Abstract
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.
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Funding
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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Choo, J.H., Han, C., Lee, D.W. et al. Molecular and functional characterization of two pyruvate decarboxylase genes, PDC1 and PDC5, in the thermotolerant yeast Kluyveromyces marxianus. Appl Microbiol Biotechnol 102, 3723–3737 (2018). https://doi.org/10.1007/s00253-018-8862-3
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DOI: https://doi.org/10.1007/s00253-018-8862-3