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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic structure and functional analysis of the PhAc-CoA catabolon
  5. The PhAc-CoA catabolon core
  6. Upper catabolic pathways of the PhAc-CoA catabolon
  7. Biotechnological applications
  8. Acknowledgements
  9. References

The term catabolon was introduced to define a complex functional unit integrated by different catabolic pathways, which are, or could be, co-ordinately regulated, and that catalyses the transformation of structurally related compounds into a common catabolite. The phenylacetyl-CoA catabolon encompasses all the routes involved in the transformation of styrene, 2-phenylethylamine, trans-styrylacetic acid, phenylacetaldehyde, phenylacetic acid, phenylacetyl amides, phenylacetyl esters and n-phenylalkanoic acids containing an even number of carbon atoms, into phenylacetyl-CoA. This common intermediate is subsequently catabolized through a route of convergence, the phenylacetyl-CoA catabolon core, into general metabolites. The genetic organization of this central route, the biochemical significance of the whole functional unit and its broad biotechnological applications are discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic structure and functional analysis of the PhAc-CoA catabolon
  5. The PhAc-CoA catabolon core
  6. Upper catabolic pathways of the PhAc-CoA catabolon
  7. Biotechnological applications
  8. Acknowledgements
  9. References

The microbial catabolism of aromatics is carried out by many degradative routes that facilitate the mineralization of these compounds. The study of these processes has provided important scientific background about new intermediates, unusual enzymes, different gene organizations and special regulatory mechanisms, thus expanding older disciplines such as biochemistry, genetics and microbiology (Timmis and Pieper, 1999). The diversity of these pathways (Wackett and Bruce, 2000) led to the introduction of new routes and concepts that comprises a new research area: biodegradation. Furthermore, the biotechnological applications of the enzymes involved in these pathways have expanded molecular biology into the powerful and varied field of metabolic engineering.

Most of these unusual pathways seem to be unrelated. They are species (or strain) specific and appear to be encoded by genes with different subcellular locations (plasmids and/or chromosome) (Timmis and Pieper, 1999). In contrast, other microbes have evolved complex catabolic units that work as a single functional element and in which the original regulatory mechanisms have developed into hierarchies, thereby facilitating their co-ordination and the efficient flux of intermediates between the different pathways. Confluence of the original independent routes forms a complex unit and leads to the metabolic specialization of a given bacterium, because its degradative potential is polarized in a particular direction.

In Pseudomonas putida U, the degradation of phenylacetaldehyde, 2-phenylethylamine, trans-styrylacetic acid, n-phenylalkanoic acid (where n is an even number of carbon atoms) or different polymers containing aromatic units (poly 3-OH-n-phenylalkanoates; PHPhAs) as monomers is carried out by specific pathways that catalyse the transformation of these molecules into either phenylacetic acid (PhAc) or phenylacetyl-CoA (PhAc-CoA) (Fig. 1). These compounds are later catabolized through a central route (phenylacetyl-CoA catabolon core), which converts them into general metabolites (Olivera et al., 1998). The combination of these routes is defined as a catabolon and its specific name (PhAc-CoA catabolon) is given as a function of the first common intermediate (PhAc-CoA).

image

Figure 1. Biochemical organization of the phenylacetyl-CoA catabolon. Sty, styrene; TA, tropic acid; EB, ethylbenzene; PhEtNH2, 2-phenylethylamine; PhAc amide, phenylacetyl amide; PhAc ester, phenylacetyl ester; PhAc, phenylacetic acid; PhAs, phenylalkanoates; PHPhAs, poly-hydroxyphenylalkanoates; StyAc, trans-styrylacetic acid; PhAc-CoA, phenylacetyl-CoA; 2′-OH-PhAc-CoA, 2′-OH-Phenylacetyl-CoA; TCA, tricarboxylic acid cycle; the box indicates the PhAc-CoA catabolon core.

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In this review, we analyse the phenylacetyl-CoA catabolon, which may provide an example of catabolic integration and may serve as a model for understanding how different unrelated routes converge in a complex catabolic unit and how bacteria become highly specialized for the degradation of a family of structurally related compounds. We discuss the genetic organization of the core in different microbes and the biotechnological applications of the enzymes belonging to the catabolon.

Genetic structure and functional analysis of the PhAc-CoA catabolon

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic structure and functional analysis of the PhAc-CoA catabolon
  5. The PhAc-CoA catabolon core
  6. Upper catabolic pathways of the PhAc-CoA catabolon
  7. Biotechnological applications
  8. Acknowledgements
  9. References

The PhAc-CoA catabolon encompasses several routes involved in the specific degradation of aromatic compounds into the PhAc or PhAc-CoA intermediates (Fig. 1). These are called upper pathways. The route of convergence (central pathway), which catalyses the transformation of PhAc and PhAc-CoA into general metabolites, is called the PhAc-CoA catabolon core. This catabolon may be the first example of a hybrid pathway because, although it is aerobic, its catabolic intermediates (aryl-CoA derivatives) were, until recently, considered to be specific to the anaerobic catabolic pathways.

The PhAc-CoA catabolon core

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic structure and functional analysis of the PhAc-CoA catabolon
  5. The PhAc-CoA catabolon core
  6. Upper catabolic pathways of the PhAc-CoA catabolon
  7. Biotechnological applications
  8. Acknowledgements
  9. References

Early reports suggested that the first step in the aerobic catabolism of PhAc was the hydroxylation of the aromatic ring, forming 2,5- or 3,4-dihydroxyPhAc intermediates (Olivera et al., 1994). However, pioneering work carried out with P. putida U strongly suggested that PhAc is mineralized through an atypical catabolic pathway involving a PhAc-CoA ligase (Martínez-Blanco et al., 1990). This enzyme, which is induced when P. putida U is cultured in PhAc, was purified, and its encoding gene was cloned, sequenced and expressed in heterologous hosts (Miñambres et al., 1996). These studies led to the isolation and characterization of the complete PhAc catabolic pathway of P. putida U using transposon mutagenesis. The gene cluster (pha) that encodes the PhAc catabolic pathway of P. putida U (Fig. 2) was located within an 18 kb fragment and is composed of 15 genes that are organized in five contiguous operons, paaABCEF, paaGHIJK, paaLMN, paaY and paaX (previously named phaABCDE, phaFOGHI, phaJKL, phaM and phaN respectively; Olivera et al., 1998). These genes encode 15 proteins grouped in six putative functional units: (i) a transport system (PaaL and PaaM); (ii) a PhAc-activating enzyme, i.e. the PhAc-CoA ligase (PaaF); (iii) a ring-hydroxylating complex (PaaG, PaaH, PaaI, PaaJ and PaaK); (iv) a ring-opening protein (PaaN); (v) a β-oxidation-like system (PaaA, PaaB, PaaC and PaaE); and (vi) two regulatory proteins (PaaX and PaaY) (Fig. 2 and Table 1).

image

Figure 2. Organization of the gene clusters encoding the phenylacetyl-CoA catabolic pathway (PhAc-CoA catabolon core) in different microbes. Colour combinations indicate the organization of these clusters in relation to those reported in P. putida U. P. putida KT2440 sequences were obtained from the Unfinished Genome Project at The Institute of Genome Research, Rockville, MD, USA. Klebsiella pneumoniae MGH78578 sequences were obtained from the Unfinished Genome Project at the Genome Sequencing Center, Washington University, St Louis, MO, USA. Preliminary sequence data for Rhodopseudomonas palustris CGA009 were obtained from the DOE Joint Genome Institute (JGI), Walnut Creek, CA, USA. The sequence data corresponding to Bordetella pertussis Tohama I, B. bronchiseptica RB50 and Burkholderia pseudomallei K96243 were from the different sequencing groups at the Sanger Centre, Cambridge, UK.

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Table 1. PhAc catabolic pathway in different microbes: correspondence between original gene denomination and protein function.
FunctionConsensus P. putida U E. coli W E. coli K-12 A. evansii KB740 B. halodurans C-125 D. radiodurans R-1
  • a

    . Accession numbers in the GenBank at NCBI.

Phenylacetyl-CoA ligase paaF phaE paaK paaK pacD phaE paaK
Ring-hydroxylating complex. Protein 1 paaG phaF paaA paaA pacE phaF paaA
Ring-hydroxylating complex. Protein 2 paaH phaO paaB paaB pacF phaO paaB
Ring-hydroxylating complex. Protein 3 paaI phaG paaC paaC pacG phaG paaC
Ring-hydroxylating complex. Protein 4 paaJ phaH paaD paaD pacH phaH paaD
Ring-hydroxylating complex. Protein 5 paaK phaI paaE paaE pacI   
Ring-opening enzyme paaN phaL paaZ paaZ pacL phaL paaZ
Enoyl-CoA hydratase 1 paaA phaA paaF paaF   phaA  
Enoyl-CoA hydratase 2 paaB phaB paaG paaG pacA phaB  
3-OH-acyl-CoA dehydrogenase paaC phaC paaH paaH pacB phaC  
Putative thioesterase paaD   paaI paaI pacC phaI  
Ketothiolase paaE phaD paaJ paaJ   phaD  
Permease paaL phaJ      
Porine paaM phaK      
Repressor protein paaX phaN paaX paaX   phaN  
Putative regulator paaY phaM paaY paaY pacM phaM  
Sourcea AF029714X97452U00096AJ278756AP001507AE002069 AE001863

The PhAc catabolic cluster of Escherichia coli W (paa) was isolated by complementation of the E. coli W Δpaa mutant (W14) with pFA2, a pUC derivative harbouring a 33 kb BamHI fragment (Ferrández et al., 1997) (Fig. 2). The paa cluster was sequenced and mapped to 31 min in E. coli K-12 and E. coli W. It is located downstream of the mao region, which is responsible for transforming 2-phenylethylamine into PhAc (Ferrández et al., 1998). The paa cluster contains 14 genes in three transcription units, paaGHIJKABCDEF, paaN and paaXY (previously called paaABCDEFGHIJK, paaZ and paaXY;Ferrández et al., 1998). The paaF gene, which is placed at the 3′ end of the largest operon, encodes a PhAc-CoA ligase. The ring-hydroxylating complex is encoded by the paaGHIJK genes, whereas the paaN gene encodes a protein that is similar to the ring-opening enzyme of P. putida U. The paaABCDE genes encode proteins that are similar to fatty acid β-oxidation enzymes. The paaXY genes are homologous to the regulatory genes of P. putida U.

Comparison of the P. putida U and E. coli W paa clusters revealed that the paaLM transport genes are not present in E. coli W. A paaD homologue has not been found P. putida U; however, this bacterium contains an open reading frame (ORF) that overlaps with paaE and is very similar to paaD. It remains to be determined whether these genes are essential for PhAc metabolism or whether other genes located outside the clusters carry out their corresponding functions.

Three promoters (P1, P2 and P3) have been identified in the P. putida U paa cluster. These control the expression of the paaABCEF, paaGHIJK and paaLMN operons respectively. The promoters were tested in E. coli by monitoring the expression of the lacZ reporter gene (Olivera et al., 1998). The promoters that control the expression of phaX and phaY have not yet been identified. PaaX behaves as a transcriptional repressor, because its blockade leads to the constitutive expression of the PhAc-CoA ligase and the transport system, which are induced by PhAc in the wild-type P. putida U. Moreover, this cluster must be subject to catabolic repression because, in the presence of 5 mM glucose, neither PhAc-CoA ligase activity nor PhAc uptake were detected, even when the culture was induced with PhAc.

The Pn (previously published as Pz), Pa and Px promoters, which drive the expression of paaN, paaGHIJKABCDEF and paaXY, respectively, have been identified in E. coli (Ferrández et al., 1998; 2000). The PaaX regulator and the homologous protein of P. putida U negatively control the expression of Pa and Pn divergent promoters. Gel retardation assays, showing that PaaX binds specifically to the Pa and Pn promoters, were complemented with in vivo experiments, which demonstrated a PaaX-mediated effect on the repression of Pa–lacZ and Pn–lacZ reporter fusions. The region within the Pa and Pn promoters, which is protected by PaaX in DNase I footprinting assays, contains a conserved 15 bp imperfect palindromic sequence motif. Mutational analysis showed that this motif is indispensable for PaaX binding and repression. In addition, PhAc-CoA, the first intermediate in the pathway, was found to be the true inducer of the pathway. Likewise, only PhAc-CoA inhibits the binding of PaaX to the target sequence. Thus, PaaX is the first example of a regulator of an aromatic catabolic pathway that responds to an aryl-CoA compound (Ferrández et al., 2000). A similar conclusion was drawn following studies of different P. putida U mutants (García et al., 2000).

A gene encoding a similar enzyme to the PhAc-CoA ligase of P. putida U and E. coli has been identified in Pseudomonas sp. Y2. This gene is located upstream of the sty cluster, which encodes the styrene upper catabolic pathway (Velasco et al., 1998). This gene belongs to a cluster that is responsible for the degradation of PhAc in Pseudomonas sp. Y2. The operons in this cluster appear to be arranged differently from those of P. putida U (J. Perera, personal communication).

Detailed analysis of the sequenced bacterial genomes has allowed the identification of putative gene clusters involved in the degradation of PhAc in E. coli K-12 (Ferrández et al., 1998), Klebsiella pneumoniae MGH78578, P. putida KT2440, Deinococcus radiodurans R-1, Azoarcus evansii KB740, Bordetella pertussis Tohama I, Bordetella bronchiseptica RB50, Burkholderia pseudomallei K96243, Rhodopseudomonas palustris CGA009 and Bacillus halodurans C-125 (Fig. 2 and Table 1). These genes are identical or very similar to those of E. coli W or P. putida U. Interestingly, E. coli C does not contain the paa cluster, and mutations in several E. coli K-12 strains (including DH1, DH5α, HB101, CC118 and JM109) may render these strains unable to grow when PhAc is the sole carbon source (Ferrández et al., 1998).

Upper catabolic pathways of the PhAc-CoA catabolon

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic structure and functional analysis of the PhAc-CoA catabolon
  5. The PhAc-CoA catabolon core
  6. Upper catabolic pathways of the PhAc-CoA catabolon
  7. Biotechnological applications
  8. Acknowledgements
  9. References

This section describes current knowledge on the different pathways involved in the modification of the side-chains of different aromatic compounds (such as styrene, 2-phenylethylamine, phenylacetaldehyde, n-phenylalkanoates, tropic acid, phenylacetyl esters and amides and ethylbenzene) to either PhAc or PhAc-CoA.

Styrene catabolic pathway

The chromosomal region involved in the conversion of styrene to PhAc has been cloned and characterized in different Pseudomonas strains (Beltrametti et al., 1997; Panke et al., 1998; Velasco et al., 1998). This region contains the styABCD catabolic operon and the stySR regulatory operon. The styAB genes encode a two-component monooxygenase that transforms styrene into epoxystyrene, which is further converted into phenylacetaldehyde and PhAc by StyC (epoxystyrene isomerase) and StyD (phenylacetaldehyde dehydrogenase) respectively. This cluster is induced by styrene through the StySR two-component signal transduction system. Expression of the styABCD operon decreases significantly in the absence of the stySR genes, suggesting that this system behaves as a positive regulator. The genes responsible for the complete mineralization of styrene (i.e. the upper pathway genes and the PhAc-CoA core genes) are clustered in the Pseudomonas sp. Y2 chromosome (see above).

2-Phenylethylamine catabolic pathway

The genes involved in the conversion of phenylethylamine to PhAc in E. coli have been characterized and located at 31 min, upstream of paaN (Ferrández et al., 1997; 1998; Hanlon et al., 1997). Phenylethylamine is transformed into phenylacetaldehyde by the monoamine oxidase encoded by maoA (Steinebach et al., 1996). Subsequently, the phenylacetaldehyde dehydrogenase encoded by padA transforms the product of monoamine oxidase into PhAc (Ferrández et al., 1997). The maoA gene is regulated by the transcriptional activator encoded by maoB (Yamashita et al., 1996). It is noteworthy that, although the PadA and the StyD dehydrogenases of E. coli and Pseudomonas sp. Y2 have the same function, their sequences are not more similar to each other than to other aldehyde dehydrogenases (Velasco et al., 1998).

P. putida U is known to grow well in chemically defined media containing 2-phenylethylamine as the sole carbon source, and this compound induces the enzymes involved in the degradation of PhAc (Martínez-Blanco et al., 1990; Olivera et al., 1998). Furthermore, analysis of the product accumulated by several paaGHIJK mutants revealed that PhAc accumulates in the presence of 2-phenylethylamine (Olivera et al., 1998), indicating that this compound is catabolized via the PhAc-CoA catabolon core. More recently, we have demonstrated the existence in P. putida cell-free extracts of a phenylacetaldehyde dehydrogenase that is induced by 2-phenylethylamine but not by PhAc (E. R. Olivera, B. Garcia, B. Miñambres, and J.M. Luengo, unpublished). This suggests that, in this bacterium, 2-phenylethylamine is catabolized by a similar pathway to that reported for E. coli.

n-Phenylalkanoates catabolic pathway

The degradation of n-phenylalkanoic acids (n-PhAs) in P. putida U was reported recently (García et al., 1999). The catabolism of these compounds requires the same enzymatic activities as those involved in the β-oxidation of medium- and long-chain n-alkanoic acids. After n-PhAs are taken up by the bacterium, they are activated to CoA thioesters by the acyl-CoA synthetase encoded by fadD. Subsequently, the acyl-CoA dehydrogenase (fadF gene product) catalyses the introduction of a double bond at position 2 of the aliphatic chain. Finally, a protein complex encoded by the fadBA operon, which has five additional enzymatic activities (enoyl-CoA hydratase, 3-OH-acyl-CoA dehydrogenase, cis3trans2-enoyl-CoA isomerase, 3-OH-acyl-CoA epimerase and 3-ketoacyl-CoA thiolase) (García et al., 1999) catalyses the release of acetyl-CoA units. This enzymatic complex comprises two subunits in α2β2 conformation. Recent studies have shown that the FadBA enzymatic complex catalyses the formation of PhAc-CoA from n-phenylalkanoates (or their derivatives) containing an even number of carbon atoms in P. putida U. However, the degradation of n-PhAs with an odd number of carbon atoms produces trans-cinnamoyl-CoA (a direct derivative of 3-phenylpropionyl-CoA), which cannot be catabolized further and, after being hydrolysed by thioesterases, is excreted as trans-cinnamic acid (Olivera et al., 2001).

Trans-Styrylacetic acid catabolism

trans-Styrylacetic acid is also catabolized through the β-oxidation pathway. In P. putida U, this compound is activated to trans-styrylacetyl-CoA, which then undergoes isomerization of the double bond from position 3 to position 2 to generate 4-phenyl-2-butenoyl-CoA, which is β-oxidized as indicated above (Fig. 2) (J. M. Luengo and M. A. Moreno, unpublished).

Tropic acid degradation

The bacterial catabolism of atropine (a tropane alkaloid synthesized by the solanaceous plant Atropa belladonna) by Pseudomonas sp. AT3 generates tropine and tropic acid (Long et al., 1997). Tropic acid is dehydrogenated to produce phenylmalonic semi-aldehyde and is then decarboxylated to phenylacetaldehyde, which is oxidized further to PhAc (Long et al., 1997). A similar pathway has been reported for the catabolism of tropic acid in Flavobacterium sp. (van den Tweel et al., 1988).

Phenylacetyl esters and amides

Phenylacetyl esters and amides can be hydrolysed to PhAc by penicillin G acylase (Pac). This enzyme forms part of a large family of enzymes known as β-lactam acylases because they are used in the semi-synthesis of β-lactam antibiotics. Although Pac has been identified in many microorganisms, E. coli W Pac has received the most attention. The pac gene of E. coli W is located in the vicinity of the cluster responsible for the mineralization of 4-OH-PhAc (Prieto et al., 1996). Although Pac catalyses the conversion of penicillin G into PhAc and 6-aminopenicillanic acid (6-APA), it does not seem to play a role in antibiotic resistance. Pac has a broad substrate range, and it can hydrolyse different esters and amides of PhAc and many of its derivatives (e.g. 4-OH-PhAc), as well as esters and amides of other aromatic and aliphatic acids (thienylacetic acid and hexanoic acid). Pac therefore appears to be the scavenger of many different natural esters and amides, which, after their hydrolysis, can be mineralized by central catabolic pathways that act on the aromatic acids released.

Ethylbenzene degradation

Ethylbenzene is widely used as a solvent and as a styrene precursor. It is also a waste product of high-octane gasoline (Corkery et al., 1994). It has been reported that the degradation of ethylbenzene by Pseudomonas fluorescens CA-4 and Pseudomonas sp. Y2 involves its oxidation to 2-phenylethanol and further transformation to PhAc via phenylacetaldehyde (Utkin et al., 1991; Corkery et al., 1994). The genes responsible for these activities remain unknown.

Biotechnological applications

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic structure and functional analysis of the PhAc-CoA catabolon
  5. The PhAc-CoA catabolon core
  6. Upper catabolic pathways of the PhAc-CoA catabolon
  7. Biotechnological applications
  8. Acknowledgements
  9. References

The PhAc-CoA catabolon offers attractive possibilities for the development of biotechnological applications. We describe the most important advances in this field and discuss future prospects.

Enzymatic synthesis of penicillins

The last step in the biosynthesis of benzylpenicillin (penicillin G) by Penicillium chrysogenum is catalysed by three enzymes (Luengo, 1998). In the first reaction, PhAc, the side-chain precursor of this β-lactam antibiotic, is taken up by the fungus from the culture broth. After PhAc has been incorporated in the mycelia, PhAc-CoA ligase (PCL) seems to be required for its activation to phenylacetyl-CoA, which is a substrate of the acyl-CoA:6-APA/isopenicillin N (IPN) acyltransferase (AT). This catalyses either the exchange of the α-aminodipoyl moiety of IPN or the direct acylation of the amino group of 6-APA with the PhAc residue present in the molecule of PhAc-CoA. Although the PhAc transport system (PTS) and AT have been characterized in P. chrysogenum, PCL remains virtually unknown. An in vitro system for the conversion of 6-APA to penicillin G was designed by coupling the PhAc-CoA ligase, encoded by the paaF gene of P. putida U, and AT from P. chrysogenum (Luengo, 1998). This system is extremely efficient (over 90% of the 6-APA is transformed into penicillin G within 30 min) and has allowed 64 different penicillins to be synthesized. These penicillins include all known natural penicillins, many of the semi-synthetic ones and others that currently can only be obtained by chemical synthesis (Luengo, 1998). As the main limitation of this system resulted from the specificity of AT, its replacement by other ATs with broader substrate ranges could facilitate the synthesis of new β-lactam antibiotics.

Improving the rate of penicillin G biosynthesis in P. chrysogenum

The in vitro behaviour of PhAc-CoA ligase suggested that cloning paaF into P. chrysogenum would improve the production of penicillin G. Transformation of P. chrysogenum with paaF allowed the isolation of recombinant strains able to produce PhAc-CoA ligase and to secrete more penicillin G (Miñambres et al., 1996). These results illustrated how the expression of a heterologous protein belonging to the bacterial PhAc-CoA catabolon core can be used to improve the biosynthetic machinery of this fungus.

Biotransformation of PhAc to 2′-OH-PhAc

2′-OH-PhAc is a common intermediate in the synthesis of many chemical compounds (Lee and Shieh, 1993). This molecule is currently synthesized chemically, and few attempts have been made to produce it using microbial systems (Staudenmaier et al., 1998). As shown above, the catabolism of PhAc involves activation to PhAc-CoA followed by hydroxylation to 2′-OH-PhAc-CoA, which is then processed further by the PaaN ring-opening enzymes of P. putida U or PaaZ of E. coli. Mutations in paaN lead to the accumulation of 2′-OH-PhAc in the culture broth when these strains are cultured in chemically defined media containing PhAc or PhAc-CoA precursors plus a carbon energy source (Ferrández et al., 1998; Olivera et al., 1998). The biotechnological interest of this biotransformation resides not only in the conversion of PhAc to 2′-OH-PhAc but also in the fact that other important molecules can be obtained in a similar manner. Expression of the gene encoding the benzoyl-CoA synthetase from Rhodopseudomonas palustris (Egland et al., 1995), together with the paaGHIJK genes from P. putida U, could lead to the production of 2-OH-benzoic acid (salicylic acid, a precursor of aspirin) if this second system can recognize benzoyl-CoA as a substrate.

Styrene biodegradation and biotransformation

Styrene, one of the most important aromatic chemicals produced industrially, is toxic in fairly low quantities. Human exposure to styrene usually occurs by inhalation in industrial settings (Warhurst and Fewson, 1994). Biological methods of air purification (biofilters) efficiently eliminate styrene from polluted air using selected and adapted microbial cells (Warhurst and Fewson, 1994). The use of natural or genetically engineered microorganisms with enhanced catabolic activity towards styrene could improve the degradation process further.

A whole-cell biocatalytic process has been developed to convert styrene to chiral (S)-styrene oxide using recombinant E. coli cells carrying the styAB genes that encode the two-component styrene monooxygenase from Pseudomonas (Panke et al., 1998; 1999; 2000).

Production of new bioplastics

When cultured in chemically defined media containing fatty acids as carbon sources, P. putida U accumulates several polymers (poly 3-hydroxyalkanoates) as reserve materials. This bacterium can also synthesize new polyesters (bioplastics) composed of different aromatic monomers (García et al., 1999). Genetic analysis has identified a three-gene operon that encodes two polymerases (PhaC1 and PhaC2) and a depolymerase (PhaZ). These enzymes are responsible for the polymerization and degradation of these polymers. This polymerization–depolymerization system is closely linked to the β-oxidation pathway that catalyses the degradation of the monomers [3-OH-acyl(aryl)-CoA derivatives] released from the polymers by PhaZ. Further genetic analysis allowed the identification of fatty acid β-oxidation genes in P. putida U as well as the isolation of different mutants. Acyl-CoA synthetase (fadD gene product) mutants cannot activate fatty acids, thus stopping its further transformation into 3-OH-acyl-CoA derivatives (plastic monomer). However, disruption or deletion of the fadB or fadA genes, which encode the β-oxidation multienzymatic complex, results in the accumulation of polyhydroxy-PhAs in the presence of PhAs and other carbon growth sources (Olivera et al., 2001). In these mutants, over 90% of the cytoplasm can be occupied by bioplastics (Fig. 3), and the structure of polymers can readily be modified (the monomer composition and length) by changing the relative proportion of phenylalkanoate precursor added to the culture broth. These polymers seem to have very promising physicochemical properties, and some of them have been used to obtain microspheres for use in pharmaceuticals or with industrial applications.

image

Figure 3. Electron micrographs. Scanning (A and B) and transmission (C and D) microphotographs of Pseudomonas putida (A and C) and its ΔfadBAβ-oxidation mutant (B and D) cultured in chemically defined solid medium containing n-phenylalkanoates. Bars correspond to 1 µm.

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Future prospects for expanding the PhAc-CoA catabolon

Our current knowledge of the PhAc-CoA catabolon allows us to envisage the creation of a single recombinant strain harbouring several upper pathways that converge into the catabolon core as described in several bacteria. Thus, the isolation of the bacterial genes encoding proteins that are involved in the two first reactions of atropine degradation or those that catalyse the transformation of ethylbenzene into phenylacetaldehyde and their expression in P. putida U could improve the rate of degradation of these compounds through the PhAc-CoA catabolon core. Moreover, the cloning of the genes involved in the catabolism of cinnamic acid in P. putida U may allow the complete degradation of n-PhAs with an odd number of carbon atoms. An additional catabolic advantage could be obtained by introducing some of the genes that encode enzymes that convert cinnamic acid into PhAc through α-oxidation into P. putida U.

Important ecological advantages could also be obtained by altering the regulatory mechanisms that control these pathways. Thus, disruption of the paaX repressor gene of the PhAc-CoA catabolon core leads to constitutive expression of this pathway, allowing the catabolism of PhAc under conditions that are repressive for the wild type. Moreover, for some applications, it might be useful to eliminate the catabolic repression of the PhAc-CoA catabolon core by placing the expression of these genes under the control of alternative promoters.

Finally, the ubiquitous nature of the PhAc-CoA catabolon and the diverse core cluster organization in microorganisms (Fig. 2) makes this catabolon an attractive model for determining how complex catabolic pathways evolve. This system is also a very useful model for studying the hierarchical regulation of the catabolic pathways involved and the optimization of the use of energy in bacteria that must choose among alternative carbon sources.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic structure and functional analysis of the PhAc-CoA catabolon
  5. The PhAc-CoA catabolon core
  6. Upper catabolic pathways of the PhAc-CoA catabolon
  7. Biotechnological applications
  8. Acknowledgements
  9. References

This investigation was supported by Fondo Europeo de Desarrollo Regional (grant 1FD97-0245) and by Dirección General de Enseñanza Superior e Investigación Científica (grant BMC2000-0125-C04).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic structure and functional analysis of the PhAc-CoA catabolon
  5. The PhAc-CoA catabolon core
  6. Upper catabolic pathways of the PhAc-CoA catabolon
  7. Biotechnological applications
  8. Acknowledgements
  9. References
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