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Applied and Environmental Microbiology, July 1999, p. 2871-2876, Vol. 65, No. 7
Department of Biology1
and Department of Industrial and Mechanic
Engineering,2 University of Rome Three,
Rome, Italy, and Division of Industrial Microbiology, Department of
Food Science, Wageningen Agricultural University, Wageningen, The
Netherlands3
Received 13 January 1999/Accepted 16 April 1999
The M1 strain, able to grow on Terpenes are important flavor and
fragrance compounds widely distributed in nature. They are isolated
from plants or chemically synthesized, but interest in processes based
on microbial transformation has greatly increased during the past
decade. In particular, attention has been focused on the production of
oxygenated derivatives of terpenes, commonly called terpenoids, which
have a stronger odor.
Several reviews concerning the opportunities for microbial
transformation of terpenes have been published recently (32, 35,
48, 52-54, 57).
Many microorganisms are able to partly oxidize terpenes by
cometabolism, often giving rise to the accumulation of different metabolites, which makes the purification of a single product very
difficult (1, 3, 12, 15, 49). Other microorganisms, mainly
belonging to the genus Pseudomonas, are able to completely mineralize terpenes, using these substrates as the only source of
carbon and energy. This opens the possibility of producing single
metabolites by the use of specific mutants or by cloning genes for
single enzymatic activities. However, information on the genetics of
terpene degradation is lacking, and only few genes have been cloned
(8, 10, 34, 44, 46, 56).
In this paper we report the characterization of a new
Pseudomonas sp. strain, called M1, able to grow on
Strains, plasmids, and media.
The bacterial strains and
plasmids used in this study are listed in Table
1.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification and Sequencing of
-Myrcene
Catabolism Genes from Pseudomonas sp. Strain M1
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-myrcene as the sole carbon and
energy source, was isolated by an enrichment culture and identified as
a Pseudomonas sp. One -myrcene-negative mutant, called
N22, obtained by transposon mutagenesis, accumulated
(E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol)
as a unique -myrcene biotransformation product. This compound was
identified by gas chromatography-mass spectrometry. We cloned and
sequenced the DNA regions flanking the transposon and used these
fragments to identify the M1 genomic library clones containing the
wild-type copy of the interrupted gene. One of the selected cosmids,
containing a 22-kb genomic insert, was able to complement the N22
mutant for growth on -myrcene. A 5,370-bp-long sequence spanning the
region interrupted by the transposon in the mutant was determined. We
identified four open reading frames, named myrA,
myrB, myrC, and myrD, which can
potentially code for an aldehyde dehydrogenase, an alcohol
dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA
hydratase, respectively. myrA, myrB, and
myrC are likely organized in an operon, since they are
separated by only 19 and 36 nucleotides (nt), respectively, and no
promoter-like sequences have been found in these regions. The
myrD gene starts 224 nt upstream of myrA and is
divergently transcribed. The myrB sequence was found to be
completely identical to the one flanking the transposon in the mutant.
Therefore, we could ascertain that the transposon had been inserted
inside the myrB gene, in complete agreement with the
accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose
a pathway for -myrcene catabolism in Pseudomonas sp. strain M1.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-Myrcene is an acyclic monoterpene found in the essential oils of
several useful plants, such as lemongrass, hops, bay, and verbena.
While this monoterpene has been extensively studied in relation to its
effects on human health (19, 21, 23), its microbial
metabolism has been little investigated. Basidiomycetes cometabolize
-myrcene, leading to the accumulation of several oxidated
derivatives (16). The only -myrcene-utilizing bacterium described so far is Pseudomonas putida S4-2, for which a
catabolic pathway has been proposed, based on the identification of
-myrcene metabolites from the culture broth (40).
-myrcene as the sole carbon and energy source and of a
-myrcene-negative mutant. The -myrcene catabolism genes were also
identified and sequenced.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids
-myrcene (Fluka)
was performed under a saturated atmosphere of this compound on both
solid and liquid media. Biotransformation experiments were performed in 1× M9 salts solution (50 mM Na2HPO4 · 7H2O, 20 mM KH2PO4, 8.5 mM NaCl, 20 mM NH4Cl) (37).
Isolation of Pseudomonas sp. strain M1.
Pseudomonas sp. strain M1 was isolated from an enrichment
culture containing a sediment sample (10 g) from the Rhine River, Wageningen, the Netherlands. The sediment sample was diluted in 30 ml
of PSMBM containing 1 mM myrcene as the sole source of carbon and
energy in a serum flask with a butyl rubber stopper (130 ml). After
incubating this culture for 2 weeks on a shaker at 30°C with two
successive transfers (3% inoculum) into fresh medium, samples of the
enrichments were plated onto PSMBM agar plates. These plates were
incubated in a desiccator in which -myrcene was supplied via the gas
phase. The colonies that developed were isolated and checked for purity
by plating them on yeast extract-glucose plates.
Construction of Pseudomonas sp. strain M1 genomic
library.
The M1 wild-type genomic library was obtained as
previously described (38) with the following modifications:
chromosomal DNA was partially digested with 0.005 U of
Sau3AI/µg for 1 h at 37°C, and the selected
fractions were ligated in a 1:3 ratio to BamHI-digested
pLAFR3. A total of 4,000 cosmid clones were stored at 
80°C in
96-well microtiter plates containing rich medium supplemented with
tetracycline (25 µg/ml).
Transposon mutagenesis.
Conjugal transfer of the
mini-Tn5 Km transposon was performed by triparental mating.
The donor strain, E. coli CC118
pir(pUT mini-Tn5 Km), was grown overnight with shaking at 37°C in
LB medium containing 100 µg of ampicillin/ml and 50 µg of
kanamycin/ml; E. coli HB101(pRK2013) was grown overnight
in LB medium containing 50 µg of kanamycin/ml. The recipient strain,
Pseudomonas sp. strain M1, was grown overnight at 30°C in
LB medium. A 1.5-ml volume of each culture was harvested, and the cells
were washed twice with 0.9% NaCl and resuspended in the same solution.
The suspensions were combined, and the mating mix was transferred onto
a 0.45-µm-pore-size cellulose filter membrane placed on the surface
of an LB medium plate. After overnight incubation at 30°C, the
bacteria were resuspended in 10 ml of 0.9% NaCl and plated (100 µl/plate) on appropriate selective medium. Kanamycin-resistant
transconjugants were screened for the loss of the ability to grow on
-myrcene (Myr
phenotype) by replica plating.
DNA manipulation. Plasmid preparation and restrictions and ligations were performed by standard procedures (37). Restriction endonucleases and T4 DNA ligase were from Boehringer Mannheim. DNA fragments were purified from agarose with the Qiaquick gel extraction kit (Qiagen). Purified DNA fragments were labeled with digoxigenin (DIG)-11-dUTP with the DIG DNA labeling kit (Boehringer). For Southern experiments, DNA was transferred onto positively charged nylon membrane (Boehringer) and the DIG labels were visualized with a chemiluminescence detection kit (Boehringer) according to the supplier's instructions.
The nucleotide sequence was determined with an Applied Biosystems automated sequencer (model 373 Stretch) with a DyeDeoxy terminator cycle sequencing kit (Perkin-Elmer). Both commercially available and synthetic primers were used for sequencing reactions. The oligonucleotides corresponding to the I and O ends of mini-Tn5 were 5'-TGTCCACTACGTGAAAGGC-3' and 5'-CGAACTTGTGTATAAGAGTCAG-3', respectively. DNA sequences were identified by similarity searches with the BLAST 2.0 (6) and FASTA 3 (42) programs. The deduced amino acid sequences were analyzed by the pattern and profile programs at the ExPASy World Wide Web Molecular Biology Server, Swiss Institute of Bioinformatics, Geneva, Switzerland (7).Culture conditions for metabolite analysis.
A 25-ml culture
of Pseudomonas sp. strain N22 was grown overnight at 30°C
with shaking in PSMBM supplemented with 0.2% sodium lactate as the
carbon source. Experimental cultures were obtained by inoculating 5%
(vol/vol) of the overnight culture into two flasks containing 50 ml of
0.2% lactate PSMBM, to one of which -myrcene was supplied via the
gas phase. When the cultures reached an optical density at 600 nm of
1.5, the cells were harvested by centrifugation at 4,000 rpm and washed
twice with 1× M9 salts solution. Finally, the pellets were resuspended
in an appropriate volume of the same salts solution to an optical
density at 600 nm of 10. -Myrcene was directly added (1 µl/ml) to
one of the two cell suspensions, and both flasks were incubated at
30°C for 1 h. As a control, a flask containing only 1× M9 salts
solution and 1 µl of -myrcene/ml was incubated under the same conditions.
Identification of the metabolites. Cells were separated by centrifugation at 8,000 rpm for 15 min. The cell-free solution was saturated with NaCl and extracted three times with one-third volume of a hexane-ether mixture (1:1), and the combined extracts were dried over anhydrous sodium sulfate and concentrated to a volume of 1 ml with a rotative evaporator (20 mm of Hg; bath temperature, 25°C).
Analysis of the extracts was performed with a gas chromatograph (GC) (Fisons GC 8000) coupled with a mass spectrometer (MS) (Fisons MD 800). A methyl-phenyl silicon (SE 52 MS) capillary column ([inside diameter]; 30 m by 0.25 mm; MEGA) was used, with a temperature program of 50°C for the first 4 min to 250°C at a heating rate of 10°C/min and a flow rate of the carrier gas, helium, of 1 ml per min; 1 µl was injected at 200°C in the splitless mode. Mass spectra were recorded in electron impact ionization mode at 70 eV by scanning the mass range of 30 to 450 m/z.Nucleotide sequence accession number. The nucleotide sequence of 5,370 bp has been deposited in GenBank under accession no. AF112883.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characterization of strain M1. The M1 strain was one of the five microorganisms isolated from an enrichment culture, as described in Materials and Methods. It is an oxidase-positive, gram-negative, motile, rod-shaped bacterium identified as Pseudomonas sp. by the API 20NE system.
The maximum growth rate on-myrcene was obtained by using
-myrcene-saturated PSMBM inoculated with 5% (vol/vol) of an
overnight culture grown on the same medium containing 0.2% lactate.
Under these conditions, the lag phase lasted approximately 2 h and
the duplication time during the exponential growth phase was about 1 h 35 min. Since the M1 strain was newly isolated, we
investigated the characteristics that are important to carry out
genetic and physiological studies.
The stability of the -myrcene-positive phenotype was studied by
replica plating after growth on lactate or glucose for 20 generations,
and we found that it was 100% stable. This is an important result in
view of the possibility of isolating mutants unable to grow on
-myrcene.
The antibiotic sensitivity test showed that the M1 strain is sensitive
to kanamycin (20 µg/ml), tetracycline (10 µg/ml), and chloramphenicol (25 µg/ml), whose resistance genes are the genetic markers of the majority of the Pseudomonas vectors. The M1
strain was also tested as the recipient strain in conjugation
experiments by using E. coli S17.1 containing the pLAFR3
cosmid as the donor strain. The resulting transconjugants contained a
plasmid whose restriction pattern was identical to the original one. As
a whole, the results obtained indicated that this newly isolated strain could be manipulated by the classical genetic and molecular techniques.
Isolation and characterization of -myrcene-negative
mutants.
In order to characterize the genes of the -myrcene
metabolic pathway, transposon mutagenesis was used to generate mutants unable to grow on -myrcene.
pir into Pseudomonas
sp. strain M1 was performed by triparental mating with the helper
plasmid pRK2013, as described in Materials and Methods. Transconjugants
were selected on glucose PSMBM in the presence of kanamycin, the
selective marker of the transposon. By a screening of 6,000 transconjugants for growth on -myrcene, we have isolated two
mutants, named N22 and N59, unable to grow on -myrcene and three
leaky mutants whose growth on this substrate was very poor.
The N22 mutant was further analyzed for the accumulation of
intermediates of -myrcene catabolism by biotransformation experiments.
-Myrcene biotransformation by N22 mutant.
N22 resting
cells, prepared as described in Materials and Methods, were incubated
in the presence and in the absence of -myrcene for 1 h at
30°C. Control experiments were performed by incubating -myrcene-containing 1× M9 salts solution without cells. Extracts were then analyzed by GC-MS. While -myrcene did not undergo
spontaneous transformation in the absence of cells (data not shown), a
comparison of the chromatograms of the N22 extracts showed a single and
abundant difference peak at a retention time of 14.24 min, present only in the extracts from the sample containing -myrcene (Fig. 1A and
B).
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-myrcene itself. The base peak at 93 m/z
(100%) and the peak at 79 m/z (45%) are in agreement with
the formation of carbocationic fragments derived from the loss of 3- and 4-carbon alcoholic subunits from the isopropyl moiety of the molecule.
These data suggested that the likely structure could be (Z)-
or (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or
myrcen-8-ol), derived from -myrcene by oxidation with oxygen
insertion in the C-8-H position.
Definitive structure and (E) stereochemistry were assigned
to the metabolite by comparison of its GC retention time and MS spectrum with that of an authentic sample of
(E)-2-methyl-6-methylen-2,7-octadien-1-ol obtained by
oxidation of myrcene with SeO2 (14). The mass
spectra and retention times of the two alcohols were identical within the limits of experimental error (not shown).
Cloning of the N22 chromosomal DNA regions flanking the mini-Tn5 insertion. The insertion of the mini-Tn5 transposon into the N22 chromosome was verified by Southern blot analysis of the chromosomal DNA digested with SacI, XhoI, and ClaI, restriction enzymes which do not cut inside the transposon. The probe used was almost all of mini-Tn5, as a 2,260-bp HindIII fragment from pUT mini-Tn5 Km. Only one hybridization band was present in each digestion, indicating that a single insertion event had occurred (data not shown).
To clone the DNA fragment containing the transposon and its flanking regions, we constructed a minilibrary of N22 chromosomal DNA digested with SacI, using the pBluescript KS(+) plasmid as the cloning vector and E. coli DH5
as the host strain.
Transformants containing the transposon were selected on kanamycin and
ampicillin plates. All the transformants analyzed contained the same
SacI fragment of approximately 7.7 kb. The restriction map
of this fragment showed that it contains a chromosomal region of 3.1 kb upstream and one of 2.2 kb downstream of the transposon. The 2.3-kb SacI-BglII fragment from the upstream region
(shown in Fig. 2A) was used as a probe in
Southern blot analysis of the SacI-digested chromosomal DNAs
from the M1 and N22 strains. The results obtained are in complete
agreement with what was expected. In fact, only one hybridization band
was present in M1, whose size (5.3 kb) corresponds to the sum of the
sizes of the two chromosomal regions flanking the transposon in the
7.7-kb SacI fragment. In N22, the hybridization band was
approximately 2.4 kb longer, consistent with the insertion of the
mini-Tn5 transposon (data not shown).
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Identification of the gene interrupted by the transposon in the N22 mutant. The transposon-containing 7.7-kb SacI fragment was used to identify the disrupted gene of the mutant. We sequenced the mini-Tn5 flanking regions with two oligonucleotides corresponding to the I and O ends of the transposon, respectively (see Materials and Methods). We obtained two sequences of approximately 300 nt each, and a comparison analysis of the deduced amino acid sequences against protein databases revealed a high level of similarity to those of alcohol dehydrogenases (see below). This result is in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the N22 mutant.
Construction of an M1 genomic library and identification of gene
bank clones containing the -myrcene degradative genes.
In order
to obtain the wild-type copy of the gene mutagenized by the
mini-Tn5 transposon in the N22 mutant, we constructed a
genomic library of strain M1 as described in Materials and Methods. We
obtained approximately 4,000 clones containing genomic inserts with an
average size of 23 kb, which means that the M1 chromosome is
represented about three times in the genomic library. Colony hybridization experiments were performed on the gene bank clones, using
the 2.3-kb SacI-BglII fragment as a probe (Fig.
2A). We identified four positive clones containing the same 5.3-kb
SacI fragment, in agreement with the size of the
hybridization band found in M1. The restriction map of the recombinant
cosmid (called pC7) of one of these clones is shown in Fig. 2A.
mutant N22 restored the -myrcene-positive
phenotype. Moreover, pC7 cosmid, transferred by conjugation into
P. putida NCIMB 8248, which is unable to grow on
-myrcene, conferred the ability to grow on this substrate on the transconjugants.
Sequencing of the M1 5.3-kb SacI fragment.
The
5.3-kb SacI fragment was subcloned and sequenced. The entire
fragment was found to be 5,370 bp long. Sequence analysis showed the
presence of three open reading frames, named myrA, myrB, and myrC, localized on one DNA strand and
one open reading frame, named myrD, on the opposite strand
(Fig. 2B). The start codon of each open reading frame is preceded by a
potential ribosomal binding site. The first open reading frame
(myrA) shows a GTG start codon at nt 1338 and ends at nt
2798. It could encode a protein of 486 amino acids (aa) (with a
theoretical molecular mass of 54.3 kDa). A database search of proteins
related to MyrA revealed a significant similarity to prokaryotic and
eukaryotic aldehyde dehydrogenases (39 to 48% identity in the first 10 best scores). The highly conserved glutamic acid and cysteine residues implicated in the catalytic activity (4) are located at
positions 232 and 266, respectively, in MyrA. The second open reading
frame (myrB), extending from nt 2818 to 3921, could encode a
polypeptide of 367 aa with an estimated molecular mass of 38 kDa. MyrB
showed homology with several alcohol dehydrogenases. The best scores (31 to 46% identity) were with zinc-containing long-chain alcohol dehydrogenases (43, 51), and a protein ScanProsite search (7) showed the presence of a zinc-binding signature at
positions 60 to 74. XylB and TerpD alcohol dehydrogenases, involved in
the degradation of xylenes (28) and -terpineol
(44), respectively, showed 45% identity with MyrB. The
myrB sequence was found to be completely identical to the
one flanking the transposon in the N22 mutant. Therefore, we could
ascertain that the transposon had been inserted between nt 3142 and
3143, inside the myrB gene, which, according to the
homologies found, could code for an alcohol dehydrogenase. The third
open reading frame, myrC, starts at position 3958 and
extends to the end of the clone without encountering a stop codon,
giving a truncated protein of 471 amino acid residues. Comparison of
MyrC with protein databases showed similarity (26 to 31% amino acid
identity) with long-chain fatty-acid CoA ligases (EC 6.2.1.3) and
4-coumarate CoA ligases (EC 6.2.1.12). Actually, the best score (47%
identity) was with Mycobacterium tuberculosis fadD4 protein
(AC Z92669), which in turn shares 30% identity with 4-coumarate CoA
ligase (P41636). All these enzymes contain the putative AMP-binding
domain signature (36, 55), which extends from position 164 to 175 in the MyrC sequence. On the complementary strand, we found an
open reading frame which starts at nt 1113 and stops at nt 277 with two
stop codons. The resulting polypeptide of 277 aa, MyrD (calculated
molecular mass, 30.4 kDa), belongs to the enoyl-CoA hydratase (ECH)
family, including an ECHH (for enoyl-CoA hydratase homolog) from
Rhodobacter capsulatus (31.8% identity; AC P24162),
possibly involved in fatty acid oxidation (11), and the
paaG product (33.8% identity; AC P77467) and PhaB (30.7%
identity; AC AF029714) from E. coli K-12 and P. putida U (41), respectively, both involved in the
phenylacetic acid catabolic pathway. Furthermore, a protein ProfileScan
search (7) showed the presence of the ECH family signature
pattern: the conserved region rich in glycine and hydrophobic residues
extending from positions 116 to 202 in the MyrD sequence. On the same
strand, downstream of myrD, we identified the 5' end of a
possible open reading frame (nt 117 to 1). In fact, the amino acid
sequence deduced from this truncated open reading frame revealed a high homology with the N terminus of positive regulators belonging to the
LysR family (47). The best score (97% identity) was with the putative regulatory protein BphR (AC D38633 [unpublished]) from
the soil bacterium Pseudomonas sp. strain KKS102, which has biphenyl- and polychlorinated biphenyl-degrading activities
(33).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study provides new information on -myrcene catabolism
genes which had not been previously described. We have identified four
open reading frames which potentially code for an alcohol dehydrogenase, an aldehyde dehydrogenase, an acyl-CoA synthetase, and
an enoyl-CoA hydratase. The relationship between these genes and
-myrcene catabolism is strongly suggested by the following facts:
(i) the pC7 M1 gene bank cosmid, containing the corresponding genes,
restored the wild-type Myr+ phenotype when transferred into
the Pseudomonas sp. strain N22 mutant, (ii) the same gene
bank clone conferred the ability to grow on -myrcene on a P. putida strain unable to utilize this substrate, and (iii) the
disruption of the alcohol dehydrogenase gene in the mutant N22 led to
the accumulation of the -myrcene oxidized derivative
(E)-2-methyl-6-methylen-2,7-octadien-1-ol. This compound was
identified by the comparison of its MS spectrum with that of the
authentic chemically synthesized compound. Although (E)-
and/or (Z)-myrcen-8-ols have already been found in insect pheromones and essential oils and recognized by their mass spectra (5, 26, 30, 31), we failed to find any reported mass spectral data in the literature. To our knowledge, this is the first
report of the mass spectrum of 2-methyl-6-methylen-2,7-octadien-1-ol. Based on this evidence, we propose the metabolic pathway of -myrcene shown in Fig. 3. The -myrcene (Fig. 3,
I) hydroxylation to 2-methyl-6-methylen-2,7-octadien-1-ol (Fig. 3, II)
is demonstrated by the accumulation of this compound by the
mutant, while the subsequent proposed degradation to
2-methyl-3-hydroxy-6-methylen-7-octenoyl-CoA (Fig. 3, VI) via
-oxidation is based on sequencing data. Experimental evidence for
2-methyl-3-keto-6-methylen-7-octenoyl-CoA (Fig. 3, VII) and
4-methylen-5-hexenoic acid (Fig. 3, VIII) formation is lacking.
However, the last compound and 2-methyl-6-methylen-2,7-octadienoic acid
(Fig. 3, IV) have been identified as -myrcene metabolites in the
-myrcene-degrading P. putida S4-2 strain (40).
Studies of biotransformation of -myrcene with fungi showed the
formation of a series of oxidized compounds derived from hydration or
epoxydation at different double bonds, and no hydroxylation of the
terminal carbon could be observed (16). Formation of
mixtures of (E)- and (Z)-myrcen-8-ols, together
with other minor components, have been described in Nocardia
and Streptomyces and in fungi only when the dienyl moiety of
-myrcene was protected by a dienophile, such as sulfur dioxide
(2). Up to now, the -myrcene catabolic pathway proposed
here has been found exclusively in M1 and S4-2, the only two
Pseudomonas strains in which -myrcene catabolism has been
studied.
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As far as the organization of the catabolic genes is concerned,
myrA, myrB, and myrC should be
organized in an operon, since they are separated by only 19 and 36 nt,
respectively, and no promoter-like sequences have been found in these
regions. The myrD gene starts 224 nt upstream of
myrA and is divergently transcribed. In the DNA region
between these two genes we have found two stretches of sequence (nt
1181 to 1200 and 1271 to 1290) with an imperfect dyad symmetry,
containing the proposed LysR motif (5'-T-N11-A-3'), involved in specific binding to LysR proteins (25).
Interestingly, a LysR-like regulator is probably present downstream of
myrD, and it could be involved in the expression of the
catabolic genes. In the sequenced fragment we did not find the
monooxygenase gene responsible for the conversion of -myrcene into
(E)-myrcen-8-ol. However, this gene could be contained in
the pC7 gene bank cosmid, which confers the ability to grow on
-myrcene on P. putida NCIMB 8248, even if the expression
of a related activity encoded by the host strain cannot be ruled out.
The N22 mutant is able to accumulate (E)-myrcen-8-ol. This compound has been used as a synthon for the production of zoapatanol or its derivatives (17), chemicals with antifertility activity obtained from plants belonging to the genus Montanoa (45). Although terpenoids containing terminal allylic alcohols [such as (E)-myrcen-8-ol] can be obtained by chemical synthesis, this oxidation step requires very toxic chemicals, difficult to separate from the product. In contrast, biotransformations have the advantage of proceeding under milder and safer conditions. Moreover, both the N22 mutant and the genes isolated in this study could represent an important opportunity for the production of fine chemicals, due to the low substrate specificity of many Pseudomonas spp. catabolic enzymes.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the European Community (BIO4-CT95-0049).
We thank D. Leak (Imperial College of Science Technology and Medicine, London, England) for providing P. putida NCIMB 8248.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, University of Rome Three, Viale G. Marconi, 446, 00146 Rome, Italy. Phone: 0039-06-55176318. Fax: 0039-06-55176321. E-mail: Zennaro{at}bio.uniroma3.it.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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