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

6 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

Fingerprint

galactose dehydrogenase
Rhizobium leguminosarum
Arabinose
Saccharomyces cerevisiae
Yeasts
Enzymes
Oxidoreductases
Caulobacter crescentus
Fucose
Peas
Lactones
Computational Biology
Galactose
NADP
NAD
Buffers
Proteins
Magnetic Resonance Spectroscopy
Bacteria

Keywords

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

Cite this

@article{f17761c92c464e7a89bfb2f13950e062,
title = "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",
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.",
keywords = "Galactose permease, l-Arabinose, l-Arabinose dehydrogenase, l-Arabinose transport, l-Arabonic acid, Platform chemical, Saccharomyces cerevisiae",
author = "Niina Aro-K{\"a}rkk{\"a}inen and Mervi Toivari and Hannu Maaheimo and Mikko Ylilauri and Pentik{\"a}inen, {Olli T.} and Martina Andberg and Merja Oja and Merja Penttil{\"a} and Wiebe, {Marilyn G.} and Laura Ruohonen and Anu Koivula",
note = "CA2: BA3111 CA2: BA3116 CA2: BA3112 CA2: BA311 CA2: BA3114 ISI: BIOTECHNOLOGY & APPLIED MICROBIOLOGY",
year = "2014",
month = "1",
day = "1",
doi = "10.1007/s00253-014-6039-2",
language = "English",
volume = "98",
pages = "9653--9665",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "Springer",
number = "23",

}

TY - JOUR

T1 - 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

AU - Aro-Kärkkäinen, Niina

AU - Toivari, Mervi

AU - Maaheimo, Hannu

AU - Ylilauri, Mikko

AU - Pentikäinen, Olli T.

AU - Andberg, Martina

AU - Oja, Merja

AU - Penttilä, Merja

AU - Wiebe, Marilyn G.

AU - Ruohonen, Laura

AU - Koivula, Anu

N1 - CA2: BA3111 CA2: BA3116 CA2: BA3112 CA2: BA311 CA2: BA3114 ISI: BIOTECHNOLOGY & APPLIED MICROBIOLOGY

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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.

AB - 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.

KW - Galactose permease

KW - l-Arabinose

KW - l-Arabinose dehydrogenase

KW - l-Arabinose transport

KW - l-Arabonic acid

KW - Platform chemical

KW - Saccharomyces cerevisiae

UR - http://www.scopus.com/inward/record.url?scp=84911806173&partnerID=8YFLogxK

U2 - 10.1007/s00253-014-6039-2

DO - 10.1007/s00253-014-6039-2

M3 - Article

C2 - 25236800

AN - SCOPUS:84911806173

VL - 98

SP - 9653

EP - 9665

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 23

ER -