Molecular and functional characterization of two pyruvate decarboxylase genes, PDC1 and PDC5, in the thermotolerant yeast Kluyveromyces marxianus
Journal
APPL MICROBIOL BIOT
Date
2018.04.02
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 in both KmPdc1p and KmPdc5p, a few amino acid sequences essential for enzymatic activity are not conserved in KmPdc5p. The deletion of KmPDC1 alone diminished most of Pdc activity, but the growth of Kmpdc1Δ strain on glucose was quite comparable to that of the wild type (WT) strain under aerobic conditions. However, different from WT, Kmpdc1Δ could not grow on glucose under anaerobic conditions. The KmPDC5 deletion did not generate any apparent change in Pdc activity and growth patterns under several tested conditions. Whereas the expression of KmPDC1 appeared to be enhanced by glucose, the basic expression levels of KmPDC5 were very low without detectable difference between glucose and non-fermentable carbon sources. Moreover, KmPDC5 overexpression was unable to complement the growth defect of Kmpdc1Δ in the presence of antimycin A, further supporting that KmPdc5p may lack Pdc activity. Noticeably, compared to WT, Kmpdc1Δ single and Kmpdc1Δpdc5Δ double mutants produced significantly less amount of glycerol, acetate, and ethanol while accumulating pyruvate. Altogether, our data indicate that the single deletion of KmPDC1 is sufficient in Crabtree-negative K. marxianus strains to generate a starting host strain to be engineered for production of high-value biomaterials derived from pyruvate without byproduct formation.
Reference
Applied Microbiology and Biotechnology (2018) 102:3723？3737