Purification and characterization of a Baeyer‒Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways

111

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. It is now well recognized that the diversity of microbial species and their metabolic capabilities constitute a tremendous source of biocatalysts (6,10,39). Only a small fraction of microorganisms in most environments can be readily isolated (1, 58); therefore, gene discovery techniques which overcome the need for strain isolation provide access to the diversity of microbial chemistry. Direct cloning approaches can be very successful (21,27,28,48), but they require a genetic selection or an easy screen as well as the efficient expression of the cloned DNA in an appropriate host (15). Other approaches, based on PCR amplification from environmental DNA, target only highly conserved gene families (50). While these techniques are powerful, they often are not applicable. Differential display (DD) is an alternate technique that can be used for the discovery of bacterial genes, requiring neither a genetic selection or screen nor the presence of highly conserved genes. This technique of DD involves the reproducible amplification of DNA fragments from the mRNA population at arbitrary sites by reverse transcription (RT) followed by PCR (RT-PCR) (36,37,57). DD is used to compare the mRNA pools from cells grown under different physiological conditions. Genes expressed at the same level in all cultures will be amplified equally from all cultures, while genes expressed only under a specific condition will give rise to RT-PCR bands only under that condition. DD is a gene discovery technique that can be applied to identify differentially expressed genes. It does not rely on prior knowledge of the genes targeted or on a genomic sequence but only on the fact that the activity that these genes encode is inducible.DD has been applied extensively to eukaryotic systems and takes advantage of the poly(A) tails of eukaryotic mRNA by using poly(dT) primers to synthesize cDNAs by RT (36,37,57). This approach of DD cannot be applied to prokaryotes, which lack stable poly(A) tails. A second variation of DD uses arbitrary oligonucleotide primers to initiate RT of the message at random sites (57) and thus can be applied to archaeal and bacterial s...

Section: Resultssupporting

confidence: 55%

mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

Brzostowicz

Walters

Thomas

et al. 2003

111

“…However, using the mlhB sequence and the recently reported protocol for gene disruption in R. erythropolis (22), an MLH-deficient mutant of R. erythropolis DCL14 can be constructed, allowing the production of economically important lactones by the resident BVMO activity (27).…”

Section: Discussionmentioning

confidence: 99%

“…A mixture of (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone was synthesized from (4R)-dihydrocarvone, by purified BVMO from R. erythropolis DCL14 (27). The reaction mixture, containing 15 mM NADPH, 50 mM glycine-NaOH buffer (pH 9.5), 1.25 mM dihydrocarvone, and 0.038 U of purified monocyclic monoterpene ketone monooxygenase per ml (27), was incubated at 30°C for 1 h. Subsequently, the reaction mixture was extracted three times with an equal volume of ethyl acetate. The organic layers were combined, dried over MgSO 4 , and evaporated to dryness with a rotary evaporator under reduced pressure.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from Rhodococcus erythropolis DCL14

Vlugt-Bergmans

Werf

2001

A monoterpene -lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.Lactones, internal cyclic monoesters, are ubiquitous in nature and have been identified in all major classes of foods, including fruits, vegetables, nuts, meat, milk products, and baked products, contributing to taste and flavor nuances (13). The organoleptically important lactones generally have ␥-or ␦-lactone structures (five-or six-membered ring structures), while a few are macrocyclic (29). Lactones are intermediates in microbial degradation pathways of alicyclic compounds (1, 5, 32) but have also been implicated in certain microbial aromatic degradation pathways (ortho cleavage pathways) (14,19). The degradation of lactones is accomplished by the activity of lactone hydrolase, which catalyzes the hydrolysis of lactones to the corresponding hydroxy acids (Fig. 1). Lactone hydrolases are commercially applied for the debittering of triterpenes present in citrus juices (15) and for the production of the vitamin D-pantothenate (T. Morikawa, K. Wada, S. Kita, K. Tuzaki, K. Sakamoto, M. Kataoka, S. Shimizu, and H. Yamada, Book Abstr. 6th Japanese-Swiss Meet. Bio/Technol., p. [34][35] 1998).The application of Baeyer-Villiger monooxygenases (BVMOs) seems an attractive option for the production of lactones (12, 31). BVMOs are enzymes catalyzing the insertion of one atom of oxygen next to an alicyclic keto group, thus forming lactones. However, BVMOs are relatively unstable enzymes that generally use the very expensive NADPH as the cofactor (31). Therefore, the application of whole cells of BMVO activity containing microorganisms is a prerequisite to ascertain in situ cofactor regeneration and to increase the stability of the enzyme. However, most microorganisms containing...

“…Some fungi (e.g., Aspergillus cellulosae, Penicillium digitatum, and Fusarium oxysporum) can detoxify limonene through C-1, C-3, C-6, or C-7 oxygenation (31,32). Further, cometabolic conversion of limonene by enzymes that degrade other compounds is also common in bacteria (25,33). It has also been suggested that other monoterpenes (e.g., ␣-and ␤-pinene) may be detoxified through pathways similar to those for limonene, since limonene was found as an intermediate product in the metabolism of these monoterpenes (27).…”

mentioning

confidence: 99%

Gene Discovery for Enzymes Involved in Limonene Modification or Utilization by the Mountain Pine Beetle-Associated Pathogen Grosmannia clavigera

Wang

Lim

Madilao

et al. 2014

bTo successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (؉)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (؉)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (؉)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.