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l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

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Abstract

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP+ but uses also NAD+ as a cofactor, and showed highest catalytic efficiency (k cat/K m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.

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Acknowledgements

Technical assistance of Tarja Laakso and CE analysis by Heidi Turkia is gratefully acknowledged. This study was financially supported by the Academy of Finland through the Centre of Excellence in White Biotechnology–Green Chemistry (grant 118573 for VTT) and the Pentoval project (grant 129174 for Merja Penttilä and grant 129175 for Olli T. Pentikäinen).

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Authors and Affiliations

  1. VTT, Technical Research Centre of Finland, P.O. Box 1000, 02044, Espoo, Finland

    Niina Aro-Kärkkäinen, Mervi Toivari, Hannu Maaheimo, Martina Andberg, Merja Oja, Merja Penttilä, Marilyn G. Wiebe, Laura Ruohonen & Anu Koivula

  2. Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, 40014, Jyväskylä, Finland

    Mikko Ylilauri & Olli T. Pentikäinen

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  1. Niina Aro-Kärkkäinen
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Correspondence to Anu Koivula.

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Aro-Kärkkäinen, N., Toivari, M., Maaheimo, H. et al. l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae . Appl Microbiol Biotechnol 98, 9653–9665 (2014). https://doi.org/10.1007/s00253-014-6039-2

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