Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it>
<p>Abstract</p> <p>Background</p> <p>Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in <it>Castellaniella defra...
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| Format: | Article |
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BMC
2012-09-01
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| Series: | BMC Microbiology |
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| Online Access: | http://www.biomedcentral.com/1471-2180/12/192 |
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| author | Lüddeke Frauke Dikfidan Aytac Harder Jens |
| author_facet | Lüddeke Frauke Dikfidan Aytac Harder Jens |
| author_sort | Lüddeke Frauke |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it> strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase <it>ldi</it>/LDI catalyzes the hydration of β-myrcene to (<it>S</it>)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase <it>geoA</it>/GeDH and a geranial dehydrogenase <it>geoB</it>/GaDH contribute to the formation of geranic acid.</p> <p>A genetic system was for the first time applied for the betaproteobacterium to prove <it>in vivo</it> the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events.</p> <p>Results</p> <p>Polar effects were absent in the in-frame deletion mutants <it>C. defragrans</it> Δ<it>ldi</it> and <it>C. defragrans</it> Δ<it>geoA</it>. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of <it>geoA</it> resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were <it>in trans</it> complemented with the broad-host range expression vector pBBR1MCS-4<it>ldi</it> and pBBR1MCS-2<it>geoA</it>, restoring in both cases the wild type phenotype.</p> <p>Conclusions</p> <p>In-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the <it>in vivo</it> function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the β-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial enzyme LDI suggests the presence of a second enzyme system activating unsaturated hydrocarbons.</p> |
| first_indexed | 2024-12-18T05:50:35Z |
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| language | English |
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| publishDate | 2012-09-01 |
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| spelling | doaj.art-5c2fd352c46a4a5a84ea7c647c416c9a2022-12-21T21:18:56ZengBMCBMC Microbiology1471-21802012-09-0112119210.1186/1471-2180-12-192Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it>Lüddeke FraukeDikfidan AytacHarder Jens<p>Abstract</p> <p>Background</p> <p>Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it> strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase <it>ldi</it>/LDI catalyzes the hydration of β-myrcene to (<it>S</it>)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase <it>geoA</it>/GeDH and a geranial dehydrogenase <it>geoB</it>/GaDH contribute to the formation of geranic acid.</p> <p>A genetic system was for the first time applied for the betaproteobacterium to prove <it>in vivo</it> the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events.</p> <p>Results</p> <p>Polar effects were absent in the in-frame deletion mutants <it>C. defragrans</it> Δ<it>ldi</it> and <it>C. defragrans</it> Δ<it>geoA</it>. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of <it>geoA</it> resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were <it>in trans</it> complemented with the broad-host range expression vector pBBR1MCS-4<it>ldi</it> and pBBR1MCS-2<it>geoA</it>, restoring in both cases the wild type phenotype.</p> <p>Conclusions</p> <p>In-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the <it>in vivo</it> function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the β-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial enzyme LDI suggests the presence of a second enzyme system activating unsaturated hydrocarbons.</p>http://www.biomedcentral.com/1471-2180/12/192β-myrcenephellandreneAnaerobic degradationGeraniol dehydrogenaseLinalool dehydratase-isomeraseGenetic system |
| spellingShingle | Lüddeke Frauke Dikfidan Aytac Harder Jens Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it> BMC Microbiology β-myrcene phellandrene Anaerobic degradation Geraniol dehydrogenase Linalool dehydratase-isomerase Genetic system |
| title | Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it> |
| title_full | Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it> |
| title_fullStr | Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it> |
| title_full_unstemmed | Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it> |
| title_short | Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in <it>Castellaniella defragrans</it> |
| title_sort | physiology of deletion mutants in the anaerobic β myrcene degradation pathway in it castellaniella defragrans it |
| topic | β-myrcene phellandrene Anaerobic degradation Geraniol dehydrogenase Linalool dehydratase-isomerase Genetic system |
| url | http://www.biomedcentral.com/1471-2180/12/192 |
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