Physiology of Deletion Mutants in the Anaerobic β-myrcene Degradation Pathway in Castellaniella Defragrans

BMC Microbiol. 2012 Sep 4;12:192. doi: 10.1186/1471-2180-12-192.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyclic Monoterpenes
  • Alcaligenaceae / genetics*
  • Alcaligenaceae / growth & development
  • Alcaligenaceae / metabolism*
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism
  • Carbon / metabolism
  • Energy Metabolism
  • Gene Deletion*
  • Genetics, Microbial / methods
  • Hydro-Lyases / genetics
  • Hydro-Lyases / metabolism
  • Metabolic Networks and Pathways / genetics*
  • Molecular Biology / methods
  • Monoterpenes / metabolism*
  • Recombination, Genetic

Substances

  • Acyclic Monoterpenes
  • Monoterpenes
  • beta-myrcene
  • Carbon
  • Alcohol Oxidoreductases
  • Hydro-Lyases
  • linalool dehydratase-isomerase, Castellaniella defragrans