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

Niina Aro-Kärkkäinen, Mervi Toivari, Hannu Maaheimo, Mikko Ylilauri, Olli T. Pentikäinen, Martina Andberg, Merja Oja, Merja Penttilä, Marilyn G. Wiebe, Laura Ruohonen, Anu Koivula (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

7 Citations (Scopus)

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 (kcat/Km) 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.

Original languageEnglish
Pages (from-to)9653-9665
Number of pages13
JournalApplied Microbiology and Biotechnology
Volume98
Issue number23
DOIs
Publication statusPublished - 1 Jan 2014
MoE publication typeA1 Journal article-refereed

Keywords

  • Galactose permease
  • l-Arabinose
  • l-Arabinose dehydrogenase
  • l-Arabinose transport
  • l-Arabonic acid
  • Platform chemical
  • Saccharomyces cerevisiae

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