Novel Biodegradable Aromatic Plastics from a Bacterial Source
1999; Elsevier BV; Volume: 274; Issue: 41 Linguagem: Inglês
10.1074/jbc.274.41.29228
ISSN1083-351X
AutoresBelén Garcı́a, Elı́as R. Olivera, Baltasar Miñambres, Martiniano Fernández-Valverde, Librada M. Cañedo, M. Auxiliadora Prieto, José L. Garcı́a, Marı́a Jesús Martı́nez, José M. Luengo,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoNovel 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 β-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the phalocus) 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 β-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the phalocus (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 eitherfadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade ofphaZ 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 β-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 thecore of the phenylacetyl-CoA catabolon is reported and discussed. 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 β-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the phalocus) 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 β-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the phalocus (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 eitherfadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade ofphaZ 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 β-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 thecore of the phenylacetyl-CoA catabolon is reported and discussed. polyhydroxyalkanoate phenylacetic acid 4-hydroxyphenylacetic acid n-alkanoic acids n-phenylalkanoic acid alkanoic acids with an odd number of carbon atoms phenylalkanoic acids with an odd number of carbon atoms alkanoic acids with an even number of carbon atoms phenylalkanoic acids with an odd number of carbon atoms polyhydroxyphenylalkanoate high pressure liquid chromatography g of dry weight cells acyl-CoA synthetase Polyhydroxyalkanoates (PHAs)1 are naturally occurring polyesters synthesized by different bacteria when cultured under a wide variety of nutritional and environmental conditions (1Byrom D. Trends Biotechnol. 1987; 5: 246-250Abstract Full Text PDF Scopus (444) Google Scholar, 2Anderson A.J. Dawes E.A. Microbiol. Rev. 1990; 54: 450-472Crossref PubMed Google Scholar, 3Brandl H. Gross R.A. Lenz R.W. Fuller R.C. 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Appl. Environ. Microbiol. 1993; 59: 1220-1227Crossref PubMed Google Scholar), and/or (iv) the existence of an additional catabolic route, linked to the β-oxidation pathway, to ensure complete assimilation of the β-oxidation products (benzoyl-CoA or phenylacetyl-CoA) generated from the monomers (3-hydroxyphenylalkanoyl-CoA derivatives) once released from the stored polymer (Fig. 1) (9Lageveen R.G. Huisman G.W. Preusting H. Ketelaar P. Eggink G. Witholt B. Appl. Environ. Microbiol. 1988; 54: 2924-2932Crossref PubMed Google Scholar). 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 (53Olivera E.R. Miñambres B. Garcı́a B. Muñiz C. Moreno M.A. Fernández A. Dı́az E. Garcı́a J.L. Luengo J.M. Proc. Nalt. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (179) Google Scholar) and its influence in the evolution of the catabolic potential of P. putida U are also discussed. 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. The strain ofP. 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 (1010bacteria). 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 (54Martı́nez-Blanco H. Reglero A. Rodrı́guez-Aparicio L.B. Luengo J.M. J. Biol. Chem. 1990; 265: 7084-7090Abstract Full Text PDF PubMed Google Scholar). 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 (54Martı́nez-Blanco H. Reglero A. Rodrı́guez-Aparicio L.B. Luengo J.M. J. Biol. Chem. 1990; 265: 7084-7090Abstract Full Text PDF PubMed Google Scholar) in which PhAc had been replaced by severaln-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 Zaidı́n, Consejo Superior de Investigaciones Cientı́ficas, 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 β-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 completepha 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. colimutant was also supplied by A. Steinbüchel and B. Rehm (Institut für Mikrobiologie, Westfälische Wilhems-Universität Münster, Münster, Germany). When required, a double mutantE. 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 (54Martı́nez-Blanco H. Reglero A. Rodrı́guez-Aparicio L.B. Luengo J.M. J. Biol. Chem. 1990; 265: 7084-7090Abstract Full Text PDF PubMed Google Scholar) PHPhAs were isolated following the procedure reported by Lageveen et al. (9Lageveen R.G. Huisman G.W. Preusting H. Ketelaar P. Eggink G. Witholt B. Appl. Environ. Microbiol. 1988; 54: 2924-2932Crossref PubMed Google Scholar). For these experiments, cells grown in a chemically defined medium (MM) (54Martı́nez-Blanco H. Reglero A. Rodrı́guez-Aparicio L.B. Luengo J.M. J. Biol. Chem. 1990; 265: 7084-7090Abstract Full Text PDF PubMed Google Scholar) 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 (49Fritzsche K. Lenz R.W. Makromol. Chem. 1990; 191: 1957-1965Crossref Google Scholar). 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. 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 (56Olivera E.R. Reglero A. Martı́nez-Blanco H. Fernández-Medarde A. Moreno M.A. Luengo J.M. Eur. J. Biochem. 1994; 221: 375-381Crossref PubMed Scopus (31) Google Scholar). In some cases, mutants were obtained by disruption of the required gene (see below). Mutants lacking a functional glyoxylic acid cycle or a β-oxidation pathway were characterized by enzyme assay (57Vanni P. Giachetti E. Pinzanti G. McFadden B.A. Comp. Biochem. Physiol. 1990; 95B: 431-458Google Scholar), 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 (8Lee Y.L. Biotechnol. Bioeng. 1996; 49: 1-14Crossref PubMed Scopus (1166) Google Scholar, 13Huisman G.W. Wonink E. Meima R. Kazemier B. Terpstra P. Witholt B. J. Biol. Chem. 1991; 266: 2191-2198Abstract Full Text PDF PubMed Google Scholar). 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 (58Miñambres B. Martı́nez-Blanco H. Olivera E.R. Garcı́a B. Dı́ez B. Barredo J.L. Moreno M.A. Schleissner C. Salto F. Luengo J.M. J. Biol. Chem. 1997; 271: 33531-33538Abstract Full Text Full Text PDF Scopus (40) Google Scholar). 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 min−1, 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 (56Olivera E.R. Reglero A. Martı́nez-Blanco H. Fernández-Medarde A. Moreno M.A. Luengo J.M. Eur. J. Biochem. 1994; 221: 375-381Crossref PubMed Scopus (31) Google Scholar, 59Luengo J.M. Moreno M.A. Anal. Biochem. 1987; 164: 559-562Crossref PubMed Scopus (10) Google Scholar, 60Carnicero D. Fernández-Valverde M. Cañedo L.M. Schleissner C. Luengo J.M. FEMS Microbiol. Lett. 1997; 149: 51-58Crossref Google Scholar). DNA manipulations, gel electrophoresis, DNA sequence analysis, primer synthesis, promoter analysis, and polymerase chain reaction amplification were performed as reported (53Olivera E.R. Miñambres B. Garcı́a B. Muñiz C. Moreno M.A. Fernández A. Dı́az E. Garcı́a J.L. Luengo J.M. Proc. Nalt. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (179) Google Scholar). 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 intoP. putida U by triparental mating as previously reported (53Olivera E.R. Miñambres B. Garcı́a B. Muñiz C. Moreno M.A. Fernández A. Dı́az E. Garcı́a J.L. Luengo J.M. Proc. Nalt. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (179) Google Scholar). Correct insertion of the pK18:mob in the desired gene was established by polymerase chain reaction amplification (53Olivera E.R. Miñambres B. Garcı́a B. Muñiz C. Moreno M.A. Fernández A. Dı́az E. Garcı́a J.L. Luengo J.M. Proc. Nalt. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (179) Google Scholar). 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. The uptake of labeled octanoic acid was carried out in bacterial cultures as previously reported (60Carnicero D. Fernández-Valverde M. Cañedo L.M. Schleissner C. Luengo J.M. FEMS Microbiol. Lett. 1997; 149: 51-58Crossref Google Scholar). When required, d
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