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Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway

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 cha...

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Autores principales: Rauter, Marion, Kasprzak, Jakub, Becker, Karin, Riechen, Jan, Worch, Sebastian, Hartmann, Anja, Mascher, Martin, Scholz, Uwe, Baronian, Kim, Bode, Rüdiger, Schauer, Frieder, Matthias Vorbrodt, H., Kunze, Gotthard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062937/
https://www.ncbi.nlm.nih.gov/pubmed/27733155
http://dx.doi.org/10.1186/s12934-016-0573-9
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author Rauter, Marion
Kasprzak, Jakub
Becker, Karin
Riechen, Jan
Worch, Sebastian
Hartmann, Anja
Mascher, Martin
Scholz, Uwe
Baronian, Kim
Bode, Rüdiger
Schauer, Frieder
Matthias Vorbrodt, H.
Kunze, Gotthard
author_facet Rauter, Marion
Kasprzak, Jakub
Becker, Karin
Riechen, Jan
Worch, Sebastian
Hartmann, Anja
Mascher, Martin
Scholz, Uwe
Baronian, Kim
Bode, Rüdiger
Schauer, Frieder
Matthias Vorbrodt, H.
Kunze, Gotthard
author_sort Rauter, Marion
collection PubMed
description 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0573-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-50629372016-10-24 Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway Rauter, Marion Kasprzak, Jakub Becker, Karin Riechen, Jan Worch, Sebastian Hartmann, Anja Mascher, Martin Scholz, Uwe Baronian, Kim Bode, Rüdiger Schauer, Frieder Matthias Vorbrodt, H. Kunze, Gotthard Microb Cell Fact Research 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0573-9) contains supplementary material, which is available to authorized users. BioMed Central 2016-10-12 /pmc/articles/PMC5062937/ /pubmed/27733155 http://dx.doi.org/10.1186/s12934-016-0573-9 Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Rauter, Marion
Kasprzak, Jakub
Becker, Karin
Riechen, Jan
Worch, Sebastian
Hartmann, Anja
Mascher, Martin
Scholz, Uwe
Baronian, Kim
Bode, Rüdiger
Schauer, Frieder
Matthias Vorbrodt, H.
Kunze, Gotthard
Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway
title Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway
title_full Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway
title_fullStr Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway
title_full_unstemmed Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway
title_short Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway
title_sort aadh2p: an arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062937/
https://www.ncbi.nlm.nih.gov/pubmed/27733155
http://dx.doi.org/10.1186/s12934-016-0573-9
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