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Characterization of alcohol dehydrogenase 3 of the thermotolerant methylotrophic yeast Hansenula polymorpha

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Abstract

In this study, we identified and characterized mitochondrial alcohol dehydrogenase 3 from the thermotolerant methylotrophic yeast Hansenula polymorpha (HpADH3). The amino acid sequence of HpADH3 shares over 70% of its identity with the alcohol dehydrogenases of other yeasts and exhibits the highest similarity of 91% with the alcohol dehydrogenase 1 of H. polymorpha. However, unlike the cytosolic HpADH1, HpADH3 appears to be a mitochondrial enzyme, as a mitochondrial targeting extension exists at its N terminus. The recombinant HpADH3 overexpressed in Escherichia coli showed similar catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The HpADH3 displayed substrate specificities with clear preferences for medium chain length primary alcohols and acetaldehyde for an oxidation reaction and a reduction reaction, respectively. Although the H. polymorpha ADH3 gene was induced by ethanol in the culture medium, both an ADH isozyme pattern analysis and an ADH activity assay indicated that HpADH3 is not the major ADH in H. polymorpha DL-1. Moreover, HpADH3 deletion did not affect the cell growth on different carbon sources. However, when the HpADH3 mutant was complemented by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium. In contrast, in a xylose medium, the ethanol production was dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain. Taken together, our results suggest that the expression of HpADH3 would be an ideal engineering target to develop H. polymorpha as a substrate specific bioethanol production strain.

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Acknowledgments

This work was supported by a grant from the KRIBB Research Initiative Program, by a Basic Science Research Program (no. 2009-0075186), and by a HTS-based Integrated Technology Development grant (no. 2008-2004174) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology. S. Suwannarangsee was supported by the International Scholar Exchange Fellowship for the academic year of 2008–2009 by the Korea Foundation for Advanced Studies.

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Authors and Affiliations

  1. Enzyme Technology Laboratory, Bioresource Technology Unit, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani, 12120, Thailand

    Surisa Suwannarangsee

  2. Applied Microbiology Research Center, Bio-Materials Research Institute, KRIBB, Jeonbuk, 580-185, South Korea

    Seonghun Kim, Jeong-Woo Seo & Chul Ho Kim

  3. Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806, South Korea

    Oh-Cheol Kim, Doo-Byoung Oh & Ohsuk Kwon

  4. Department of Medical Biotechnology, Soonchunhyang University, Asan, Chungnam, 336-745, South Korea

    Sang Ki Rhee

  5. Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, 156-756, South Korea

    Hyun Ah Kang

  6. Biofuel Production by Biocatalyst Research Unit and Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Warawut Chulalaksananukul

  7. Systems Biotechnology Program, School of Science, University of Science and Technology (UST), Daejeon, 305-350, South Korea

    Ohsuk Kwon

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  1. Surisa Suwannarangsee
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  2. Seonghun Kim
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Correspondence to Ohsuk Kwon.

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Suwannarangsee, S., Kim, S., Kim, OC. et al. Characterization of alcohol dehydrogenase 3 of the thermotolerant methylotrophic yeast Hansenula polymorpha . Appl Microbiol Biotechnol 96, 697–709 (2012). https://doi.org/10.1007/s00253-011-3866-2

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  • DOI: https://doi.org/10.1007/s00253-011-3866-2

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