Molecular and functional characterization of two pyruvate decarboxylase genes, PDC1 and PDC5, in the thermotolerant yeast Kluyveromyces marxianus
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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.
KeywordsKluyveromyces 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.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 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. https://doi.org/10.1186/s13068-016-0653-4 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1111/j.1432-1033.1990.tb15442.x CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1007/s00253-013-4760-x CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1111/j.1567-1364.2008.00429.x CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1128/Ec.00260-12 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1002/(SICI)1097-0061(19980330)14:5<459::AID-YEA248>3.0.CO;2-O CrossRefPubMedGoogle Scholar
- 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
- Pronk JT, Yde Steensma H, Van Dijken JP (1996) Pyruvate metabolism in Saccharomyces cerevisiae. Yeast 12(16):1607–1633. https://doi.org/10.1002/(SICI)1097-0061(199612)12:16<1607::AID-YEA70>3.0.CO;2-4 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1093/nar/22.22.4673 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1128/AEM.70.1.159-166.2004 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.ymben.2015.07.008 CrossRefPubMedGoogle Scholar