From the Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del Consejo Superior de Investigaciones Científicas, Velázquez 144, Madrid, 28006, Spain
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ABSTRACT |
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Aspergillus nidulans utilizes phenylacetate as a carbon source via homogentisate, which is degraded to fumarate and acetoacetate. Mutational evidence strongly suggested that phenylacetate is converted to homogentisate through two sequential hydroxylating reactions in positions 2 and 5 of the aromatic ring. Using cDNA substraction techniques, we have characterized a gene, denoted phacA, whose transcription is strongly induced by phenylacetate and which putatively encodes a cytochrome P450 protein. A disrupted phacA strain does not grow on phenylacetate but grows on 2-hydroxy- or 2,5-dihydroxyphenylacetate. Microsomal extracts of the disrupted strain are deficient in the NADPH-dependent conversion of phenylacetate to 2-hydroxyphenylacetate. We conclude that PhacA catalyzes the ortho-hydroxylation of phenylacetate, the first step of A. nidulans phenylacetate catabolism. The involvement of a P450 enzyme in the ortho-hydroxylation of a monoaromatic compound has no precedent. In addition, PhacA shows substantial sequence divergence with known cytochromes P450 and defines a new family of these enzymes, suggesting that saprophytic fungi may represent a source of novel cytochromes P450.
Phenylacetate is a precursor for benzylpenicillin production.
phacA disruption increases penicillin production 3-5-fold,
indicating that catabolism competes with antibiotic biosynthesis for
phenylacetate and strongly suggesting strategies for Penicillium
chrysogenum strain improvement by reverse genetics.
Aerobic degradation of aromatic hydrocarbons by microbes involves
the action of oxygenases (enzymes that incorporate one or two atoms
from dioxygen into substrates) acting at two different levels in
specific catabolic pathways (1, 2). First, oxygenase enzymes acting at
the upstream segment of these pathways incorporate one (monooxygenases,
aromatic ring hydroxylases) or two (aromatic ring dioxygenases) oxygen
atoms into the aromatic substrate as hydroxyl groups, preparing the
ring for a subsequent ring-opening step. In this second step, the
dihydroxylated aromatic ring is opened by ring-cleavage dioxygenases.
Monooxygenases are a mechanistically diverse group of enzymes (1)
including, for example, flavoproteins such as
p-hydroxybenzoate hydroxylase (3), multicomponent enzymes
such as Pseudomonas mendocina toluene 4-monoxygenase, in
which one of the terminal hydroxylase polypeptides contains a binuclear
iron cluster (4), or heme-containing cytochrome P450 systems.
Monooxygenases of the cytochrome P450 superfamily (5, 6) are widely
distributed among living organisms and catalyze a multiplicity of
biosynthetic and catabolic reactions, usually with narrow substrate
specificity, including the hydroxylation of a variety of lipophylic drugs.
In common with other saprophytic microbes, the genetically amenable,
obligate aerobic fungus Aspergillus nidulans shows notable metabolic versatility. For example, it can use the aromatic hydrocarbon compound phenylacetate
(PhAc)1 as sole carbon
source. Despite the abundant information available on the catabolic
pathways of other aromatic compounds, which have been extensively
studied in bacteria, our understanding of PhAc degradation pathways is
scarce. In Pseudomonas putida U, it is known that PhAc is
degraded through phenylacetyl-CoA (7), although the ring cleavage steps
remain uncharacterized. In A. nidulans, PhAc degradation
proceeds through 2,5-dihydroxy-PhAc (homogentisate, see Fig. 1). The
three structural genes mediating the conversion of homogentisate to
Krebs cycle intermediates (i.e. the "lower" PhAc
pathway) have been characterized (8-10,) but the steps leading to
homogentisate have not yet been reported. We describe here mutational
and molecular analysis showing that A. nidulans PhAc catabolism proceeds via homogentisate through two sequential
hydroxylating steps, of which the first is the 2-hydroxylation of the
ring catalyzed by a novel cytochrome P450. Targeted inactivation of
this gene results in penicillin overproduction.
Fungal Strains, Media, and Growth Conditions--
A
nidulans strains carried markers in standard use (11). Standard
media for A. nidulans (12) were used for strain maintenance, growth tests, and transformation. Complementation tests were carried out in constructed diploids.
A biA1 strain was the source of cDNA, and a biA1
methG1 argB2 strain was the recipient strain for phacA
gene disruption. A biA1 methG1 strain was used as wild type
control in experiments with the disrupted strains. Culture conditions
inducing high levels of expression of the PhAc catabolic genes have
been described (13) and were used to grow mycelia for protein
extraction. PhAc and its monohydroxy and dihydroxy derivatives were
used as sole carbon source at 10 mM (although homogentisate
was occasionally used at 25-50 mM), and 10 mM
ammonium chloride was used as sole nitrogen source.
Isolation of PhAc Nonutilizing Mutants--
2-, 3-, and
4-fluorophenylacetate at a 5 mM concentration were
shown to prevent growth of A. nidulans in the presence of a derepressing carbon source, indicating that their catabolism was toxic
for the mold. However, some residual growth was observed when plates
were incubated for more than 3 days at 37 °C. Sectors of markedly
more vigorous mycelia frequently arose after prolonged incubation.
Although the reason why catabolism of these PhAc derivatives results in
toxicity is not clear, preliminary tests showed that mutations
preventing the toxicity of 2- and 3-fluorophenylacetate also
prevented the catabolism of PhAc. Therefore, conidiospores of a
yA2 pantoB100 strain were plated to obtain
isolated colonies, which were transferred to minimal medium with 0.05%
lactose (w/v) as carbon source in the presence of 5 mM 2- or 3-fluorophenylacetate. Sectors with more vigorous growth were
purified and tested for the utilization of different PhAc derivatives
as sole carbon source. Two major classes were found, which were denoted
class I and II (see "Results").
phacA Gene Disruption--
A pUC18-based plasmid denoted
pPhacA::argB was constructed by standard techniques. This
plasmid contains (starting from the lacZ promoter in pUC18)
0.94 kbp of the phacA upstream region sequentially followed
by its genomic coding region up to codon 297, a genomic, 3.2-kbp
fragment containing an argB+ allele, the genomic
sequence of phacA corresponding to codons 393-518, and
finally 1.2 kbp of the phacA 3'-downstream region. This
insert was purified from the plasmid after digestion with EcoRI and used for transformation (14). Transformed
(arginine-independent) clones in which the resident phacA
gene had been replaced by the transforming fragment were identified by
Southern analysis. The mutated allele would encode a protein truncated
at residue 297 and therefore would lack the predicted region involved
in heme binding.
Characterization of phacA cDNA and Genomic
Clones--
phacA cDNA clones were obtained by
differential screening of a cDNA library enriched in PhAc-induced
transcripts, as described (8-10). Seven cDNA clones were obtained.
The insert of one such clone was used to isolate genomic clones from a
standard Preparation of Microsomes and Enzyme Assays--
Mycelia from
the isogenic
PhAc 2-hydroxylase was assayed by measuring the formation of
2-hydroxy-PhAc in a reaction that required PhAc (added at 1 mM) and NADPH (also at 1 mM) in the presence of
a microsomal fraction. 2-Hydroxy-PhAc was chemically determined using
diazotized 4-nitroaniline essentially as described (17). Absorbance was
read at 550 nm and converted to nmol of 2-hydroxy-PhAc using a
reference plot. The range of linear response was 1-100 nmol of
2-hydroxy-PhAc. This method also detected 3-hydroxy-PhAc, and
therefore, the identity of the reaction product as 2-hydroxy-PhAc was
confirmed by direct HPLC analysis of the reaction mixtures (see
"Results"). Proteins were precipitated in the presence of 5% (w/v)
trichloroacetic acid, and 10 µl samples were injected into a
Nucleosil 300-5 C18 column (250 × 4 mm) coupled to a 11 × 4-mm precursor column of this support, using as mobile phase (at 0.6 ml/min) a solution containing 50 mM monohydrogen potassium
phosphate, 0.1 mM EDTA, 100 mM trifluoroacetic
acid, and 8% (v/v) acetonitrile. Detection was at 220 nm.
Penicillin Production--
Cultures for penicillin production
were inoculated with spores of the The "Upper" Phenylacetate Degradation Pathway--
We found
that 2-, 3-, or 4-fluoro-PhAc prevents A. nidulans growth on
0.05% (w/v) lactose as carbon source. We therefore selected mutations
resulting in fluorophenylacetate resistance, assuming that they would
prevent PhAc utilization and following Apirion (19), who used
fluoroacetate resistance to select acetate nonutilizing mutants.
Mutations preventing PhAc utilization (phac) were
efficiently selected with 2-fluorophenylacetate. They were recessive in
diploids, indicating that they represent loss-of-function mutations.
They were classified in two major classes. Class I mutants did not grow
on PhAc but grew on 2-hydroxy-PhAc or 2,5-dihydroxy-PhAc. By contrast,
class II mutants did not grow on either PhAc or 2-hydroxy-PhAc but grew
on 2,5-dihydroxy-PhAc. Class I or II mutations did not affect growth on
acetate, Phe, Tyr, 3- or 4-hydroxy-PhAc, and 3,4-dihydroxy-PhAc,
showing that they specifically prevented PhAc catabolism. Mutations in
class I complemented class II mutations. As 2,5-dihydroxy-PhAc is known
to be an intermediate of PhAc catabolism (8-10), these data are
consistent with the pathway shown in Fig. 1 in which PhAc is converted into
homogentisate through two hydroxylating reactions, prevented by class I
and class II mutations, respectively.
Molecular Cloning of phacA, a Gene Encoding a Novel Cytochrome
P450--
We have previously used a differential screening procedure
of a subtracted cDNA library to isolate cDNA clones
representing transcripts induced by PhAc (8-10). This collection of
cDNAs included clones for the three genes (fahA,
maiA, and hmgA) of the lower PhAc pathway (Fig.
1; Refs. 8-10). Among the remaining cDNA clones, those that did
not represent these previously described genes were identified and
classified by restriction enzyme mapping and/or partial cDNA
sequencing. Seven overlapping cDNAs represented a novel
PhAc-induced transcript whose gene was named phacA and
which contained an open reading frame putatively encoding a
518-residue polypeptide (Mr 58,495). DNA
sequencing of genomic clones showed that the phacA-coding
region is interrupted by three introns, 65, 56, and 53 nucleotides
long. The nucleotide sequence of phacA and the amino acid
sequence of its derived protein product have been deposited in the
DDBJ/EMBL/GenBank data bases under accession number AJ132442. Blastp
searches against nonredundant Swissprot+Translation of EMBL nucleotide
sequence data bases revealed that all 20 entries showing the highest
amino acid sequence identity to PhacA were cytochrome P450 proteins of
the CYP1 family (of which 19 were CYP1A1 P450s), including mammalian
and fish proteins but only a single human CYP. Identity levels were in
the 25% range, with the highest identity (27.4% in a 465 residue
overlap) shown by a Sparus aurata (gilthead sea bream) P450
protein. Cytochromes P450 are heme-thiolate enzymes, and PhacA residues
431 to 439 contain the peptide motif
Gly-Xaa-Gly-Xaa-Xaa-Xaa-Cys-Xaa-Gly (where Xaa indicates any amino
acid), which is involved in heme binding in proteins of this class (6).
We conclude that phacA encodes a cytochrome P450. The above
levels of identity are below those required to place it in an already
existing family (>40% identity required (5)). Therefore, PhacA
defines a new family of these
proteins.2 It has been
denoted CYP504 using the current P450 nomenclature system.
phacA Is Strongly Induced by Phenylacetate--
phacA
transcript levels were analyzed by Northern analysis in cells grown in
glucose and subsequently transferred to minimal media, each containing
a different carbon source. This analysis (Fig.
2) showed that phacA
transcription is strongly induced by PhAc and largely repressed by
glucose (PhAc plus glucose, Fig. 2). The transcript was absent in cells
transferred to glucose alone or to gluconeogenic substrates acetate or
glutamate. 2-Hydroxy-PhAc was also a strong inducer (although less so
than PhAc), and 3-hydroxy-PhAc was a weak inducer. 4-Hydroxy-PhAc,
2,5-, or 3,4-dihydroxy-PhAc did not induce phacA
transcription. Notably, Phe (but not Tyr) induced phacA
transcription to some extent. These results support the contention that
phacA is involved in PhAc catabolism.
The Phenotype of a Disruption Strain Is Consistent with phacA
Encoding a PhAc 2-Hydroxylase--
To confirm the involvement of PhacA
in PhAc catabolism, we constructed a disruption-deletion
phacA mutation by reverse genetics. We transformed an
argB2 arginine-requiring strain with a linear DNA fragment
carrying a mutated phacA gene in which a 3.2-kbp fragment
containing an argB+ allele replaced a 289-base
pair NaeI-KpnI phacA genomic fragment including codons 298-392 (see Fig. 3).
This mutant phacA gene would encode a PhacA protein
truncated at residue 297 and therefore lacking the 221 C-terminal
residues, which include the essential Cys-containing peptide motif
involved in heme binding. Homokaryotic transformant clones were
purified after repeated streaking on medium lacking arginine and
analyzed by Southern blot hybridization. Two transformants showing an
identical hybridization pattern consistent with the integration event
shown in Fig. 3 were chosen for further analyses. Both grew normally on
0.05% (w/v) lactose but, in contrast to the wild type, showed residual
growth (similar to that observed in the absence of a carbon source) on
PhAc-minimal medium (Fig. 3), showing that PhacA is indeed involved in
PhAc catabolism. By contrast, both transformants were able to grow on
2-hydroxy-PhAc (Fig. 3). These data indicate that PhacA is involved in
the ortho-hydroxylation of PhAc. The disruption-deletion
mutation (denoted
Following incubation in vitro of wild type microsomes with
PhAc and NADPH, we detected the formation of a monohydroxylated PhAc
derivative using a chemical detection method ((Fig. 4A; see "Experimental Procedures"). Direct HPLC analysis of the reaction mixture showed that this compound was 2-hydroxy-PhAc and that its
formation was absolutely dependent on the presence of both PhAc and
NADPH (Fig. 4B). This showed that A. nidulans
microsomes contain a phenylacetate ortho-hydroxylating
activity. This activity was markedly and reproducibly reduced but not
abolished by the phacA Disruption Results in Increased Penicillin
Production--
Penicillin-producing filamentous fungi use PhAc
(activated as a CoA thioester) as a precursor for penicillin G
biosynthesis. PhAc is exchanged with the L-aminoadipyl
moiety of isopenicillin N to yield penicillin G in a reaction catalyzed
by acyl-CoA::isopenicillin N-acyltransferase (see
Ref. 21 for review). PhAc cannot be synthesized by fungi, and therefore
it has to be fed to penicillin cultures. We reasoned that the PhAc
degradation pathway would compete with the penicillin biosynthetic
pathway for PhAc, suggesting that interrupting the initial step of the
degradation pathway may improve the incorporation of PhAc into
penicillins. This prediction was confirmed with the two above
We have analyzed the "upper pathway" of PhAc catabolism
(i.e. the conversion of PhAc to homogentisate, Fig. 1) in
the filamentous fungus A. nidulans. Mutational and molecular
evidence strongly indicates that this conversion proceeds via two
sequential hydroxylating steps, the first of which is a 2-hydroxylation
of the aromatic ring catalyzed by a cytochrome P450 monoxygenase
encoded by the phacA gene. First, we have isolated a class
of mutations preventing growth on PhAc but allowing growth on
2-hydroxy-PhAc or homogentisate as sole carbon source. Second, we
describe a gene, denoted phacA, whose inactivation results
in the same phenotype as the above mutations. This The Higher eukaryotes have multiple cytochrome P450 monooxygenases
catalyzing a variety of oxidative reactions. Such abundance is not
found in the microbial world. For example, only three CYP genes are
found in the genome of S. cerevisiae (23). The marked metabolic versatility of filamentous fungi would suggest a greater variety of CYP enzymes in their proteomes. In addition to
phacA, four stc genes of the A. nidulans sterigmatocystin biosynthetic cluster encode CYP enzymes
(24). The closely related organism A. niger has a very
specific benzoate-4 hydroxylase enzyme, encoded by the bphA
gene (25). Notably, PhacA and BphA each define a new CYP family,
suggesting that metabolically versatile saprophytic fungi may represent
an as yet unexplored source of variability for CYP enzymes catalyzing
novel metabolic reactions.
Industrial penicillin production by Penicillium chrysogenum
strains requires the addition of PhAc, which is the side-chain precursor for the synthesis of penicillin G. Part of the added PhAc is
oxidized (26), and it is not transformed into penicillin. Therefore,
strain improvement programs using mutation and selection techniques
have been directed to prevent such oxidation (27). Engineered
expression in P. chrysogenum of a bacterial phenylacetyl-CoA ligase leading to increased levels of phenylacetyl-CoA available for
penicillin biosynthesis resulted in penicillin overproduction (28). We
show here that targeted disruption of the gene mediating the first step
of A. nidulans PhAc catabolism results in a 3- to 5-fold
increase in penicillin production and makes the recombinant strain less
dependent on the external supply of PhAc. This presumably results from
increased availability of phenylacetate for penicillin G biosynthesis.
A P. chrysogenum phacA homologue has been identified. Therefore, these results pave the way for the improvement of P. chrysogenum industrial strains using a similar methodology.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EMBL4 library. Cross-hybridizing sequences were mapped to
two contiguous BamHI fragments, 2.4 kb and 1.9 kb long (a
BamHI site was shown to split the phacA open
reading frame). Nucleotide sequencing and comparison of genomic and
cDNA clones revealed the intron-exon organization of the gene and
showed that all seven cDNA clones were incomplete at the N-terminal
coding region, the longest of which (at the 5'-end) ended within
predicted codon 6. cDNAs including the predicted initiation codon
were obtained by direct polymerase chain reaction amplification of the
cDNA library using an internal phacA primer and
gt10-specific primers followed by a second polymerase chain reaction
reaction primed with the above internal oligonucleotide and a second
oligonucleotide ending 1 nucleotide upstream of the ATG codon. The
presence of the ATG codon in the resulting cDNA product (and the
absence of introns between codons 1 and 6) was confirmed by sequencing.
phacA and phacA strains were
pregrown in glucose minimal medium, washed, transferred to 10 mM PhAc minimal medium, and incubated for an additional
4 h at 37 °C to induce transcription of genes for PhAc
catabolism (13). Cells were collected by filtration, washed,
resuspended in 100 mM potassium phosphate buffer, pH 7.0, and disrupted with a glass bead beater (Braun, 0.5-mm glass beads) at
4 °C. Crude extracts were clarified after centrifugation at
22,000 × g for 15 min. Microsomal pellets were
recovered after centrifugation at 100,000 × g for
1 h and resuspended in 100 mM potassium phosphate
buffer, pH 7.0. These extracts contained 1-4 mg/ml protein. Enzyme
activities of the microsomal extract were determined using standard
procedures (15, 16) with minor modifications. NADPH-cytochrome P450
reductase was assayed in 1-ml reactions at 25 °C with 0.1 M phosphate buffer, pH 7.0, following the
NADPH-dependent reduction of cytochrome c (0.05 mM initial concentration;
550 = 21 mM
1 cm
1) or ferrycyanide (0.5 mM initial concentration;
420 = 1.02) by the
decrease of absorbance at 550 nm and 420 nm, respectively.
phacA or the
phacA+ strain in penicillin production broth
(18) with 2% lactose as the main carbon source, 2.5% (w/v) corn steep
liquor, and the indicated concentrations of sodium phenylacetate.
Flasks were shaken at 250 rpm at 37 °C. Samples were taken at
different time points and used to measure penicillin using a bioassay
with Micrococcus luteus, with penicillin G as standard
(18).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The A. nidulans
phenylacetate degradation pathway. Shown are the steps
required for PhAc degradation to Krebs cycle intermediates. Enzymes in
the upper pathway are specific for PhAc. Enzymes in the lower pathway
(gene names italized) are common to PhAc and Phe/Tyr catabolism
(indicated with horizontal arrows). Phe and Tyr are also
degraded through homogentisate.
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Fig. 2.
Northern analysis of phacA
transcript levels. Cells were grown on minimal medium with
0.3% (w/v) glucose as sole carbon source for 16 h at 37 °C and
transferred to media with the indicated carbon sources (glucose at 1%
(w/v), all aromatic compounds, and glutamate at 10 mM and
potassium acetate at 30 mM; carbon indicates
no carbon source added). These secondary cultures were incubated for a
further 1 h at 37 °C. Mycelia were then harvested and used to
isolate RNA (18). The probe was a 1.2 kb phacA cDNA
clone (4FG4). Actin transcript was used as loading control.
phacA) was recessive in diploids (in
agreement with its predicted loss-of-function phenotype) and did not
complement phac-4, a prototype of classical class I
mutations that, in common with
phacA, leads to inability to use PhAc but allows growth on 2-hydroxy-PhAc. phac-4 is
therefore a phacA allele that we renamed
phacA4.
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Fig. 3.
Disruption of phacA.
The phacA gene was replaced by a mutant version after
transformation with a linear DNA fragment in which phacA
codons 298-392 had been replaced by a 3.2-kbp DNA fragment containing
the argB+ gene (see "Experimental
Procedures"). This mutant allele encodes a PhacA protein truncated
after residue 297.Two transformants (denoted phacA #3 and
#4) carrying the expected disruption-deletion mutation were purified
and tested for growth on minimal medium with 0.05% (w/v) lactose, 10 mM PhAc, or 10 mM 2-hydroxy-PhAc as sole carbon
source, as indicated. A wild type strain and a strain carrying a null
(
hmgA) mutation in the homogentisate dioxygenase gene
(see Fig. 1) were used as controls. Plates were incubated for 4 days at
37 °C before being photographed.
phacA Microsomes Are Deficient in P450 PhAc 2-Hydroxylating
Activity--
Most eukaryotic cytochromes P450 are microsomal enzymes.
Electrons are transferred to their catalytic heme center from
(NADPH)-cytochrome P450 reductase, a microsomal enzyme containing FAD
and FMN (see Fig. 4A). We
prepared microsomal fractions from wild type and
phacA
cells induced with PhAc, which showed similar (NADPH)-cytochrome P450
reductase activity, as assayed by the NADPH-dependent
reduction of artificial electron acceptors such as ferricyanide or
cytochrome c (20) (Fig. 4A). The corresponding
phacA+ and
phacA soluble fractions
showed similar, high levels of homogentisate dioxygenase (a soluble
enzyme of the PhAc degradation pathway, data not shown), indicating the
equivalent induction of PhAc catabolism in both strains.
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Fig. 4.
phacA disruption results in a marked
reduction of microsomal PhAc 2-hydroxylase activity. A,
a cytochrome P450 monooxygenase catalyzes the incorporation of one of
the atoms from dioxygen (as an hydroxyl group) into the aromatic ring
of PhAc. The source of electrons for such reaction is a NAPH-cytochrome
P450 oxidoreductase, an enzyme that transfers electrons from NADPH
through two flavin redox centers (20). In vitro, the
activity of the reductase can be monitored by using artificial electron
acceptors such as ferricyanide and cytochrome c, as
indicated. Shown below are PhAc 2-hydroxylase and NAPH-cytochrome P450
oxidoreductase activities in the microsomal fractions of
phacA and phacA+ mycelia.
Formation of 2-hydroxy-PhAc was monitored with a chemical method (17).
B, HPLC analysis of PhAc 2-hydroxylase in the above
microsomal fractions. The positions of standards were indicated by
roman numbers as follows: I, 2,5-dihydroxy-PhAc;
II, 3,4-dihydroxy-PhAc; III, 4-hydroxy-PhAc;
IV, 3-hydroxy-PhAc; V, 2-hydroxy-PhAc;
VI, PhAc. The retention time for authentic 2-hydroxy-PhAc
was 11.82 min, whereas the product formed in the complete
phacA+ and
phacA reactions showed
retention times of 11.81 and 11.86 min, respectively.
phacA mutation (Fig. 4, A and
B). Finally, wild type mycelia pregrown in glucose and
transferred to media containing PhAc secreted 2-hydroxy-PhAc to the
culture supernatant (Fig. 5). In
agreement with the above in vitro assays, secretion of
2-hydroxy-PhAc was also markedly reduced, but not abolished, by the
phacA mutation (Fig. 5). All these data, together with
the growth characteristics of the
phacA strain (see
above), strongly support the conclusion that phacA encodes a
phenylacetate 2-hydroxylase and that a second, minor enzyme showing
this activity is present in A. nidulans microsomes (see
"Discussion").
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Fig. 5.
Reduced secretion of 2-hydroxyphenylacetate
in a phacA strain. Culture supernatants of
phacA+ (circles) and
phacA (triangles) strains were assayed for the
presence of 2-hydroxy-PhAc. Mycelia were pregrown in glucose minimal
medium and transferred to PhAc. Samples were taken at the indicated
time-points after the transfer.
phacA strains, which in independent experiments
reproducibly showed a marked elevation in penicillin production over
the wild type. Fig. 6 shows one such
experiment, illustrating how the increase is already evident in
cultures supplemented with 0.125% (w/v) PhAc (1.8 µg/ml in the wild
type, 5.2 µg/ml in the
phacA strain). Moreover,
reduction of PhAc to 0.0625% (w/v) resulted in nearly a 40% decrease
in wild type penicillin production but had no decreasing effect in
mutant strain production (Fig. 6; for simplicity, results with only one
of the two disrupted strains are shown; see the legend). Under such
conditions, the
phacA mutation resulted in a 4.9-fold
increase in penicillin production.
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Fig. 6.
Increased penicillin production resulting
from phacA disruption. Time course of penicillin
production in a phacA (denoted here as
phacA::argB) and a phacA+ strain.
Media contained the indicated PhAc concentrations. PhAc is used as
side-chain precursor for penicillin G biosynthesis (see text). Shown
are data for transformant #3. Data for transformant #4 (not shown) were
nearly indistinguishable in this and other penicillin production
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phacA
mutation does not complement with a prototypical classical mutation of
the above class, indicating that both affect the same gene. Third,
phacA encodes a CYP (cytochrome P450 monooxygenase). CYP
enzymes are usually involved in a variety of biosynthetic and catabolic
hydroxylating reactions (5, 6), in agreement with the predicted PhacA
function. Fourth, phacA+ microsomal extracts
catalyze the PhAc- and NADPH-dependent synthesis of
2-hydroxy-PhAc. By contrast, a mutant
phacA microsomal
fraction is markedly deficient in this reaction. An A. niger
microsomal, NADPH-dependent phenylacetate-2-monooxygenase
activity previously reported by others (17) is possibly encoded by a
phacA homologue. In agreement with our Northern analysis,
such activity was detected in phenylacetate-grown cells and absent from
glucose-grown cells (17).
phacA mutation constructed here is almost certainly a
null mutation, which strongly suggested that the residual PhAc 2-hydroxylase activity that we detected with
phacA
microsomal extracts is encoded by a different gene. We have identified
the gene (denoted pshA) encoding this minor
activity.3 In agreement with
our prediction, a double
phacA
pshA
mutation abolished microsomal PhAc 2-hydroxylation.3 A
pshA strain grows on PhAc but does not grow on
3-hydroxy-PhAc (which in the wild type is also catabolized through
homogentisate), indicating that pshA encodes a
3-hydroxy-PhAc 6-hydroxylase (i.e. and
ortho-hydroxylase), converting 3-hydroxy-PhAc to
2,5-dihydroxy-PhAc. This enzyme has been previously described in the
fungus Trichosporum cutaneum, which converts PhAc to
homogentisate through sequential hydroxylation of positions 3 and 6 of
the ring (22).
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ACKNOWLEDGEMENTS |
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We thank E. Reoyo for technical assistance, Brian Nowak-Thompson, Eduardo Díez, and José Luis García for critical reading of the manuscript, Beatriz Galán and Auxi Prieto for their advice with HPLC analysis, and David R. Nelson for his assignment of an standarized cytochrome P450 designation to PhacA.
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FOOTNOTES |
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* This work was supported by Spanish Comisión Interministerial de Ciencia y Tecnología Grants BIO94-932 and BIO97-348 and Antibióticos S. A. U.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ132442.
To whom correspondence should be addressed. Fax: (34 91) 5627518;
E-mail: cibp173{at}fresno.csic.es.
§ Present address: Dept. of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., L103 Portland, OR 97201-3098.
2 David R. Nelson, personal communication.
3 J. M. Mingot, M. A. Peñalva, and J. M. Fernández-Cañón, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PhAc, phenylacetate; CYP, cytochrome P450; kbp, kilobase pair(s); HPLC, high performance liquid chromatography.
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