title: Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in Castellaniella defragrans.
abstract: BACKGROUND
  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 Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of β-myrcene to (S)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid.A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events.
  
  
  RESULTS
  Polar effects were absent in the in-frame deletion mutants C. defragrans Δldi and C. defragrans ΔgeoA. 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 geoA 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 in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype.
  
  
  CONCLUSIONS
  In-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the in vivo 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.
authors.affiliation: Dep, of Microbiology, Max Planck Institute for Marine Microbiology, Bremen, Germany.
authors.forename: Frauke
authors.initials: F
authors.lastname: Lüddeke
authors.forename: Aytac
authors.initials: A
authors.lastname: Dikfidan
authors.forename: Jens
authors.initials: J
authors.lastname: Harder
mesh_terms: D000080462:Acyclic Monoterpenes; D042401:Alcaligenaceae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D004734:Energy Metabolism; D017353:Gene Deletion; D005827:Genetics, Microbial / Q000379:methods; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods; D039821:Monoterpenes / Q000378:metabolism*; D011995:Recombination, Genetic
chemical_list: D000080462:Acyclic Monoterpenes; D039821:Monoterpenes; C008574:beta-myrcene; D002244:Carbon; D000429:Alcohol Oxidoreductases; D006836:Hydro-Lyases; C561837:linalool dehydratase-isomerase, Castellaniella defragrans