Polyhydroxyalkanoic Acid Research Articles

A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds.

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 microgram/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed.

Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid beta-oxidation by acrylic acid.

Heterologous expression of the phaC1 gene from Pseudomonas aeruginosa, which encodes one of the polyhydroxyalkanoic acid synthases, in Escherichia coli impaired in fatty acid beta-oxidation results in polyhydroxyalkanoic acid accumulation when cells were cultivated on fatty acids. We evaluated the application of the fatty acid beta-oxidation inhibitor acrylic acid as a tool to channel intermediates of beta-oxidation to polyhydroxyalkanoic acid synthesis. Various E. coli strains affected in fatty acid metabolism and the wild-type strain harboring plasmid pBHR71 were analyzed with respect to polyhydroxyalkanoic acid accumulation in the presence of acrylic acid. The E. coli fadR mutant RS3097 revealed the strongest polyhydroxyalkanoic acid accumulation. The optimum inhibitory concentration of acrylic acid was 0.24 mg ml-1 and caused efficient channeling of intermediates of beta-oxidation to polyhydroxyalkanoic acid synthesis. Under these conditions and grown on decanoate E. coli RS3097 harboring plasmid pBHR71 revealed a polyhydroxyalkanoic acid accumulation contributing to about 60% of cellular dry weight.

Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli ( fadR): inhibition of fatty acid β-oxidation by acrylic acid

Heterologous expression of the phaC1 gene from Pseudomonas aeruginosa, which encodes one of the polyhydroxyalkanoic acid synthases, in Escherichia coli impaired in fatty acid β-oxidation results in polyhydroxyalkanoic acid accumulation when cells were cultivated on fatty acids. We evaluated the application of the fatty acid β-oxidation inhibitor acrylic acid as a tool to channel intermediates of β-oxidation to polyhydroxyalkanoic acid synthesis. Various E. coli strains affected in fatty acid metabolism and the wild-type strain harboring plasmid pBHR71 were analyzed with respect to polyhydroxyalkanoic acid accumulation in the presence of acrylic acid. The E. coli fadR mutant RS3097 revealed the strongest polyhydroxyalkanoic acid accumulation. The optimum inhibitory concentration of acrylic acid was 0.24 mg ml −1 and caused efficient channeling of intermediates of β-oxidation to polyhydroxyalkanoic acid synthesis. Under these conditions and grown on decanoate E. coli RS3097 harboring plasmid pBHR71 revealed a polyhydroxyalkanoic acid accumulation contributing to about 60% of cellular dry weight.

A New Metabolic Link between Fatty Acid de NovoSynthesis and Polyhydroxyalkanoic Acid Synthesis: THE PHAG GENE FROM PSEUDOMONAS PUTIDAKT2440 ENCODES A 3-HYDROXYACYL-ACYL CARRIER PROTEIN-COENZYME A TRANSFERASE

To investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino acids with a molecular mass of 33,876 Da, and the amino acid sequence exhibited 44% amino acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201. S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon. Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source. No complementation of the rhlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressing phaG gene under lac promoter control, was obtained. Heterologous expression of phaG in Pseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source. Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P. oleovorans-harboring plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.

Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria.

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB-4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction.

Biological basis of enzyme‐catalyzed polyester degradation: 59 C‐terminal amino acids of poly(3‐hydroxybutyrate) (PHB) depolymerase a from pseudomonas lemoignei are sufficient for PHB binding

AbstractBiodegradable polyesters such as biologically produced poly[(R)‐3‐hydroxybutyric acid], (PHB) other polyhydroxyalkanoic acids and related chemosynthetic polyesters have attracted industrial interest, and bacterial produced PHB is commercially available since 1990. A large variety of polyester degrading microorganisms have been found to be present in environment. The microorganisms decompose the polymers by secretion of extracellular polyester depolymerases and utilize low molecular weight degradation products for growth. Microbial polyester depolymerases have the unique property to be water soluble and to be able to bind specifically to polyester surfaces. The objective of this contribution is a functional analysis of a bacterial PHB depolymerase polyester binding domain. In addition, a detailed summary of the present knowledge on the biochemistry of enzymatic polyester hydrolysis is provided.

Biosynthesis of novel copolyesters containing 3-hydroxypivalic acid by Rhodococcus ruber NCIMB 40126 and related bacteria

Rhodococcus ruber and related Gram-positive bacteria synthesized and accumulated novel copolyesters containing 3-hydroxypivalic acid as constituent if the cells were cultivated in a mineral salts medium containing 3-hydroxypivalic acid and glucose as carbon sources. The copolyesters contributed 0.4–10% of the cellular dry mass, and they contained up to 78 mol% of 3-hydroxypivalic acid in addition to 3-hydroxybutyric acid and 3-hydroxyvaleric acid; a homopolyester of 3-hydroxypivalic acid was also synthesized under certain conditions. The presence of 3-hydroxypivalic acid in the accumulated copolyesters was confirmed by nuclear magnetic resonance spectrometry as well by coupled gas chromatography/mass spectrometry. This is the first time that the incorporation of 3-hydroxypivalic acid and therefore of a hydroxyalkanoic acid with two methyl group substituents at the α-carbon atom in a naturally occurring copolyester is reported. It indicates that 3-hydroxypivalic acid-coenzyme A is accepted by polyhydroxyalkanoic acid synthase as a substrate.

Biosynthesis of novel copolyesters containing 3-hydroxypivalic acid byRhodococcus ruberNCIMB 40126 and related bacteria

Rhodococcus ruber and related Gram-positive bacteria synthesized and accumulated novel copolyesters containing 3-hydroxypivalic acid as constituent if the cells were cultivated in a mineral salts medium containing 3-hydroxypivalic acid and glucose as carbon sources. The copolyesters contributed 0.4–10% of the cellular dry mass, and they contained up to 78 mol% of 3-hydroxypivalic acid in addition to 3-hydroxybutyric acid and 3-hydroxyvaleric acid; a homopolyester of 3-hydroxypivalic acid was also synthesized under certain conditions. The presence of 3-hydroxypivalic acid in the accumulated copolyesters was confirmed by nuclear magnetic resonance spectrometry as well by coupled gas chromatography/mass spectrometry. This is the first time that the incorporation of 3-hydroxypivalic acid and therefore of a hydroxyalkanoic acid with two methyl group substituents at the α-carbon atom in a naturally occurring copolyester is reported. It indicates that 3-hydroxypivalic acid-coenzyme A is accepted by polyhydroxyalkanoic acid synthase as a substrate.

Microbial degradation of polyesters: a review on extracellular poly(hydroxyalkanoic acid) depolymerases

Thermoplastic polyesters such as biologically produced poly[( R)-3-hydroxy-butyric acid], other polyhydroxyalkanoic acids and related chemosynthetic polyesters, have properties which make them valuable for many applications. In addition, these materials can be biodegraded to water and carbon dioxide. Many aerobic and anaerobic polyester-degrading microorganisms have been found in all moderate ecosystems. The microorganisms decompose the polymers by secretion of extracellular polyester depolymerases and utilize low molecular weight degradation products. This review summarizes the present knowledge on microbial extracellular polyester depolymerases.

A new Comamonas testosteroni steroid-inducible gene: Cloning and sequence analysis

Comamonas testosteroni can grow on a variety of steroid compounds as the sole carbon and energy source. In a previous study, we cloned and sequenced the testosterone-inducible β hsd gene from C. testosteroni (Genti-Raimondi, S., Tolmasky, M., Patrito, L., Flury, A. and Actis, L., Molecular cloning and expression of the β-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni. Gene, 1991, 105, 43–49.). Herein we report the cloning and characterization of another steroid-inducible gene ( stdC), located 2400 bp upstream of β hsd. Nucleotide sequencing of a region encompassing the stdC gene revealed an open reading frame 546 bp long including the stop codon TGA with significant similarity to the orf4, orf1 and orf4 of unknown function described in the polyhydroxyalkanoic acid (PHA) cluster of Chromatium vinosum, Rhizobium meliloti and Thiocystis violacea, respectively. The aminoacid sequence deduced from the nucleotide sequence predicts a putative protein of 181 amino acids with a molecular weight of 20 715 Da. Northern blot experiments indicate that the stdC gene was transcribed as a monocistronic mRNA with an apparent molecular size of 670 nt. The stdC transcript was abundant in C. testosteroni cells grown with different steroid carbon sources harvested in the exponential phase and was found to be under catabolite repression.

Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003.

Cosmids from the 1A3-1A10 region of the complete miniset were individually subcloned by using the vector M13 mp18. Sequences of each cosmid were assembled from about 400 DNA fragments generated from the ends of these phage subclones and merged into one 189-kb contig. About 160 ORFs identified by the CodonUse program were subjected to similarity searches. The biological functions of 80 ORFs could be assigned reliably by using the WIT and Magpie genome investigation tools. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Most of the ORFs lacking significant similarity with reference databases also were grouped. There are two large clusters of these ORFs, one located between 45 and 67 kb of the map, and the other between 150 and 183 kb. Nine of the loosely identified ORFs (of 15) of the first of these clusters match ORFs from phages or transposons. The other cluster also has four ORFs of possible phage origin.

Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli

The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli. Wild-type strains and other widely used non-recombinant strains of E. coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source. Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene ( phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase. A 3.5-kb genomic SmaI/ ApaI fragment from A. eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/ EcoRI fragment from C. kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS − and pSK −, respectively. The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E. coli XL1-Blue. Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources. If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated. The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources. Poly(4HB) could amount up to approximately 80% (w/w) of the cell dry matter if E. coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen. 4HB was also incorporated into PHA if γ-butyrolactone was used as carbon source. If levulinic acid, 4-hydroxyvaleric acid or γ-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents.

Biosynthesis and biotechnological production of degradable polyhydroxyalkanoic acid

The use of biodegradable polymers is one of the key solution to environmental problems and the development of biocompatible material. The impact of such a large commercial opportunity is one of the primary reasons for much interest in the field of microbial polyester, polyhydroxyalkanoic acid (PHA). Its valuable properties of biodegradability, biocompatibility and thermoplasticity have attracted considerable commercial interest, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has been launched as the first market product. Recent advances in molecular genetics and microbial physiology of PHA biosynthesis have been uncovering the biosynthetic mechanics at molecular level, and extensive efforts for the developments of practical applications and cost-effective mass production of PHA will profell the commercialization of PHA towards the commodity market for biodegradable plastics. The biosynthesis of new members of PHA family with new monomer or unusual composition will also lead to the biotechnological production of tailor-made biopolymer for various applications.

Lipid storage compounds in marine bacteria

Forty psychrophile or psychrotrophic crude-oil-utilizing marine bacteria were investigated for their ability to accumulate lipid storage compounds in the cytoplasm during cultivation under nitrogen-limiting conditions. Most of them (73%) were able to accumulate specialized lipids like polyhydroxyalkanoic acids (PHA) while other lipids such as wax esters occurred in two isolates. Accumulation of PHA occurred predominantly at low temperatures (4–20 °C) as demonstrated for three isolates. Electron microscopy revealed polyphosphate inclusions occurring in two isolates in addition to PHA. Cells of the isolate Acinetobacter sp. 211 were able to synthesize and accumulate lipid inclusions during growth on acetate, ethanol, olive oil, hexadecanol and heptadecane. The composition of the lipid inclusions depended on the compounds provided as carbon source. Wax esters and acylglycerols occurred mainly during the cultivation on olive oil; in contrast, wax esters and free alcohols occurred during cultivation on hexadecanol. Total fatty acids in cells of the Acinetobacter sp. 211 amounted to 25% of the cellular dry weight in olive-oil-grown cells. Palmitic acid was the main fatty acid in the lipids when the cells were cultivated on acetate or ethanol (44% and 32% of total fatty acids respectively). In contrast, fatty acids occurring in the lipids during cultivation on hexadecanol, heptadecane or olive oil were related to the carbon source. The fatty acids present in the accumulated lipids consisted predominantly of saturated and unsaturated straight-chain fatty acids with a chain length ranging from 12 to 18 carbon atoms. Analysis of the lipid-granule-associated proteins in cells of Acinetobacter sp. 211 revealed a protein of 39 kDa as the predominant protein species.

Structural identification of polyhydroxyalkanoic acid (PHA) containing 4-hydroxyalkanoic acids by gas chromatography-mass spectrometry (GC-MS) and its application to bacteria screening

The methanolysis products of polyhydroxyalkanoic acids (PHAs) containing 4-hydroxybutyric acid (4HB), 4-hydroxyvaleric acid (4HV), and 4-hydroxyhexanoic acid (4HHx), when analyzed by GC-MS, showed two major chromatographic peaks with characteristic retention times of each methyl ester of 4-hydroxyalkanoic acid and the corresponding g-lactone (γ-butyrolactone, γ-valerolactone, γ-caprolactone, respectively). The method and results of GC-MS could be incorporated into an efficient screening procedure for isolation of bacterial strains which could accumulate a PHA containing 4-hydroxyalkanoic acid as the principal monomer from structurally related carbon substrates.

Determination of the thickness of the boundary layer surrounding bacterial PHA inclusion bodies, and implications for models describing the molecular architecture of this layer

AbstractThe thickness of the boundary layer surrounding polyhydroxyalkanoic acid (PHA) inclusion bodies in bacteria was determined from electron micrographs of ultrathin cell sections. The obtained values were compared with the thickness of boundary layers of other kinds of prokaryotic inclusion bodies known to be monolayers (sulfur globule, hydrocarbon‐containing inclusion, carboxysome, gas vesicle, chiorobium vesicle) and the thickness of typical bilayer membranes (cytoplasmic membrane, photosynthetic vesicle). The data are discussed in view of contradicting models published recently for the molecular architecture of the boundary layer of PHA inclusion bodies.

Biodegradation of polyhydroxyalkanoic acids.

Stimulated by the commercial availability of bacteriologically produced polyesters such as poly[(R)-3-hydroxybutyric acid], and encouraged by the discovery of new constituents of polyhydroxyalkanoic acids (PHA), a considerable body of knowledge on the metabolism of PHA in microorganisms has accumulated. The objective of this essay is to give an overview on the biodegradation of PHA. The following topics are discussed: (i) general considerations of PHA degradation, (ii) methods for identification and isolation of PHA-degrading microorganisms, (iii) characterization of PHA-degrading microorganisms, (iv) biochemical properties of PHA depolymerases, (v) mechanisms of PHA hydrolysis, (vi) regulation of PHA depolymerase synthesis, (vii) molecular biology of PHA depolymerases, (viii) influence of the physicochemical properties of PHA on its biodegradability, (ix) degradation of polyesters related to PHA, (x) biotechnological aspects of PHA and PHA depolymerases.

Identification of 5-hydroxyhexanoic acid, 4-hydroxyheptanoic acid and 4-hydroxyoctanoic acid as new constituents of bacterial polyhydroxyalkanoic acids

A recombinant strain of Pseudomonas putida GPp104 (pHP1014::E146), which expressed the polyhydroxyalkanoic acid (PHA) synthase of Thiocapsa pfennigii exhibiting an unusual substrate specificity at a high level was incubated in two-stage batch or fed-batch accumulation experiments with 5-hydroxyhexanoic acid (5HHx) as carbon source in the second cultivation phase, copolyesters of 3-hydroxybutyric acid (3HB) plus 5HHx, or of 3HB, 3-hydroxyhexanoic acid (3HHx) plus 5HHx were accumulated as revealed by gas-chromatographic and 13C-NMR spectroscopic analysis. When the recombinant P. putida GPp104 was incubated with 4-hydroxyheptanoic acid (4HHp) as carbon source in the second cultivation phase, a copolyester consisting of 3HB, 3-hydroxyvaleric acid and 3- and 4-hydroxyheptanoic acid accumulated. Providing 4-hydroxyoctanoic acid as carbon source in the second cultivation phase led to the accumulation of a polyester that contained 1–2 mol% 4-hydroxyoctanoic acid besides 3-hydroxyoctanoic acid, 3HHx, 3-hydroxyvaleric acid and 3HB. In addition to PHA containing these new constituents, PHA with 4-hydroxyvaleric acid was accumulated from laevulinic acid. Eleven strains from five genera have been also analysed for their ability to utilize different carbon sources for colony growth, which might serve as potential precursors for the biosynthesis of PHA with unusual constituents. Although most of the carbon sources were utilized by some strains for colony growth, accumulation experiments gave no evidence for the accumulation of new PHA by these wild-type strains.

Evaluation of soil gram-negative bacteria yielding polyhydroxyalkanoic acids from carbohydrates and propionic acid

A screening programme was developed leading to the isolation of 75 strains of soil gram-negative bacteria which are able to produce polyhydroxyalkanoic acids (PHA) from sugar-cane derivatives. The evaluation of these strains was performed with regard to their efficiency in converting carbohydrates or propionic acid into PHA constituents. Several strains were able to use sucrose as well as glucose and fructose to grow and afterwards to accumulate poly-(3-hydroxybutyric acid) (PHB) with promising yields. Seven strains were found to have more than 80% of the theoretical value when converting carbohydrates into PHB and accumulated at least 50% of the cell dry weight as PHB. Ten strains incorporated 3-hydroxyvaleric acid units into the polymer from propionic acid of which 3 gave yields comparable to those of Alcaligenes eutrophus.

Incorporation of 2-methyl-3-hydroxybutyric acid into polyhydroxyalkanoic acids by axenic cultures in defined media

Alcaligenes eutrophus and Burkholderia cepacia synthesized and accumulated a terpolyester consisting of 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 2-methyl-3-hydroxybutyric acid (2Me3HB) if the cells were cultivated in a mineral salts medium containing tiglic acid as the sole carbon source or in combination with gluconic acid. The presence of 1–2 mol% of 2Me3HB in the polyester was confirmed by comparison with chemically synthesized methyl ester of 2Me3HB and by nuclear magnetic resonance spectrometry as well as by gas chromatography/mass spectrometry. This is the first report of the incorporation of 2Me3HB by axenic cultures cultivated under defined conditions.