| Summary: | <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>
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