title: Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway.
abstract: BACKGROUND
  The non-conventional yeast Arxula adeninivorans uses 1-butanol as a carbon source and has recently attracted attention as a promising organism for 1-butanol production. Alcohol dehydrogenases (adhp) are important catalysts in 1-butanol metabolism, but only Aadh1p from Arxula has been characterized. This enzyme is involved in ethanol synthesis but has a low impact on 1-butanol degradation.
  
  
  RESULTS
  In this study, we identified and characterized a second adhp from A. adeninivorans (Aadh2p). Compared to Saccharomyces cerevisiae ADHs' (ScAdh) protein sequences it originates from the same ancestral node as ScAdh6p, 7p and 4p. It is also localized in the cytoplasm and uses NAD(H) as cofactor. The enzyme has its highest activity with medium chain-length alcohols and maximum activity with 1-butanol with the catalytic efficiency of the purified enzyme being 42 and 43,000 times higher than with ethanol and acetaldehyde, respectively. Arxula adeninivorans strain G1212/YRC102-AADH2, which expresses the AADH2 gene under the control of the strong constitutive TEF1 promoter was constructed. It achieved an ADH activity of up to 8000 U/L and 500 U/g dry cell weight (dcw) which is in contrast to the control strain G1212/YRC102 which had an ADH activity of up to 1400 U/L and 200 U/g dcw. Gene expression analysis showed that AADH2 derepression or induction using non-fermentable carbon-sources such as ethanol, pyruvate, glycerol or 1-butanol did occur. Compared to G1212/YRC102 AADH2 knock-out strain had a slower growth rate and lower 1-butanol consumption if 1-butanol was used as sole carbon source and AADH2-transformants did not grow at all in the same conditions. However, addition of the branched-chain amino acids leucine, isoleucine and valine allowed the transformants to use 1-butanol as carbon source. The addition of these amino acids to the control strain and Δaadh2 mutant cultures had the effect of accelerating 1-butanol consumption.
  
  
  CONCLUSIONS
  Our results confirm that Aadh2p plays a major role in A. adeninivorans 1-butanol metabolism. It is upregulated by up to 60-fold when the cells grow on 1-butanol, whereas only minor changes were found in the relative expression level for Aadh1p. Thus the constitutive overexpression of the AADH2 gene could be useful in the production of 1-butanol by A. adeninivorans, although it is likely that other ADHs will have to be knocked-out to prevent 1-butanol oxidation.
authors.affiliation: Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany.
authors.forename: Marion
authors.initials: M
authors.lastname: Rauter
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
authors.forename: Jakub
authors.initials: J
authors.lastname: Kasprzak
authors.affiliation: Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Becker
authors.affiliation: Jäckering Mühlen-und Nährmittelwerke GmbH, Vorsterhauser Weg 46, 59007, Hamm, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Riechen
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Worch
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Mascher
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Scholz
authors.affiliation: School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
authors.forename: Kim
authors.initials: K
authors.lastname: Baronian
authors.affiliation: Institute of Microbiology, University of Greifswald, Jahnstr. 15, 17487, Greifswald, Germany.
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Bode
authors.affiliation: Institute of Microbiology, University of Greifswald, Jahnstr. 15, 17487, Greifswald, Germany.
authors.forename: Frieder
authors.initials: F
authors.lastname: Schauer
authors.affiliation: Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Matthias Vorbrodt
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany. kunzeg@ipk-gatersleben.de.
authors.forename: Gotthard
authors.initials: G
authors.lastname: Kunze
mesh_terms: D020001:1-Butanol / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D015870:Gene Expression; D055786:Gene Knockout Techniques; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009243:NAD / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D009243:NAD; D000431:Ethanol; D002244:Carbon; D020001:1-Butanol; D000426:Alcohol Dehydrogenase
keywords: 1-butanol; Aadh2p; Alcohol dehydrogenase; Arxula adeninivorans