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J Biol Chem, Vol. 274, Issue 41, 29228-29241, October 8, 1999
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From the Departamento de Bioquímica y Biología
Molecular, Facultades de Biología y Veterinaria, Campus de
Vegazana s/n, Universidad de León, 24007 León,
España, ¶ Instituto Biomar, 24231 Onzonilla, León,
España, and Departamento de Microbiología
Molecular, Centro de Investigaciones Biológicas, Consejo Superior
de Investigaciones Científicas,
28006 Madrid, España
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Novel biodegradable bacterial plastics, made up
of units of 3-hydroxy-n-phenylalkanoic acids, are
accumulated intracellularly by Pseudomonas putida U due to
the existence in this bacterium of (i) an acyl-CoA synthetase (encoded
by the fadD gene) that activates the aryl-precursors; (ii)
a Polyhydroxyalkanoates
(PHAs)1 are naturally
occurring polyesters synthesized by different bacteria when cultured
under a wide variety of nutritional and environmental conditions
(1-8). Some species accumulate these polymers only when an essential
nutrient becomes limiting, whereas others store these compounds from
the very early exponential phase of growth (9-11). All PHAs known to
date are accumulated intracellularly as reserve materials or as a sink
for reducing equivalents (2), and they start to be mobilized when the
carbon or energy source is exhausted from the culture broths (4, 8,
12). Regarding the monomer content, the structure of PHAs is quite
variable (more than 80 molecules have been reported as constituents of
PHAs), and their composition depends on the bacterial strain and growth
conditions (12). Although PHAs are commonly stored as heteropolymers or
copolymers (1-8, 13), in some cases only homopolymers
(poly-3-hydroxybutyric, poly-3-hydroxyhexanoic, and 3-hydroxyheptanoic
acid) are accumulated (13-14). Generally speaking, most PHAs reported
to date can be included in two different groups: those containing units
of 3-hydroxybutyrate or close derivatives (short chain length
hydroxyfatty acids) (called PHBs) (4, 15-18) and those consisting of
medium chain length hydroxyfatty acids
(C5-C12) or related compounds (2-4, 9, 19-28). The general structure of both species of polymers, is as shown
in Scheme 1.
R O
-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a
polymerization-depolymerization system (encoded in the pha
locus) integrated by two polymerases (PhaC1 and PhaC2) and a
depolymerase (PhaZ). The complete assimilation of these compounds
requires two additional routes that specifically catabolize the
phenylacetyl-CoA or the benzoyl-CoA generated from these polyesters
through -oxidation. Genetic studies have allowed the cloning,
sequencing, and disruption of the genes included in the pha
locus (phaC1, phaC2, and phaZ) as
well as those related to the biosynthesis of precursors
(fadD) or to the catabolism of their derivatives
(acuA, fadA, and paa genes).
Additional experiments showed that the blockade of either
fadD or phaC1 hindered the synthesis and
accumulation of plastic polymers. Disruption of phaC2
reduced the quantity of stored polymers by two-thirds. The blockade of
phaZ hampered the mobilization of the polymer and decreased
its production. Mutations in the paa genes, encoding the
phenylacetic acid catabolic enzymes, did not affect the synthesis or
catabolism of polymers containing either 3-hydroxyaliphatic acids or
3-hydroxy-n-phenylalkanoic acids with an odd number of carbon atoms as monomers, whereas the production of polyesters containing units of 3-hydroxy-n-phenylalkanoic acids with
an even number of carbon atoms was greatly reduced in these bacteria. Yield-improving studies revealed that mutants defective in the glyoxylic acid cycle (isocitrate lyase
) or in the
-oxidation pathway (fadA), stored a higher amount of
plastic polymers (1.4- and 2-fold, respectively), suggesting that
genetic manipulation of these pathways could be useful for isolating
overproducer strains. The analysis of the organization and function of
the pha locus and its relationship with the
core of the phenylacetyl-CoA catabolon is reported and discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Here R represents an aliphatic chain ranging between 1 and 9 carbon atoms.
In recent years, physicochemical studies on PHAs (mechanical properties and general characteristics) have received considerable attention due to their biotechnological applications and industrial relevance. In this sense, these polyesters are used as biodegradable and as biocompatible thermoplastic materials (29-31). Furthermore, the interest in their occurrence in nature (1-8), their physiological role (32-33), and their molecular organization and polymer architecture (34-39) has led to the accumulation of an impressive body of knowledge about this topic (2-7). As a consequence, genetic, biochemical, and engineering approaches have focused on reconstituting in vitro the main enzymatic steps involved in PHA biosynthesis and/or degradation (14, 40-45) as well as on expressing the biosynthetic genes in different prokaryotic or eukaryotic systems (2, 7, 14, 43-48).
Although dozens of different bacterial PHAs have been reported in the
last decade (1-6), to the best of our knowledge, only two species,
Pseudomonas oleovorans (49-51) and Pseudomonas
putida BM01 (52), synthesize plastic polyesters containing an
aromatic ring in the monomer. These unusual polymers,
poly-(3-hydroxyphenoxyalkanoates), were accumulated when this strain
was cultured in a chemically defined medium containing
-phenoxyalkanoates as carbon sources (50). However, when other
structurally related compounds (n-phenylalkanoates, PhAs)
were tested as polymer precursors, it was observed that P. oleovorans was able to synthesize only a homopolymer of
3-hydroxy-5-phenylpentanoic acid when grown in the presence of
5-phenylpentanoic acid (49-51).
The restricted capacity of plastic-producing bacteria to store
polyesters bearing a phenyl group suggests that the synthesis of these
unusual compounds requires (i) a specific uptake system for the
transport of the aromatic precursors, (ii) a specific acyl-CoA
synthetase (ACS), (iii) a specific polymerase or a mutated enzyme with
broader substrate specificity (14), and/or (iv) the existence of an
additional catabolic route, linked to the -oxidation pathway, to
ensure complete assimilation of the -oxidation products (benzoyl-CoA
or phenylacetyl-CoA) generated from the monomers
(3-hydroxyphenylalkanoyl-CoA derivatives) once released from the stored
polymer (Fig. 1) (9). This complete assimilation could reduce the
possibility that intermediate metabolites might inhibit the
biosynthetic process.
In the present work, we describe the existence of novel natural
aromatic plastics and analyze their structure for the first time.
Genetic and biochemical studies were also performed in order to
establish the sequence and characteristics of the genes and enzymes
specifically involved in the synthesis and degradation of these
polyesters (PHPhAs). The inclusion of this pathway in the
phenylacetyl-CoA catabolon (53) and its influence in the evolution of
the catabolic potential of P. putida U are also discussed.
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EXPERIMENTAL PROCEDURES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Materials-- n-Phenylalkanoic acids, n-alkanoic acids, and [1-14C]phenylacetic acid were supplied by Lancaster Synthesis or by Sigma. [1-14C]Octanoic acid was from American Radiolabeled Chemicals. All other products were of analytical quality or HPLC grade.
Microorganisms and Culture Conditions-- The strain of P. putida (U) (Colección Española de Cultivos Tipo 4848) used in all of the experiments was from our collection. It was maintained on Trypticase Soy Agar (Difco), and growth slants (8 h at 30 °C) were used to inoculate liquid medium. Each 2000-ml Erlenmeyer flask containing 500 ml of the required medium was inoculated with 10 ml of a bacterial suspension (1010 bacteria). Incubations were carried out in a rotary shaker (250 rpm) at 30 °C for the time required in each set of experiments. The medium (MM) used for the growth of P. putida U and its mutants was a chemically defined one (54). When required, the carbon source (phenylacetic acid, PhAc) was replaced by a different one (several aromatic analogues or fatty acids). The concentration of the molecule used as carbon source was indicated in each set of experiments. The synthesis of plastic polymers was studied in bacteria grown for different times in a MM (54) in which PhAc had been replaced by several n-alkanoic (As) or PhAs at the required concentrations (between 5 and 15 mM). In some experiments, the carbon source was 4-hydroxyphenylacetic acid (4-HPhAc). This compound, which is efficiently assimilated by P. putida U, cannot be used as plastic precursor by this bacterium.
Escherichia coli HB101 containing the plasmid pGS9, which
includes the transposon Tn5, was kindly supplied by J. L. Ramos (Estación Experimental del Zaidín, Consejo
Superior de Investigaciones Científicas, Granada,
España). E. coli XL1-Blue (Stratagene) was supplied by
the commercial firm, and it was used for overexpressing different
proteins. E. coli fadR (a strain defective in the
-oxidation transcriptional repressor) and fadB (mutated
in the gene encoding the enoyl-CoA hydratase and the 3-hydroxyacyl-CoA
dehydrogenase) mutants, which were used to study the functional
expression of phaC1, phaC2, or the complete
pha locus
(phaC1-phaZ-phaC2) from P. putida U, were kindly supplied by C. C. DiRusso (Department of Biochemistry and Molecular Biology, Albany Medical College, Albany,
NY). A different culture of the fadB E. coli
mutant was also supplied by A. Steinbüchel and B. Rehm (Institut
für Mikrobiologie, Westfälische Wilhems-Universität
Münster, Münster, Germany). When required, a double mutant
E. coli JMU 193 (fadR, fadB) supplied by Q. Ren, Institut Für Biotechnologie, ETH, Zürich,
Switzerland) was used. Micrococcus luteus (ATCC 9341) was
used for the evaluation of acyl-CoA synthetase by bioassay (54)
Characterization of PHPhAs-- PHPhAs were isolated following the procedure reported by Lageveen et al. (9). For these experiments, cells grown in a chemically defined medium (MM) (54) containing different concentrations of n-phenylalkanoic acids as the sole carbon sources (see above) were used. The contents and composition of PHPhAs were determined by gas chromatography as previously reported (49).
NMR spectral analyses were recorded at 18 °C on a Varian Unity 300 NMR spectrometer at 300 (1H) and 75 (13C) MHz, using tetramethylsilane as internal standard. Spectra were measured in CD3OD.
Isolation of Mutants-- Mutants of P. putida unable to degrade octanoic acid or phenylacetic acid or those affected in the production of poly-(3-hydroxyphenylalkanoic) acids were selected by mutagenesis with the transposon Tn5 as reported (56). In some cases, mutants were obtained by disruption of the required gene (see below).
Mutants lacking a functional glyoxylic acid cycle or a -oxidation
pathway were characterized by enzyme assay (57), metabolically (studying their ability to grow in MM containing acetate or octanoate as the sole carbon source), and by location of the insertion. Mutants
handicapped in the biosynthesis of plastic polymers were identified by the different contrast of the colonies (translucent) when
properly cultured (8, 13).
The strains unable to assimilate phenylacetic acid (indicated as
PhAc) were classified according to the intermediate
accumulated in the culture broth (see below) or as a function of the
presence or absence of phenylacetyl-CoA ligase activity in their
cell-free extracts (58).
HPLC Equipment and Chromatographic Procedure--
To determine
the rate of utilization of the carbon sources and to identify the
catabolic intermediates accumulated by certain mutants, samples of
culture broth (50 µl) were taken at different times, centrifuged, and
filtered through a Millipore filter (pore size, 0.45 µm). Aliquots
were analyzed on a HPLC apparatus (SP8800; Spectra Physics) equipped
with a variable wavelength UV-visible detector (Waters 486), Millenium
software (Waters 2010), and a microparticulate (particle size 10 µm;
pore size 100 nm) reverse-phase column (Nucleosil C-18, 4.6 (inner
diameter) by 250 nm; Phenomenex Laboratories). The mobile phase was as
follows: A, 0.2 M KH2PO4 (pH 4.5);
B, CH3CN in a linear gradient ranging from 95% A:5% B to
50% A:50% B over 1 h. Flow rate was 1 ml min1, and
the eluate was monitored at 254 nm. Column temperature was 30 °C.
Under these conditions, the retention times of 4-HPhAc, 2-hydroxyphenylacetic acid, phenylacetic acid (PhAc), 6-phenylhexanoic acid, and 8-phenyloctanoic acid were 19, 25, 30, 45, and 56 min, respectively. HPLC analysis of other products was carried out as
reported elsewhere (56, 59-60).
DNA Manipulations and Sequencing-- DNA manipulations, gel electrophoresis, DNA sequence analysis, primer synthesis, promoter analysis, and polymerase chain reaction amplification were performed as reported (53). To analyze the function of the genes encoding the polymerases and the depolymerase involved in the biosynthesis and mobilization of the plastic polymers, each particular gene was disrupted by homologous recombination. Thus, an internal fragment (400-900 base pairs) belonging to the gene to be mutated was cloned in pK18:mob, and the resulting construct was introduced into P. putida U by triparental mating as previously reported (53). Correct insertion of the pK18:mob in the desired gene was established by polymerase chain reaction amplification (53). When required, overexpression of the different genes and proteins was carried out using the plasmid pQE-32 (Qiagen Inc.) according to the manufacturer's instructions.
[14C]Octanoic Acid Transport Studies-- The uptake of labeled octanoic acid was carried out in bacterial cultures as previously reported (60). When required, different phenylalkanoates were added to the transport system in order to analyze their effect on the uptake of octanoic acid. Unlabeled octanoic acid was analyzed by gas liquid chromatography as described elsewhere (61).
Enzymatic Assays-- Phenylacetyl-CoA ligase was measured colorimetrically or by HPLC as reported elsewhere (54, 58).
The ACS from P. putida U was assayed as indicated for other analogous enzymes (62-63). When the molecules tested as substrates were longer than C5, a different assay method (bioassay against M. luteus) was used (64).
PhaC1 and PhaC2 activities were assayed in vivo by analyzing the monomeric composition of the plastics stored by mutants of P. putida U specifically blocked in one or both enzymes. The substrate specificities of these enzymes were also studied in recombinant E. coli JMU193 containing either the phaC1 or the phaC2 genes (see "Results and Discussion").
Isocitrate lyase was measured spectrophotometrically in cell-free
extracts (54) of P. putida U as indicated previously
(65).
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Strategy Followed for Producing Novel PHAs
The collection of unusual bacterial plastics (those containing
units of PHPhAs) was approached under the assumption that bacteria able
to accumulate aliphatic plastics would also be able to synthesize aromatic polyesters whenever the substrate specificity of the polymerases is broad enough and when the effective degradation of the
final -oxidation product (phenylacetyl-CoA or benzoyl-CoA) is
produced by a catabolic route. Accordingly, we first selected bacterial
species able to (i) produce aliphatic plastics and (ii) grow in a
chemically defined medium (MM) containing either benzoic acid or
phenylacetic acid as the sole carbon source (54). These microbes would
have (i) the biosynthetic pathway necessary for aliphatic polymer
production and (ii) the catabolic enzymes required for the degradation
of the final -oxidation products: benzoyl-CoA (or benzoic acid) and
phenylacetyl-CoA (or phenylacetic acid). This second requirement seems
to be essential, since if n-phenylalkanoic acids are
catabolized, as expected, through the -oxidation pathway (Fig.
1) (66), the final product would be
benzoyl-CoA or phenylacetyl-CoA if they have an odd or an even number
of carbon atoms, respectively. Accordingly, only those microbes able to
assimilate phenylacetic acid (PhAc) or benzoic acid via a CoA
intermediate would be able to grow efficiently in MM containing
n-phenylalkanoic acids as the sole carbon source.
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Taking into account all the above considerations, we selected a bacterial strain, P. putida U, that meets the following metabolic conditions: (i) it is able to catabolize PhAc and benzoic acid aerobically (54-56), and (ii) it synthesizes different polyesters when cultured in MM containing C6-C12 alkanoic acids (60).
Ultrastructural studies revealed that P. putida U is able to synthesize biodegradable plastics when cultured in a chemically defined medium containing several PhAs whose aliphatic moiety ranges between 6 and 10 carbon atoms as carbon sources (see Table I). These compounds, which appeared as electron-transparent granules, were stored intracellularly as reserve materials (Fig. 2).
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Although certain bacterial species only accumulate PHAs when an essential nutrient (e.g. nitrogen, phosphorus, sulfur, magnesium) becomes limiting (8, 10), P. putida U synthesizes plastic polyesters from the very early logarithmic phase of growth (Fig. 2), suggesting that the regulatory mechanisms that control the biosynthesis of these polymers in both types of bacteria are quite different.
Bacterial growth and polymer contents were higher when PhAs containing
acyl chains longer than six carbon atoms were used as carbon sources
(Fig. 3 and Table I), the highest values
being detected when 10-phenyldecanoic acid was supplied to the medium. When other PhAs (containing an acyl chain of less than six carbon atoms) were tested, no polyesters were produced, since P. putida cannot assimilate these compounds or does so very poorly
(Fig. 3A).
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Structural Analysis of the PHPhAs
The general structure of these novel polyesters, established by
NMR studies,2 is shown in
Fig. 4. A 1H-13C correlation
spectrum allowed the specific
13C assignments reported, whereas the multiplicities and
connectivities in the 1H NMR spectra were established by
distortionless enhancement by polarization transfer and
1H-1H COSY experiments, respectively (67).
These analyses allowed us to conclude that the structures of the
monomers are 3-hydroxy-5-phenylvaleric acid, 3-hydroxy-6-phenylhexanoic
acid, 3-hydroxy-8-phenyloctanoic acid, and 3-hydroxy-10-phenyldecanoic
acid (see Table I). This is the first description of the existence of
natural plastics containing units of 3-hydroxyphenyl derivatives with
an even number of carbon atoms. Thus, when P. putida U was
cultured in MM containing 6-phenylhexanoic acid as carbon source, a
homopolymer of 3-hydroxy-6-phenylhexanoate was produced. However, when
the carbon source was 8-phenyloctanoic or 10-phenyldecanoic acid,
copolymers were formed. In the first case, study of the monomer
composition revealed that the polymer contained two different
molecules: 3-hydroxy-6-phenylhexanoate and 3-hydroxy-8-phenyloctanoate
at relative proportions of 62.5 and 37.5%, respectively. However, when
the carbon source was 10-phenyldecanoate, the polymer contained units
of 3-hydroxy-6-phenylhexanoate (48.2%), 3-hydroxy-8-phenyloctanoic
acid (31.2%), and 3-hydroxy-10-phenyldecanoate (20.6%), respectively
(see Table I). Interestingly, when the molecules used as carbon sources
were PhAs containing an odd number of carbon atoms, the only plastic
produced was a homopolymer of 3-hydroxyphenylvaleric acid (see Table
I). These results allowed us to conclude that the polymerases (see
below) responsible for the condensation of aromatic monomers into
plastic polymers recognize CoA derivatives of 3-hydroxyalkanoic acids
containing acyl chains longer than four carbon atoms as substrates.
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Analysis of the polymers stored by P. putida U when this microbe was cultured in MM containing different As (from butyric acid to dodecanoic acid) as carbon sources revealed that the minimum length of the monomers was six carbon atoms and that, depending on the carbon source used for growth, different polymers (or copolymers) were produced (see Table II). In sum, when C2n+2PhAs or C2n+2As are used as polymer precursors, the plastics synthesized contain monomers that have the same length in the acyl moiety, regardless of whether or not they are linked to a benzene ring. However, when the precursors are molecules with an odd number of carbon atoms (aromatics or aliphatics) the monomer length is not the same. Thus, when 6-phenylhexanoic, 8-phenyloctanoic, or 10-phenyldecanoic acids were used as carbon sources, the monomeric units of these polymers were the same as those obtained when hexanoic, octanoic, and decanoic acid were employed but linked to a benzene ring (see Tables I and II). However, when 7-phenylheptanoic or 9-phenylnonanoic acids were supplied to the medium, polymers containing 3-hydroxy-7-phenylheptanoic acid or 3-hydroxy-9-phenylnonanoic acids were not found, a homopolymer of poly-3-hydroxy-5-phenylvaleric acid being the only polyester synthesized. Although the explanation for the absence of polymers containing units of 3-hydroxyphenylheptanoic or 3-hydroxyphenylnonanoic acids is unknown, it could be speculated that the physicochemical differences between structurally related substrates (containing an odd or an even number of carbon atoms) might be the cause of this structural difference. Moreover, we have never observed monomers with a size longer than the fatty acid precursor, indicating that in P. putida U the precursors of these polyesters are not obtained through fatty acid synthesis.
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Analysis of PHPhAs Biosynthetic and Catabolic Enzymes
Although certain bacteria such as Alcaligenes eutrophus
or P. oleovorans accumulated high amounts of plastic
polymers containing 3-hydroxy-n-alkanoic acids as monomers
(2, 8, 50-51), to the best of our knowledge they do not produce
polyesters when n-phenylalkanoic acids containing an even
number of carbon atoms are used as plastic precursors. It could
therefore be speculated that the ability of P. putida U to
synthesize these aromatic polyesters could be due to the existence in
this bacterium of (i) a transport system with broader substrate
specificity that could recognize both kinds of polymer precursors (or
two different ones, specifically involved in the uptake of
phenylalkanoic acids and alkanoic acids, respectively), (ii) two (or
more) acyl-CoA synthetases that activate PhAs and As separately or a
single enzyme with broader substrate specificity that would activate
both kinds of precursors, (iii) a different polymerizing-depolymerizing
system, (iv) two different -oxidation pathways (or only one but
including some duplicate, less specific or channeling steps) required
for the accumulation and partial catabolism of both kinds of plastic
polymers, and/or (v) specific routes that ensure the complete
catabolism of the final -oxidation products (acetyl-CoA,
propionyl-CoA, phenylacetyl-CoA, or benzoyl-CoA). To establish which of
these possibilities were true, the following experiments were performed.
Transport System-- When the uptake of [14C]octanoic acid was studied (60), we observed that 5-phenylpentanoic acid, 6-phenylhexanoic acid, 7-phenylheptanoic acid, and 8-phenyloctanoic acid strongly inhibited the uptake of octanoic acid (70, 85, 98, and 97%, respectively) when added at the same concentration (154 µM), whereas other related compounds with a lower molecular weight such as 3-phenylpropionic acid and 4-phenylbutyric acid did not cause any significant effect (zero inhibition and 10%, respectively). Furthermore, an efficient transport of octanoic acid (98%) was observed when P. putida U was cultured in MM containing phenyloctanoic acid. We therefore suggest that the uptake of n-phenylalkanoic and n-alkanoic acids are at least tightly linked and very probably brought about through the same mechanism. Although a definitive conclusion could only be obtained after analyzing the uptake of radioactive n-phenylalkanoic acids, the lack of commercially available labeled compounds and the difficulty involved in synthesizing them did not allow further and more accurate analyses of this particular point. However, taking into account that a blockade in acyl-CoA synthetase (which activates medium chain-length fatty acids; see below) also handicapped the cellular incorporation of octanoic acid and other polyester precursors (as shown after HPLC analysis of the culture broths), it seems reasonable to propose that the same transport system could be involved in the uptake of all of these compounds (Fig. 1). In fact, it has been suggested (68-70) that the acyl-CoA synthetase from E. coli activates fatty acids concomitant with transport, and it has been proposed that this enzyme would either be a component of the transport apparatus involved in the uptake of fatty acid or that it would interact, inside the inner membrane, with a hypothetical fatty acid cotransporter. More recently, several authors (71-75) have provided evidence indicating that the uptake of long chain fatty acid in E. coli requires the direct participation of an outer membrane protein and acyl-CoA synthetase.
Acyl-CoA-activating Enzyme--
The analysis of the
acyl-CoA-activating enzyme(s) was carried out. It could be speculated
that two different enzymes (or more) would be involved in the
activation of alkanoic and phenylalkanoic acids and that, therefore,
the absence of this enzyme that activates phenylalkanoic acids would
explain why certain bacteria do not synthesize aromatic polyesters. To
clarify this point, we analyzed different mutants unable to grow in
chemically defined media (MM) containing 6-, 7-, 8-, or
9-phenylalkanoic acids (or the equivalent alkanoic acids) as carbon
sources. These mutants, which were selected by random insertion of the
transposon Tn5 (76), were separated into two different
groups: those unable to catabolize octanoate and those that do not grow
in MM containing acetate as the sole carbon source. The first class of
mutants corresponded to bacteria in which the Tn5 had been
inserted into some of the genes (or their regulatory elements) encoding
a general acyl-CoA-activating enzyme or some of the proteins belonging
to the -oxidation pathway (66). In the second group, we included
bacteria in which the transposon has altered the expression of a gene
encoding an acetyl-CoA-activating enzyme (acetyl-CoA synthetase or
acetate kinase) or some of the proteins involved in the glyoxylic acid
cycle (65). Among the strains included in the first group we identified
a mutant (Oct1 ACS,
henceforth called ACS) in which Tn5 insertion
occurred in a gene encoding a protein that shows strong homology
(accession number AF150669) with the long chain fatty acid ACSs from
E. coli and Pseudomonas fragi (77-79). This gene
(fadD) was cloned, sequenced, and expressed in E. coli DH5
'.
The ACS mutant efficiently catabolized acetate and
butyrate but was unable to assimilate n-alkanoic or
n-phenylalkanoic acids with an acyl moiety ranging between
C5 and C10, undoubtedly indicating that a
single enzyme is responsible for the activation of all of these
compounds and that, as expected, this reaction is not catalyzed by
acetyl-CoA synthetase or by the acetate kinase (EC 6.2.1.1 and EC
2.7.2.1, respectively). Furthermore, study of the polyesters stored
revealed that this mutant does not accumulate plastic polymers when
cultured in the appropriate conditions (MM plus 4-HPhAc plus PhAs or As
as carbon sources) (Fig. 2 and Table III). Additionally, we observed that the
ACS mutant was unable to transport
[14C]octanoic acid (0.3% of the final amount transported
by the wild type), suggesting that, as mentioned above, the mechanism
involved in the uptake of this acid and the activation to its CoA
thioester comprises a single event or at least two closely related
events (Fig. 1). Similar results were obtained when the transport of [14C]phenylacetic acid was analyzed in mutants in which
the gene encoding phenylacetyl-CoA ligase (EC 6.2.1.30), the first
enzyme of the specific route involved in the catabolism of phenylacetic acid (56), had been disrupted. We observed that the P. putida U mutants lacking this enzymatic activity were unable to
take up phenylacetic acid from the
broths.3 Thus, it can be
concluded that in the absence of this single acyl-CoA synthetase
activity the uptake of all of the substrates recognized by this enzyme,
which are the precursors of the monomers used for the synthesis of
polyesters, will be blocked, hence avoiding an unnecessary loss of
energy, since once transported these compounds cannot be catabolized.
As a consequence of this blockade, polyesters cannot be synthesized. In
light of the above results, it seems that this single acyl-CoA
synthetase is responsible for activation of alkanoates and
phenylalkanoates.
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On the other hand, it is interesting to draw attention to the lack of
plastic storage in the ACS mutant when grown in MM plus
acetate plus citrate (Fig. 2), since this result indicated that
P. putida U, unlike other related bacteria (48), does not
synthesize polyesters (or does so very poorly) via fatty acid
biosynthesis. This assumption is supported by the fact that when this
mutant or the wild type was cultured in minimal media containing either
glucose or gluconate as carbon source, synthesis of plastic polymer was
not detected (data not shown).
The Polymerizing-Depolymerizing Enzymatic System--
We have
analyzed the sequence and organization of the genes and proteins
involved in the polymerization and depolymerization of these polyesters
in P. putida U. For these experiments, mutants blocked in
the genes involved in the synthesis of poly-3-hydroxyphenylalkanoates were isolated (13). Sequence analysis of the DNA fragment in which the
transposon Tn5 had been inserted revealed that (i) in P. putida U, three genes (phaC1, phaZ,
and phaC2 included in the pha locus; see Fig.
5) are involved in the
synthesis and mobilization of these polymers, (ii) the sequences of
these proteins are similar to the enzymes reported in P. oleovorans and in Pseudomonas aeruginosa (13, 21), and
(iii) they also have a similar organization (PhaC1-PhaZ-PhaC2) to that
reported in such bacteria (see Fig. 6).
The contribution of each particular enzyme in the biosynthesis and
catabolism of these polymers was analyzed by studying different mutants
in which specific disruptions (by homologous recombination; Ref. 53) of
the genes encoding PhaC1, PhaC2, and PhaZ (see Fig. 5) had been done.
We revealed for the first time in Pseudomonads of rRNA homology group I
that a blockade on the phaC1 prevented accumulation of
plastic polymers, whereas a mutation in the phaC2 reduced
the intracellular amount of polymers by two-thirds. Furthermore, disruption of the gene encoding PhaZ prevented the mobilization of the
polymer accumulated intracellularly and decreased the total PHAs
content, suggesting that PhaC2 is not synthesized, since it must be
expressed from a promoter located upstream the gene encoding the
depolymerase (see Table IV).
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An analysis based on the amounts of polymer stored by the different
mutants allowed us to obtain interesting conclusions about the genetic
organization of the pha locus. Thus, when P. putida U (wild type) was cultured in MM plus octanoic acid until
the stationary phase of growth, the amount of PHAs extracted was 365 mg/g of dry weight cells (dwc). This quantity represents a balance of
the biosynthetic activities of PhaC1 and PhaC2 (additive) and the
mobilizing activity corresponding to PhaZ (subtractive). This balance
could be represented as follows: PhaC1 + PhaC2 
PhaZ = 365. When PhaC2 was mutated, the quantity of plastic synthesized (PhaC1 PhaZ) was reduced to 115 mg/dwc; therefore, we conclude that the
contribution of PhaC2 to the synthesis of plastic should be about 250 mg/dwc. However, when a PhaZ mutant was cultured in the same
conditions, we obtained only 250 mg/dcw. This later quantity suggests
that in this mutant only the PhaC1 is working, since otherwise total
synthesis should have been greater. Although these data suggest that
there is not an active promoter downstream from the phaZ
gene, we cannot rule out the possibility that a promoter could exist
between the phaC1 and the phaZ genes. Moreover, we observed that mutants in which the phaC1 gene was
disrupted did not synthesize plastics at all (Table IV), which should
not have happened if there had been a promoter downstream from the phaC1 gene. It is worth noting that although the
depolymerase activity was lacking in the PhaZ mutant (and therefore
mobilization of the polymer did not occur; see Table IV), the quantity
of polymer accumulated (250 mg/dwc) was considerably lower than that
found in the control (365 mg/dwc), suggesting that the polymerases and probably all three enzymes (PhaC1, PhaC2, and PhaZ) must be organized in a complex to function efficiently.
Genetic studies revealed that whereas the sequences of the genes included in the pha locus are well conserved, further differences were found when the promoter and the two intergenic regions reported in the pha loci of P. oleovorans, P. aeruginosa, and P. putida U were compared (Fig. 6). Thus, in P. putida U the lengths of the intergenic regions are shorter than in other species and lack the inverted repeat sequences found in P. oleovorans and in P. aeruginosa (13, 21). This genetic organization suggested that in P. putida U the three pha genes are organized in an operon and that translational coupling may exist.
On the other hand, the study of the putative promoter regions in the three bacterial species revealed that the sequence of P. aeruginosa is quite different from the other two species (Fig. 6A). This variation could account for a different regulation of the three pha loci. When genetic studies, using a plasmid specifically designed for testing promoters (53), were performed, we did not find any functional promoter sequences within the fragment of DNA containing the structural sequences (PhaC1, PhaZ, PhaC2) from the ATG of phaC1 to the TGA of phaC2, whereas a promoter sequence was found in a DNA fragment of 200 base pairs located upstream from the ATG of phaC1.
All of these results allow us to conclude the following: (i) only one promoter (located 5'-upstream from phaC1) exists in the pha locus; (ii) the three genes are expressed under the control of this promoter; (iii) the two polymerases (PhaC1 and PhaC2) have fairly similar polymerizing rates; (iv) the activity of PhaZ released 26% of the total polymer synthesized; (v) the same enzymatic system (PhaC1-PhaZ-PhaC2) is involved in the synthesis and mobilization of all of the plastic polymers (aliphatic and aromatic) found in P. putida U; and (vii) the lack of depolymerase does not imply a greater accumulation of polymers but rather the impossibility of mobilizing them (see Table IV and Fig. 2).
Next, the general specificity of both polymerases was analyzed
independently by studying the structure of the polymers synthesized and
stored in the different mutants reported above. We observed that the
polyesters accumulated by the wild-type strain and by the PhaZ and by
the PhaC2 mutants were similar in monomer composition and in the
percentage of monomers present in the polymer, indicating that the two
polymerases recognize the same substrates with similar efficiency (see
Tables IV and V). We attempted to confirm
the substrate specificity of these enzymes by cloning phaC1
and phaC2 separately in a E. coli fadB mutant
handicapped in fatty acid -oxidation (75), as reported for the PHA
synthetases from P. aeruginosa (48, 80). Unfortunately, we
failed to observe the synthesis of plastics in this strain even when
the complete pha locus was cloned. It could be that the
production of plastic polymers would require the existence of a
-oxidation constitutive pathway, so we cloned each independent gene
(phaC1 or phaC2) as well as the entire
pha locus in the E. coli fadR mutant (a strain
defective in the -oxidation transcriptional repressor but which is
sensitive to carbon catabolite repression) (74, 75), which were then cultured in minimal media containing different PHA or PHPhA precursors as the sole carbon source. Ultrastructural and chemical analysis revealed that plastic polymers did not accumulate in these recombinant strains. Furthermore, when these bacteria were cultured in media containing different fatty acids, a cellular size lower than the wild
types (about one-fifth) was observed, suggesting that, under these
conditions, overexpression of the polymerases causes cellular damage in
the recipient strain. Accordingly, taking into account that when
studies on overexpression of PhaC1 in E. coli DH5' were
performed, an abnormal division, due to the absence of septum formation
(see Fig. 7), was observed, we used
E. coli JMU193 for cloning the pha biosynthetic
genes in a monocopy construction, under the control of a inducible
promoter, as reported by other authors (55, 81-83). With these
experiments, we attempted simulate the genetic organization (monocopy
of each different gene and strict control of their expression) required
for the synthesis of these plastics in the wild type (P. putida U), thus avoiding excess synthesis of the polymerase(s)
that could cause harmful effects on the recombinant strain. We observed
that when these genetically engineered strains were cultured in minimal
medium containing glycerol (20 mM) and palmitic acid (3 mM) as carbon sources, the limited expression of the
polymerases led to lower synthesis of aliphatic plastics (10% of
bacterial dry weight). However, synthesis of aromatic polymers was not
observed when palmitic acid was replaced by 8-phenyloctanoic acid.
These data suggest that the low yield of synthesis could be due to the
fact that either even the low expression of these genes is harmful for
the recipient bacteria or that additional proteins (probably an
R-specific enoyl-CoA hydratase or the granule-associated proteins) would be required for a more efficient synthesis of such polymers (7,
45, 47, 84). Although the quantity of the aliphatic polymers
accumulated was very low, their analysis revealed that, as indicated
above, PhaC1 and PhaC2 showed identical substrate specificity.
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Yield Improvement Studies
Since the overexpression of the pha locus from P. putida U in E. coli does not lead to a higher
production of PHAs (or PHPhAs) in the recombinant strains, certain
biochemical studies were also performed in order to improve the
production of plastic polymers in P. putida U. It is well
established that the degradation of n-alkanoic acids and
phenylalkanoic acids through -oxidation generates several units of
acetyl-CoA (66) that are later catabolized by the glyoxylic cycle (65).
It could therefore be expected that the introduction of a mutation in
some of the genes encoding enzymes belonging to the glyoxylic acid
cycle could slow down the -oxidation route (the source of
acetyl-CoA) and hence cause the accumulation of some catabolic
intermediates (polyester precursors), thus channeling them to the
polymeric system and increasing the amount of stored polymers. To check
this hypothesis, we isolated a mutant in which the transposon
Tn5 (76) had been inserted into a gene belonging to the
glyoxylic acid cycle (in the aceA gene encoding the
isocitrate lyase gene, accession number AF150671). This mutant, which
shows an Oct Ac phenotype, was unable to
grow in MM containing either acetate or n-alkanoic acids
whose carbon length was an even number of carbon atoms, whereas it grew
well in MM containing either several n-alkanoic acids with
an odd number of carbon atoms or n-phenylalkanoic acids with
an even or odd number. These results indicate that unlike the
catabolism of alkanoic acids containing an even number of carbon atoms,
those other compounds do not require the glyoxylic acid cycle to be
assimilated (see below). Moreover, when this isocitrate
lyase mutant was cultured in MM plus citrate plus acetate
plus octanoate (or 8-phenyloctanoate), a higher percentage of PHA (or
PHPhA) content was observed (see Table
VI). To explain this result, we assume
that although some octanoic acid is used as the carbon source and
catabolized through -oxidation, the presence of citrate and acetate
slows down the metabolic flux of this catabolic pathway, thus
channeling the intermediates (3-OH-acyl-CoAs) to the synthesis of
polymer.
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Taking into account that a blockade in -oxidation could lead to the
accumulation of 3-OH-acyl-CoA derivatives that could be used for the
synthesis of plastic polymers, we analyzed the accumulation of PHAs and
PHPhAs when P. putida U was cultured in MM containing 15 mM octanoic acid or 8-phenyloctanoic acid as the source of
intermediates, 5 mM 4-OH-phenylacetic acid for supporting
bacterial growth, and the ketothiolase inhibitor acrylic acid (0, 5 mM) (85, 86). We observed that, as Steinbüchel and
co-workers reported in a E. coli recombinant strain (86), accumulation of polymer increased 2-fold (60 and 38% of bacterial dry
weight, respectively) with respect to a control cultured in the absence
of acrylic acid.
When a PhaZ mutant (handicapped in the mobilization of
the plastic once synthesized, see Table IV) was cultured in MM
supplemented with acrylic acid, the quantity of polymer accumulated was
double that observed in the control (without acrylic acid) but lower (65%) than the amount of plastic accumulated by the wild-type P. putida U. These results again support the hypothesis that the polyhydroxyalkanoate biosynthetic enzymes work more efficiently as a
complex enzymatic system.
To further analyze the overproduction of PHAs, the fadA gene
encoding the 3-oxoacyl-CoA thiolase (accession number AF150672) involved in the -oxidation pathway (see below) was disrupted by
homologous recombination by using an internal fragment of the gene,
obtained by polymerase chain reaction amplification using primers of
the homologous gene of P. fragi (87). We observed that the
quantity of PHAs accumulated in this mutant, when it was cultured in MM
supplemented with 15 mM octanoic acid and 5 mM
4-HPhAc, was higher (68% dwc) than that observed in the parental strain, showing that a blockade in the -oxidation contributes efficiently to improve the synthesis of plastic polymers.
PhA-specific Catabolic Pathways: The Link with the Phenylacetyl-CoA Catabolon
According to the results presented above, we assumed that the
biosynthesis and mobilization of PHPhAs and PHAs in P. putida U must be carried out by the same enzymatic system and that
it was similar to those reported in P. oleovorans and in
P. aeruginosa (13, 21). However, the synthesis of aromatic
polyesters is not a common event among the bacteria, although many
species are able to accumulate high amounts of aliphatic plastics
intracellularly (1). This difference cannot be explained by the
requirement of a different acyl-CoA synthetase or different
polymerases, since, as we have shown above, the same enzymes
participate in the activation and in the polymerization of aromatic and
aliphatic monomers. However, the degradation of PhAs containing an even
number of carbon atoms (C2n+2PhAs) by
P. putida U seems to require two different pathways: the
first, a -oxidation route, that catabolizes PhAs to
phenylacetyl-CoA, and a route that must transform this thioester into
assimilable metabolites. Under this assumption, it could be argued that
only bacteria able to metabolize PhAs would be able to synthesize
aromatic plastics (PHPhAs). In fact, the accumulation of these polymers
would not represent any advantage if the microbe were unable to
catabolize them. Thus, phenyl derivatives containing either an odd or
an even number of carbon atoms will be catabolized through
-oxidation to benzoyl-CoA (odd) or phenylacetyl-CoA (even), which
could be further degraded to the intermediates of general metabolism.
Bacteria lacking these additional pathways would only be able to
partially catabolize these compounds (releasing some units of
acetyl-CoA), and this incomplete degradation would afford scant
energetic benefit for the microbe. However, it is not clear whether a
single -oxidation pathway exists in P. putida U or
whether different pathways might be involved in the catabolism of PhAs
and alkanoates in this bacterium. To clarify this point, the different
mutants unable to catabolize octanoic acids (Oct) were
classified into three groups: (i) those lacking an specific acyl-CoA
activating activity (ACS also called
Oct1; see above); (ii) those mutants
lacking a functional glyoxylic acid cycle
(Oct2), and (iii) a different group
affected in the -oxidation pathway (Oct3) obtained by genetic disruption
of the ketothiolase gene (the fadA mutant). We observed that
all these strains efficiently catabolized phenylacetic acid and benzoic
acid, whereas those named Oct1 and
Oct3 were unable to grow in MM
containing either aliphatic or phenylalkanoic acids with a number of
carbon atoms (even or odd) higher than 4 or did so very poorly. These
data allow us to conclude that in P. putida U a single
acyl-CoA synthetase and single -oxidation pathway are involved in
the catabolism of alkanoic and arylalkanoic acids with the above
indicated carbon length.
Recently, an additional pathway required for the specific catabolism of
phenylacetic acid (via phenylacetyl-CoA) (56) has been discovered (53).
We identified a piece of DNA of about 18 kilobase pairs containing 15 open reading frames that are required for the catabolism of PhAc and
phenylacetyl-CoA in P. putida U (53). This catabolic
pathway, called the phenylacetyl-CoA catabolon core, appears to be
organized in three contiguous operons that contain five different
functional units and several regulatory elements (Fig.
8). Using mutagenesis with the transposon
Tn5, different mutants of P. putida U unable to
grow in MM containing PhAc as the carbon source (PhAc
strains) were selected. We observed that all of the PhAc
mutants were able to catabolize n-As and n-PhAs
containing an odd number of carbon atoms
(C2n+1PhAs), whereas they assimilated
C2n+2PhAs with an even number very poorly, since they were only able to utilize the acetyl-CoA molecules released by -oxidation (Fig. 3C). Analysis of plastic
polymer accumulation in these mutants grown in MM containing
4-hydroxyphenylacetic acid for supporting bacterial growth and
8-phenyloctanoic acid as a plastic precursor revealed that whereas
mutations in all of the catabolic genes involved in the specific route
of phenylacetic acid did not affect the production of aliphatic
polymers (PHAs), most of them (with the exception of the mutants
lacking phenylacyl-CoA ligase, an enzyme that is not required for the
catabolism of phenylalkanoic acids) (53) accumulated lower quantities
of aromatic polymers (PHPhAs) (Table III). These results suggest that
both pathways (PHPhAs biosynthetic/degradative route and PhAc catabolic
pathway) are closely related, since a blockade in the second one causes the accumulation of some intermediate(s) that negatively affect(s) (probably by feedback control) the activities of the enzymes involved in the process of polymerization (Table III).
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We also observed that all the PhAc mutants were able to
grow in MM containing benzoic acid as the sole carbon source (not shown), indicating that the -oxidation final products of both types
of PhAs (benzoyl-CoA/phenylpropionyl-CoA and phenylacetyl-CoA) are
catabolized through different pathways. Similar data had been reported
in E. coli W, since two unrelated pathways are involved in
the degradation of phenylacetic acid and phenylpropionic acid in this
bacterium (88, 89).
Conclusions
In sum, from the present findings the following conclusions can be
drawn. (i) P. putida U synthesizes and accumulates novel biodegradable plastic polymers containing as monomers
3-hydroxy-n-phenyl derivatives (10 
n 
5).
(ii) A single acyl-CoA synthetase seems to be involved in the
activation of phenylalkanoics and alkanoic acids to their CoA
thioesters (which are the precursors of the different polymers), and a
similar transport system seems to be required for the uptake of
alkanoic and phenylalkanoic acids. The disruption of the
fadD gene handicapped the uptake of precursors, their
activation, and, therefore, the synthesis of plastics. (iii) Polymerization of the monomers is carried out by two polymerases (PhaC1
and PhaC2), which are also involved in the synthesis of polyhydroxyalkanoates. These enzymes are encoded by two genes (phaC1 and phaC2) organized in a single operon,
which also includes a depolymerase gene
(phaC1-phaZ-phaC2) and which is very
similar to the pha locus reported for P. oleovorans and P. aeruginosa. Both enzymes (PhaC1 and
PhaC2) showed a similar substrate specificity and polymerizing rate,
whereas the depolymerizing rate shown by PhaZ is about one half of the
activity shown by each of these polymerases. Expression of these
enzymes in recombinant E. coli fadB or fadR
mutants does not permit the synthesis of polymers, whereas the
synthesis and accumulation of PHAs occurred at a very low rate when a
monocopy construction was used. (iv) A blockade in a -oxidation gene
affecting the expression of ketothiolase (fadA mutant)
greatly improves the accumulation of these polymers. Similar results
were obtained when the ketothiolase was inhibited with acrylic acid as
well as when the metabolic flux through the glyoxylic acid was altered
(disruption on the gene encoding the isocitrate lyase). (v) Finally, at
least two specific catabolic routes linked to a single -oxidation
pathway are required for the complete catabolism of the
3-hydroxyphenylalkanoates containing an even number of carbon atoms.
Both are integrated in a complex catabolic unit (phenylacetyl-CoA
catabolon) responsible for the degradation of several structurally
related aromatic compounds (53).
Deeper knowledge of these biodegradable plastics could be important not
only to establish the physicochemical properties of novel, nonpolluting
compounds but also to obtain derivatives with new or broader
applications. Moreover, expression of the genes encoding the plastic
polymerases and depolymerase in other microbes, via genetic engineering
(incorporation of genes encoding R-specific enoyl-CoA to the
pha locus in order to efficiently channel -oxidation intermediates to the polymeric system), could help to enhance both
their biosynthetic and their catabolic potential as well as increase
the quantity of biodegradable plastics that could be stored (45).
Furthermore, a biochemical approach to the regulatory mechanisms
controlling the expression of these genes and study of the proteins
participating in both the synthesis and in the stabilization of such
polymers could help in the design of genetically engineered microbes
that could be employed to eliminate certain aromatic contaminants from
the biosphere under a broad range of metabolic and environmental
conditions. Thus, we have recently shown that the disruption of the
paaN gene, encoding the repressor of the PhAc catabolic
pathway (Fig. 8) (53), elicits a dual effect, i.e. the
constitutive expression of the pathway and the suppression of the
carbon catabolite repression of this route. This result shows that
genetically engineered strains could be efficiently used to degrade
PhAc and certain aromatic precursors even from complex media containing
readily metabolizable carbon sources that in the wild-type strain would
strongly repress the PhAc catabolic pathway.
<
), 3-phenylpropionic acid (
),
5-phenylvaleric acid (
), 7-phenylheptanoic acid (
), and
9-phenylnonanoic acid (
). B, aryl-alkanoic acids with an
even number of carbon atoms: phenylacetic acid (
