycin A, and the purified recombinant KmPdc5p was inactive in Pdc activity assay, supporting the notion that KmPdc5p may lack Pdc enzymatic activity. Notably, compared to the WT, Kmpdc1Δ single and Kmpdc1Δpdc5Δ double mutants produced significantly less glycerol, acetate, and ethanol while accumulating pyruvate. Altogether, our data indicate that a single deletion of KmPDC1 is sufficient in Crabtree-negative K. marxianus strains to generate a starting host strain for engineering of production of high-value biomaterials derived from pyruvate without byproduct formation.


Kluyveromyces marxianus Pyruvate decarboxylase PDC1 PDC5 Crabtree effect 



This work was supported by the National Research Foundation of Korea (NRF), grant no. NRF-2017M3C1B5019295 (STEAM Research Project). Gyu Hun Sim was supported by Chung-Ang University Research Scholarship Grants in 2016.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8862_MOESM1_ESM.pdf (404 kb)
ESM 1 (PDF 404 kb)


  1. Agarwal PK, Uppada V, Noronha SB (2013) Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris). Appl Microbiol Biotechnol 97(21):9439–9449. CrossRefPubMedGoogle Scholar
  2. Baburina I, Dikdan G, Guo F, Tous GI, Root B, Jordan F (1998) Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions. Biochemistry 37(5):1245–1255. CrossRefPubMedGoogle Scholar
  3. Bianchi MM, Tizzani L, Destruelle M, Frontali L, Wésolowski-Louvel M (1996) The ‘petite-negative’ yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity. Mol Microbiol 19(1):27–36. CrossRefPubMedGoogle Scholar
  4. Chen D, Toone WM, Mata J, Lyne R, Burns G, Kivinen K, Brazma A, Jones N, Bahler J (2003) Global transcriptional responses of fission yeast to environmental stress. Mol Biol Cell 14(1):214–229. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Choo JH, Han C, Kim JY, Kang HA (2014) Deletion of a KU80 homolog enhances homologous recombination in the thermotolerant yeast Kluyveromyces marxianus. Biotechnol Lett 36(10):2059–2067. CrossRefPubMedGoogle Scholar
  6. Choo JH, Hong CP, Lim JY, Seo JA, Kim YS, Lee DW, Park SG, Lee GW, Carroll E, Lee YW, Kang HA (2016) Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid. Biotechnol Biofuels 9:246. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Dickinson JR, Lanterman MM, Danner DJ, Pearson BM, Sanz P, Harrison SJ, Hewlins MJ (1997) A 13C nuclear magnetic resonance investigation of the metabolism of leucine to isoamyl alcohol in Saccharomyces cerevisiae. J Biol Chem 272(43):26871–26878CrossRefPubMedGoogle Scholar
  8. Dyda F, Furey W, Swaminathan S, Sax M, Farrenkopf B, Jordan F (1993) Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution. Biochemistry 32(24):6165–6170CrossRefPubMedGoogle Scholar
  9. Erasmus DJ, van der Merwe GK, van Vuuren HJ (2003) Genome-wide expression analyses: metabolic adaptation of Saccharomyces cerevisiae to high sugar stress. FEMS Yeast Res 3(4):375–399CrossRefPubMedGoogle Scholar
  10. Fonseca GG, Heinzle E, Wittmann C, Gombert AK (2008) The yeast Kluyveromyces marxianus and its biotechnological potential. Appl Microbiol Biotechnol 79(3):339–354. CrossRefPubMedGoogle Scholar
  11. Franzblau SG, Sinclair NA (1983) Induction of pyruvate decarboxylase in Candida utilis. Mycopathologia 83(1):29–33CrossRefPubMedGoogle Scholar
  12. Fredlund E, Blank LM, Schnurer J, Sauer U, Passoth V (2004) Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala. Appl Environ Microbiol 70(10):5905–5911. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fredlund E, Beerlage C, Melin P, Schnurer J, Passoth V (2006) Oxygen and carbon source-regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala. Yeast 23(16):1137–1149. CrossRefPubMedGoogle Scholar
  14. Gounaris AD, Turkenkopf I, Buckwald S, Young A (1971) Pyruvate decarboxylase. I. Protein dissociation into subunits under conditions in which thiamine pyrophosphate is released. J Biol Chem 246(5):1302–1309PubMedGoogle Scholar
  15. Heo P, Yang TJ, Chung SC, Cheon Y, Kim JS, Park JB, Koo HM, Cho KM, Seo JH, Park JC, Kweon DH (2013) Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome. J Biotechnol 167(3):323–325. CrossRefPubMedGoogle Scholar
  16. Hill J, Donald KA, Griffiths DE (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res 19(20):5791. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hohmann S, Cederberg H (1990) Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5. Eur J Biochem 188(3):615–621. CrossRefPubMedGoogle Scholar
  18. Holloway P, Subden RE (1993) The isolation and nucleotide sequence of the pyruvate decarboxylase gene from Kluyveromyces marxianus. Curr Genet 24(3):274–277. CrossRefPubMedGoogle Scholar
  19. Hong J, Wang Y, Kumagai H, Tamaki H (2007) Construction of thermotolerant yeast expressing thermostable cellulase genes. J Biotechnol 130(2):114–123. CrossRefPubMedGoogle Scholar
  20. Hua Q, Yang C, Shimizu K (1999) Metabolic flux analysis for efficient pyruvate fermentation using vitamin-auxotrophic yeast of Torulopsis glabrata. J Biosci Bioeng 87(2):206–213. CrossRefPubMedGoogle Scholar
  21. Ida Y, Hirasawa T, Furusawa C, Shimizu H (2013) Utilization of Saccharomyces cerevisiae recombinant strain incapable of both ethanol and glycerol biosynthesis for anaerobic bioproduction. Appl Microbiol Biotechnol 97(11):4811–4819. CrossRefPubMedGoogle Scholar
  22. Iding H, Siegert P, Mesch K, Pohl M (1998) Application of alpha-keto acid decarboxylases in biotransformations. Biochim Biophys Acta 1385(2):307–322. CrossRefPubMedGoogle Scholar
  23. Ikushima S, Fujii T, Kobayashi O, Yoshida S, Yoshida A (2009) Genetic engineering of Candida utilis yeast for efficient production of L-lactic acid. Biosci Biotechnol Biochem 73(8):1818–1824. CrossRefPubMedGoogle Scholar
  24. Ishchuk OP, Voronovsky AY, Stasyk OV, Gayda GZ, Gonchar MV, Abbas CA, Sibirny AA (2008) Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose. FEMS Yeast Res 8(7):1164–1174. CrossRefPubMedGoogle Scholar
  25. Jeong H, Lee DH, Kim SH, Kim HJ, Lee K, Song JY, Kim BK, Sung BH, Park JC, Sohn JH, Koo HM, Kim JF (2012) Genome sequence of the thermotolerant yeast Kluyveromyces marxianus var. marxianus KCTC 17555. Eukaryot Cell 11(12):1584–1585. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Johnson CW, Beckham GT (2015) Aromatic catabolic pathway selection for optimal production of pyruvate and lactate from lignin. Metab Eng 28:240–247. CrossRefPubMedGoogle Scholar
  27. Kellermann E, Seeboth PG, Hollenberg CP (1986) Analysis of the primary structure and promoter function of a pyruvate decarboxylase gene (PDC1) from Saccharomyces cerevisiae. Nucleic Acids Res 14(22):8963–8977. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kiers J, Zeeman AM, Luttik M, Thiele C, Castrillo JI, Steensma HY, van Dijken JP, Pronk JT (1998) Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359. Yeast 14(5):459–469.<459::AID-YEA248>3.0.CO;2-O CrossRefPubMedGoogle Scholar
  29. Kim S, Hahn JS (2015) Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing. Metab Eng 31:94–101. CrossRefPubMedGoogle Scholar
  30. Konig S (1998) Subunit structure, function and organisation of pyruvate decarboxylases from various organisms. Biochim Biophys Acta 1385(2):271–286. CrossRefPubMedGoogle Scholar
  31. Lane MM, Morrissey JP (2010) Kluyveromyces marxianus: a yeast emerging from its sister’s shadow. Fungal Biol Rev 24(1–2):17–26. CrossRefGoogle Scholar
  32. Lane MM, Burke N, Karreman R, Wolfe KH, O'Byrne CP, Morrissey JP (2011) Physiological and metabolic diversity in the yeast Kluyveromyces marxianus. Antonie Van Leeuwenhoek 100(4):507–519. CrossRefPubMedGoogle Scholar
  33. Lobell M, Crout DHG (1996) Pyruvate decarboxylase: a molecular modeling study of pyruvate decarboxylation and acyloin formation. J Am Chem Soc 118(8):1867–1873. CrossRefGoogle Scholar
  34. Lu P, Davis BP, Jeffries TW (1998) Cloning and characterization of two pyruvate decarboxylase genes from Pichia stipitis CBS 6054. Appl Environ Microbiol 64(1):94–97PubMedPubMedCentralGoogle Scholar
  35. Mann S, Melero CP, Hawksley D, Leeper FJ (2004) Inhibition of thiamin diphosphate dependent enzymes by 3-deazathiamin diphosphate. Org Biomol Chem 2(12):1732–1741. CrossRefPubMedGoogle Scholar
  36. Merico A, Sulo P, Piskur J, Compagno C (2007) Fermentative lifestyle in yeasts belonging to the Saccharomyces complex. FEBS J 274(4):976–989. CrossRefPubMedGoogle Scholar
  37. Moller K, Langkjaer RB, Nielsen J, Piskur J, Olsson L (2004) Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri. Mol Gen Genomics 270(6):558–568. CrossRefGoogle Scholar
  38. Nosaka K (2006) Recent progress in understanding thiamin biosynthesis and its genetic regulation in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 72(1):30–40. CrossRefPubMedGoogle Scholar
  39. Nosaka K, Esaki H, Onozuka M, Konno H, Hattori Y, Akaji K (2012) Facilitated recruitment of Pdc2p, a yeast transcriptional activator, in response to thiamin starvation. FEMS Microbiol Lett 330(2):140–147. CrossRefPubMedGoogle Scholar
  40. Passoth V, Zimmermann M, Klinner U (1996) Peculiarities of the regulation of fermentation and respiration in the crabtree-negative, xylose-fermenting yeast Pichia stipitis. Appl Biochem Biotechnol 57-58:201–212CrossRefPubMedGoogle Scholar
  41. Porro D, Bianchi MM, Brambilla L, Menghini R, Bolzani D, Carrera V, Lievense J, Liu CL, Ranzi BM, Frontali L, Alberghina L (1999) Replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts. Appl Environ Microbiol 65(9):4211–4215PubMedPubMedCentralGoogle Scholar
  42. Pronk JT, Yde Steensma H, Van Dijken JP (1996) Pyruvate metabolism in Saccharomyces cerevisiae. Yeast 12(16):1607–1633.<1607::AID-YEA70>3.0.CO;2-4 CrossRefPubMedGoogle Scholar
  43. Romagnoli G, Luttik MA, Kotter P, Pronk JT, Daran JM (2012) Substrate specificity of thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae. Appl Environ Microbiol 78(21):7538–7548. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Rosche B, Sandford V, Breuer M, Hauer B, Rogers PL (2002) Enhanced production of R-phenylacetylcarbinol (R-PAC) through enzymatic biotransformation. J Mol Catal B Enzym 19–20:109–115. CrossRefGoogle Scholar
  45. Schaaff I, Green JB, Gozalbo D, Hohmann S (1989) A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations. Curr Genet 15(2):75–81. CrossRefPubMedGoogle Scholar
  46. Seeboth PG, Bohnsack K, Hollenberg CP (1990) pdc1 0 mutants of Saccharomyces cerevisiae give evidence for an additional structural PDC gene: cloning of PDC5, a gene homologous to PDC1. J Bacteriol 172(2):678–685CrossRefPubMedPubMedCentralGoogle Scholar
  47. Skory CD (2003) Induction of Rhizopus oryzae pyruvate decarboxylase genes. Curr Microbiol 47(1):59–64. CrossRefPubMedGoogle Scholar
  48. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680. CrossRefPubMedPubMedCentralGoogle Scholar
  49. van Maris AJ, Geertman JM, Vermeulen A, Groothuizen MK, Winkler AA, Piper MD, van Dijken JP, Pronk JT (2004) Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast. Appl Environ Microbiol 70(1):159–166. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Van Urk H, Voll WS, Scheffers WA, Van Dijken JP (1990) Transient-state analysis of metabolic fluxes in crabtree-positive and crabtree-negative yeasts. Appl Environ Microbiol 56(1):281–287PubMedPubMedCentralGoogle Scholar
  51. Vemuri GN, Eiteman MA, McEwen JE, Olsson L, Nielsen J (2007) Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 104(7):2402–2407. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Xu QW, Vu H, Liu LP, Wang TC, Schaefer WH (2011) Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells. J Biomol NMR 49(3–4):207–219. CrossRefPubMedGoogle Scholar
  53. Zhang J, Zhang B, Wang D, Gao X, Sun L, Hong J (2015) Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway. Metab Eng 31:140–152. CrossRefPubMedGoogle Scholar
  54. Zhang B, Zhu Y, Zhang J, Wang D, Sun L, Hong J (2017) Engineered Kluyveromyces marxianus for pyruvate production at elevated temperature with simultaneous consumption of xylose and glucose. Bioresour Technol 224:553–562. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jin Ho Choo
    • 1
  • Changpyo Han
    • 1
  • Dong Wook Lee
    • 1
  • Gyu Hun Sim
    • 1
  • Hye Yun Moon
    • 1
  • Jae-Young Kim
    • 2
  • Ji-Yoon Song
    • 2
  • Hyun Ah Kang
    • 1
    Email author
  1. 1.Department of Life Science, College of Natural ScienceChung-Ang UniversitySeoulRepublic of Korea
  2. 2.Biomaterials Laboratory, Material Research CenterSamsung Advanced Institute of TechnologyGyeonggi-doRepublic of Korea

Personalised recommendations