Biosynthesis of multicomponent polyhydroxyalkanoates by <Emphasis Type="Italic">Wautersia eutropha</Emphasis>

The effect of carbon supply on polyhydroxyalkanoate (PHA) synthesis by bacteria Wautersia eutropha was studied. Synthesis of multicomponent PHA composed of short-and long-chain monomers (C₄–C₈) by two natural strains (H16 and B5786) under mixotrophic conditions (CO₂ + alkanoic acids as cosubstrates) was demonstrated for the first time. The PHA composition was shown to be dependent on the cosubstrate type. In the presence of odd fatty acids, four-and five-component polymers were synthesized; hydroxybutyrate, hydroxyvalerate, and hydroxyheptanoate were the major monomers, while hydroxyhexanoate and hydroxyoctanoate were minor. In the presence of even fatty acids, PHA contained not only the corresponding molecules (hydroxyhexanoate and hydroxyoctanoate), but also hydroxyvalerate; synthesis of four-component PHA which contain mainly hydroxybutyrate and hydroxyhexanoate (up to 18 mol %) is therefore possible. A series of four-and five-component PHA was synthesized and their physicochemical characteristics were determined.     

Physicochemical properties of multicomponent poly(hydroxyalkanoates)

The properties of new five-component poly(hydroxyalkanoates) (PHA) formed by short-and medium-chain monomers synthesized by the bacterium Wautersia eutropha B5786 were studied by X-ray diffraction, IR spectroscopy, differential thermal analysis, and viscometry. The degree of crystallinity of PHA decreased from 72 to 57% as the molar fraction of hydroxyhexanoate increased from 2.5 to 18.0 mol%. The melting temperature (T _m) and decomposition temperature (T _d) of the multicomponent PHA are lower than those for poly(hydroxybutyrate), whose T _m and T _d are 168–170 and 260–265°C, respectively. Both parameters of the multicomponent PHA decrease to 156 and 252°C, respectively, as the hydroxyhexanoate mole fraction is raised. The effect of hydroxyhexanoate on the physicochemical properties of the PHA is similar to that of hydroxyvalerate observed previously.     

Physicochemical properties of multicomponent polyhydroxyalkanoates

X-ray structure analysis, IR spectrometry, differential thermal analysis, and viscosimetry have been used to investigate the properties of novel five-component polyhydroxyalkanoates formed by short- and medium-chain-length monomers synthesized by the bacterium Wautersia eutropha B5786. As the molar fraction of hydroxyhexanoate contained in polyhydroxyalkanoates samples increased from 2.5 to 18.0 mol%, their degree of crystallinity decreased from 72 to 57%. The melting temperature of multicomponent polyhydroxyalkanoates (Tm) and their temperature for the onset of decomposition (Td) are lower than those of polyhydroxybutyrate, whose Tm is 168-170 degrees C and Td 260-265 degrees C. In multicomponent polymers (PHA(SC+MC)), both parameters decrease as the molar fraction of hydroxyhexanoate grows to 156 and 252 degrees C, respectively, in the range of hydroxyhexanoate content studied. Hydroxyhexanoate influences the physicochemical properties of polyhydroxyalkanoates similarly to hydroxyvalerate; as the fraction of either of these medium-chain-length monomers in polyhydroxyalkanoates increases, the crystallinity of the polymer decreases, but its thermostability remains unchanged.     

Production of polyhydroxyalkanoates by Pseudomonas nitroreducens

A strain coded AS 1.2343 was isolated from oil-contaminated soil in an oil-field in North China Tianjian City and it was identified as Pseudomonas nitroreducens. The strain demonstrated some unusual ability to synthesize polyhydroxybutyrate (PHB) homopolymer from medium-chain-length (mcl) fatty acids including hexanoate and octanoate. While polyhydroxyalkanoates (PHA) consisting of mcl hydroxyalkanoate (HA) monomers such as hydroxyoctanoate (HO) and hydroxydecanoate (HD) were the major compositions when butyrate, decanoate, lauric acid and tetradecanoic acid were used as substrates for the cell growth, respectively. PHA was accumulated up to 77% of the cell dry weight when growth was conducted in lauric acid, it appeared that the HA contents in the PHA would not be much affected by the changing of the lauric acid concentration. Varying the concentration ratio of butyrate to octanoate could change the composition of PHA accumulated by the strain. Yet PHB homopolymer was always the only polyester synthesized by the strain, regardless of the octanoate concentration change. Additionally, the ratio of carbon to nitrogen (C/N) in butyrate media was found to have effects on the PHA monomer content, as C/N increased from 2 to 100, content of HB decreased from 100% to 7%. PHA polyester synthesized by cells of Pseudomonas nitroreducens AS 1.2343 was a blend polymers consisting of acetone-insoluble HB and acetone-soluble mcl HA monomers.     

Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater

Production of polyhydroxyalkanoates (PHAs) in activated sludge treating wastewater represents an economical and environmental promising alternative to pure culture fermentations. A process for production of PHA from a paper mill wastewater was examined at laboratory scale. The three stage process examined consisted of acidogenic fermentation to convert wastewater organic matter to volatile fatty acids (VFAs), an activated sludge system operating under feast/famine conditions to enrich for PHA producing organisms and accumulation of PHA in batch experiments. After fermentation of the wastewater, 74% of the soluble COD was present as VFA (acetate, propionate, butyrate and valerate) and the resulting PHA after batch accumulation consisted of 31-47 mol% hydroxybutyrate and 53-69 mol% hydroxyvalerate. The maximum PHA content achieved was 48% of the sludge dry weight and the three stage process exhibited a potential to produce 0.11 kg of PHA per kg of influent COD treated.     

Hyperproduction of polyesters consisting of medium-chain-length hydroxyalkanoate monomers by strain Pseudomonas stutzeri 1317

Pseudomonas stutzeri strain 1317 was found to grow on various fatty acids, alcohols, diols, as well as glucose and gluconate for the synthesis of polyhydroxyalkanoates (PHA) with various monomer units. The PHA monomer structures were dependent on the type of fatty acids and alcohols, as well as the diols in the culture media. Only even number monomers, such as 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD), were accumulated when even numbered fatty acids, alcohols, glucose and gluconate, as well as diol were used as carbon sources. Odd numbered fatty acids and odd numbered alcohols led to the formation of odd numbered monomers, such as 3-hydroxyvalerate (HV), 3-hydroxyheptanoate (HHp), 3-hydroxynonanoate (HN) and 3-hydroxyundecanoate (HU). The strain tolerated up to 1.5% of ethanol and made 8.3% of PHA when growth was conducted in 1.2% of ethanol. PHA formed up to 77% of cell dry weight when the strain was grown in tridecanoate. PHA synthesis was highly dependent on the nitrogen source. A depletion in nitrogen supply immediately resulted in PHA accumulation in cells grown in the glucose mineral medium.     

A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria as inclusion bodies, and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. In this paper, Fourier-transform infrared spectroscopy (FT-IR) was used to carry out rapid qualitative analysis of PHA in intact bacterial cells. The FT-IR spectra of pure PHA containing short-chain-length monomers, such as hydroxybutyrate (HB), medium-chain-length hydroxyalkanoate (mclHA) monomers including hydroxyoctanoate (HO) and hydroxydecanoate (HD), or both HB and mclHA monomers, showed their strong characteristic band at 1728 cm⁻¹, 1740 cm⁻¹ or 1732 cm⁻¹ respectively. Other accompanying bands near 1280 cm⁻¹ and 1165 cm⁻¹ helped identify the types of PHA. The intensity of the methylene band near 2925 cm⁻¹ provided additional information for PHA characterization. In comparison, bacterial cells accumulating the above PHA also showed strong marker bands at 1732 cm⁻¹, 1744 cm⁻¹ or 1739 cm⁻¹, corresponding to intracellular PHB, mclPHA and P(HB + mclHA) respectively. The accompanying bands visible in pure PHA were also observable in the intact cells. The FT-IR results were further confirmed by gas chromatography analysis. Received: 14 October 1998 / Received revision: 29 December 1998 / Accepted: 30 December 1998     

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.     

Biopolymers production from mixed cultures and pyrolysis by-products

Polyhydroxyalkanoates (PHAs) production from low value substrates and/or byproducts represents an economical and environmental promising alternative to established industrial manufacture methods. Bio-oil resulting from the fast-pyrolysis of chicken beds was used as substrate to select a mixed microbial culture (MMC) able to produce PHA under feast/famine conditions. In this study a maximum PHA content of 9.2% (g/g cell dry weight) was achieved in a sequencing batch reactor (SBR) operated for culture selection. The PHA obtained with bio-oil as a carbon source was a copolymer composed by 70% of hydroxybutyrate (HB) and 30% of hydroxyvalerate (HV) monomers. Similar results have been reported by other studies that use real complex substrates for culture selection indicating that bio-oil can be a promising feedstock to produce PHAs using MMC. To the best of our knowledge this is the first study that demonstrated the use of bio-oil resulting from fast pyrolysis as a possibly feedstock to produce short chain length polyhydroxyalkanoates.     

Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through beta-oxidation

Transgenic plants producing peroxisomal polyhydroxy- alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the beta-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the beta-oxidation of these fatty acids. Tween-laurate was a stronger inducer of beta-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween-oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards beta-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the beta-oxidation cycle as well as removal and degradation of these unusual fatty acids through beta-oxidation. Such enhanced flow of fatty acids through beta-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through beta-oxidation as well as to analyse the degradation pathway of unusual fatty acids.     

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.     

Synthesis of Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae by Using Intermediates of Fatty Acid β-Oxidation

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the β-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.     

Enhanced biosynthesis of polyhydroxyalkanoates in a mutant strain of Rhizobium meliloti

Strains of Rhizobium spp. isolated from leguminous plants and standard strains accumulated 27% to 57% polyhydroxyalkanoate (PHA) of their cell biomass. Among these cultures, one strain of Rhizobium meliloti synthesized 10–30% more PHA than others and contained 3% hydroxyvalerate (HV) when grown on sucrose as carbon substrate. The occurrence of hydroxybutyrate (HB) and HV was confirmed by GC and ¹H NMR analysis. Treatment of the culture with 4-N-piperidinobutyl-2-chlorophenoxazine resulted in a mutant which synthesized upto 69%, PHA of the cell biomass with an improved yield of 11 to 47% under different carbon and nitrogen ratios, compared to the parent strain.     

Effect of carbon source on the formation of polyhydroxyalkanoates (PHA) by a phosphate-accumulating mixed culture

In the present work, attention was devoted to understand how different carbon substrates and their concentration can influence the production of PHA by polyphosphate-accumulating bacteria. Acetate, propionate, and butyrate were tested independently. The composition of the polymers formed was found to vary with the substrate used. Acetate leads to the production of a copolymer of hydroxybutyrate (HB) and hydroxyvalerate (HV) with the HB units being dominant. With propionate, HV units are mainly produced and only a small amount of HB is synthesized. When butyrate is used, the amount of polymer formed is much lower with the HB units being produced to a higher extent. The yield of polymer produced per carbon consumed (Y_P/S) was found to diminish from acetate (0.97) to propionate (0.61) to butyrate (0.21). Using a mixture of acetate, propionate, and butyrate and increasing the carbon concentration, although maintaining the relative concentration of each substrate, propionate is primarily consumed and consequently, PHA synthesized was enriched in HV units. The polymers obtained in all experiments were copolymers with the average molecular weight of the most representative fraction higher when hydroxybutyrate units were present in considerable amounts. All the polymers synthesized were found to be quite homogeneous and their average molecular weight is of the same order of magnitude as the ones commercially available.     

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ?foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ?scdA?echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ?scdA?echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.     

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.     

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.     

Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Direct utilization of palm oil for the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids was demonstrated using Pseudomonas aeruginosa IFO3924. By secreted lipase, palm oil was hydrolyzed into glycerol and fatty acids. Fatty acids became favorable carbon sources for cell growth and PHA production via β-oxidation and glycerol for rhamnolipid production via de novo fatty acid synthesis. Both PHA and rhamnolipid syntheses started after the nitrogen source was exhausted and cell growth ceased. PHA synthesis continued until all fatty acids were exhausted, and at that time, PHA content in the cells reached a maximum, but stopped despite the remaining glycerol (<2g/l). In contrast, rhamnolipid synthesis continued until glycerol was exhausted.     

Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads

The fluorescent pseudomonads are classified as a group, one characteristic of which is that they do not accumulate poly-3-hydroxybutyrate (PHB) during nutrient starvation in the presence of excess carbon source. In this paper we show that prototype strains from this subclass, such as Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens, do accumulate poly-3-hydroxyalkanoates (PHA) when grown on fatty acids. These PHAs are composed of medium-chain-length (C6 to C12) 3-hydroxy fatty acids. The ability to form these polyesters does not depend on the presence of plasmids. A specificity profile of the enzymes involved in the biosynthesis of PHA was determined by growing Pseudomonas oleovorans on fatty acids ranging from C4 to C18. In all cases, PHAs were formed which contained C6 to C12 3-hydroxy fatty acids, with a strong preference for 3-hydroxyoctanoate when Ceven fatty acids were supplied and 3-hydroxynonanoate when Codd fatty acids were the substrate. These results indicate that the formation of PHAs depends on a specific enzyme system which is distinct from that responsible for the synthesis of PHB. While the fluorescent pseudomonads are characterized by their inability to make PHB, they appear to share the capacity to produce PHAs. This characteristic may be helpful in classifying pseudomonads. It may also be useful in the optimization of PHA production for biopolymer applications.     

Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. Identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA

Pseudomonas oleovorans accumulates poly(3-hydroxyalkanoates) (PHAs) after growth on medium chain length hydrocarbons. Large amounts of this polyester are synthesized when cells are grown under nitrogen-limiting conditions. When nitrogen is resupplied in the medium, the accumulated PHA is degraded. In this paper, we describe mutants which are defective in the synthesis or in the degradation of PHA. These mutants were used to select DNA fragments which encode PHA polymerases and a PHA depolymerase. A 25-kilobase (kb) DNA fragment was isolated from P. oleovorans that complements a Pseudomonas putida mutant unable to accumulate PHA. Subcloning resulted in the assignment of a 6.4-kb EcoRI fragment as the pha locus, containing genetic information for PHA synthesis. Mutants in the PHA degradation pathway were also complemented by this fragment, indicating that genes encoding PHA biosynthetic and degradative enzymes are clustered. Analysis of the DNA sequence of the 6.4-kb fragment revealed the presence of two open reading frames encoding PHA polymerases based on homology to the poly(3-hydroxybutyrate) polymerase from Alcaligenes eutrophus. A third open reading frame complemented the PHA degradation mutation and is likely to encode a PHA depolymerase. The presence of two PHA polymerases is due to a 2098-base pair DNA duplication. The PHA polymerases are 53% identical and show 35-40% identity to the poly(3-hydroxybutyrate) polymerase. No clear difference in specificity was found for the PHA polymerases. However, with the pha locus cloned on a multicopy vector, a polymer was accumulated that contains a significantly higher amount of substrate-derived monomers. An increase in the rate of polyester synthesis versus oxidation of the monomers in the beta-oxidation explains these findings.