Crystal structure of multi-functional enzyme FadB from Cupriavidus necator: Non-formation of FadAB complex.

Cupriavidus necator H16 is a gram-negative chemolithoautotrophic bacterium that has been extensively studied for biosynthesis and biodegradation of polyhydroxyalkanoate (PHA) plastics. To improve our understanding of fatty acid metabolism for PHA production, we determined the crystal structure of multi-functional enoyl-CoA hydratase from Cupriavidus necator H16 (CnFadB). The predicted model of CnFadB created by AlphaFold was used to solve the phase problem during determination of the crystal structure of the protein. The CnFadB structure consists of two distinctive domains, an N-terminal enol-CoA hydratase (ECH) domain and a C-terminal 3-hydroxyacyl-CoA dehydrogenase (HAD) domain, and the substrate- and cofactor-binding modes of these two functional domains were identified. Unlike other known FadB enzymes that exist as dimers complexed with FadA, CnFadB functions as a monomer without forming a complex with CnFadA. Small angle X-ray scattering (SAXS) measurement further proved that CnFadB exists as a monomer in solution. The non-sequential action of FadA and FadB in C. necator appears to affect ß-oxidation and PHA synthesis/degradation.

In vitro evidence of chain transfer to tetraethylene glycols in enzymatic polymerization of polyhydroxyalkanoate.

A polyhydroxyalkanoate (PHA) was enzymatically synthesized in vitro, and the end structure of PHA associated with a chain transfer (CT) reaction was investigated. In the CT reaction, PHA chain transfers from PHA synthase (PhaC) to a CT agent, resulting in covalent bonding of CT agent to the PHA chain at its carboxyl end. In vitro CT reaction has never been demonstrated because of relatively low yields of in vitro synthesized poly[(R)-3-hydroxybutyrate)] (P(3HB)), which makes it difficult to characterize the end structures of the polymers by nuclear magnetic resonance (NMR). To overcome these difficulties, a novel in vitro synthesis method that produced relatively larger amounts of P(3HB) was developed by employing PhaCDa from Delftia acidovorans and two enantioselective enoyl-coenzyme A (CoA) hydratases which were R-hydratase (PhaJAc) from Aeromonas caviae and S-hydratase (FadB1x) from Pseudomonas putida KT2440 with ß-butyrolactone and CoA as starting materials. Using this method, P(3HB) synthesis was performed with tetraethylene glycols (TEGs) as a discriminable CT agent, and the resultant P(3HB) was characterized by (1)H-NMR. NMR analysis revealed that the carboxylic end of P(3HB) was covalently linked to TEGs, providing the first direct evidence of in vitro CT reaction.

Engineered Escherichia coli for short-chain-length medium-chain-length polyhydroxyalkanoate copolymer biosynthesis from glycerol and dodecanoate.

Short-chain-length medium-chain-length polyhydroxyalkanoate (SCL-MCL PHA) copolymers are promising as bio-plastics with properties ranging from thermoplastics to elastomers. In this study, the hybrid pathway for the biosynthesis of SCL-MCL PHA copolymers was established in recombinant Escherichia coli by co-expression of ß-ketothiolase (PhaARe) and NADPH-dependent acetoacetyl-CoA reductase (PhaBRe) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaCRe), Aeromonas hydrophila (PhaCAh), and Pseudomonas putida (PhaC2Pp) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1Pp and PhaJ4Pp), and A. hydrophila (PhaJAh). When glycerol supplemented with dodecanoate was used as primary carbon source, E. coli harboring various combinations of PhaABCJ produced SCL-MCL PHA copolymers of various monomer compositions varying from C4 to C10. In addition, polymer property analysis suggested that the copolymers produced from this recombinant source have thermal properties (lower glass transition and melting temperatures) superior to polyhydroxybutyrate homopolymer.

Biosynthesis and characterization of 3-hydroxyalkanoate terpolyesters with adjustable properties by Aeromonas hydrophila.

Polyhydroxyalkanoates (PHA) terployesters P(3HB-co-3HV-co-3HHx) consisting of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) were produced by wild-type Aeromonas hydrophila 4AK4, its recombinant harboring PHA synthesis genes phaPCJ encoding PHA binding protein phasin, PHA synthase, and enoyl-CoA hydratase, and another its recombinant harboring phaAB encoding beta-ketothiolase and acetoacetyl-CoA reductase, respectively, when grown in lauric acid and/or valerate. The terpolyesters produced by A. hydrophila 4AK4 (phaAB) grown in velarate were found to produce copolymers P(3HB-co-3HV) containing high 3HV fractions with a maximum of 99 mol% 3HV. In terpolyesters, 3HV ranged from 9 to 32 mol% depending on the valerate concentration and strain used. A maximal terpolyester P(3HB-co-3HV-co-3HHx) content in dry cells was 71 wt%. Transmission electron microscopy study of A. hydrophila 4AK4 harboring phaPCJ revealed the full occupation of terpolyester P(3HB-co-3HV-co-HHx) in the cellular spaces. Terpolyesters with various monomer compositions showed changing thermal and mechanical properties. Those with higher 3HV fractions demonstrated an improved property over the lower HV containing ones.

Poly[(R)-3-hydroxybutyrate] formation in Escherichia coli from glucose through an enoyl-CoA hydratase-mediated pathway.

In this study, a new metabolic pathway for the synthesis of poly[(R)-3-hydroxybutyrate] [P(3HB)] was constructed in a recombinant Escherichia coli strain that utilized forward and reverse reactions catalyzed by two substrate-specific enoyl-CoA hydratases, R-hydratase (PhaJ) and S-hydratase (FadB), to epimerize (S)-3HB-CoA to (R)-3HB-CoA via a crotonyl-CoA intermediate. The R-hydratase gene (phaJ(Ac)) from Aeromonas caviae was coexpressed with the PHA synthase gene (phaC(Re)) and 3-ketothiolase gene (phaA(Re)) from Ralstonia eutropha in fadR mutant E. coli strains (CAG18497 and LS5218), which had constitutive levels of the beta-oxidation multienzyme FadB(Ec). When grown on glucose as the sole carbon source, the cells accumulated P(3HB) up to an amount 6.5 wt% of the dry cell weight, whereas the control cells without phaJ(Ac) or fadR mutation accumulated significantly smaller amounts of P(3HB). These results suggest that PhaJ(Ac) and FadB(Ec) played an important role in supplying monomers for P(3HB) synthesis in the pathway. Furthermore, by using this pathway, a P(3HB)-concentration-dependent fluorescent staining screening technique was developed to rapidly identify cells that possess active R-hydratase.

Identification of a fabZ gene essential for flexirubin synthesis in Cytophaga hutchinsonii.

Cytophaga hutchinsonii, an aerobic soil bacterium which could degrade cellulose, produces yellow flexirubin pigments. In this study, fabZ, annotated as a putative ß-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase gene, was identified by insertional mutation and gene deletion as an essential gene for flexirubin pigment synthesis. The availability of a FabZ mutant that fails to produce flexirubin allowed us to investigate the biological role of the pigment in C. hutchinsonii. Loss of flexirubin made the FabZ mutant more sensitive to UV radiation, oxidative stress and alkaline stress than the wild type.

Butyrate production under aerobic growth conditions by engineered Escherichia coli.

Butyrate is an important industrial platform chemical. Although several groups have reported butyrate production under oxygen-limited conditions by a native producer, Clostridium tyrobutylicum, and by a metabolically engineered Escherichia coli, efforts to produce butyrate under aerobic growth conditions have met limited success. Here, we constructed a novel butyrate synthetic pathway that functions under aerobic growth conditions in E. coli, by modifying the 1-butanol synthetic pathway reported previously. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, and endogenous thioesterase(s) of E. coli. To evaluate the potential of this pathway for butyrate production, culture conditions, including pH, oxygen supply, and concentration of inorganic nitrogen sources, were optimized in a mini-jar fermentor. Under the optimal conditions, butyrate was produced at a concentration of up to 140 mM (12.3 g/L in terms of butyric acid) after 54 h of fed-batch culture.

Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 A resolution.

beta-Oxidation of amino acyl coenzyme A (acyl-CoA) species in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. MFE-2 has a modular organization of three domains. The function of the C-terminal domain of the mammalian MFE-2, which shows similarity with sterol carrier protein type 2 (SCP-2), is unclear. Here, the structure of the SCP-2-like domain comprising amino acid residues 618-736 of human MFE-2 (d Delta h Delta SCP-2L) was solved at 1.75 A resolution in complex with Triton X-100, an analog of a lipid molecule. This is the first reported structure of an MFE-2 domain. The d Delta h Delta SCP-2L has an alpha/beta-fold consisting of five beta-strands and five alpha-helices; the overall architecture resembles the rabbit and human SCP-2 structures. However, the structure of d Delta h Delta SCP-2L shows a hydrophobic tunnel that traverses the protein, which is occupied by an ordered Triton X-100 molecule. The tunnel is large enough to accommodate molecules such as straight-chain and branched-chain fatty acyl-CoAs and bile acid intermediates. Large empty apolar cavities are observed near the exit of the tunnel and between the helices C and D. In addition, the C-terminal peroxisomal targeting signal is ordered in the structure and solvent-exposed, which is not the case with unliganded rabbit SCP-2, supporting the hypothesis of a ligand-assisted targeting mechanism.

Co-expression of polyhydroxyalkanoate synthase and (R)-enoyl-CoA hydratase genes of Aeromonas caviae establishes copolyester biosynthesis pathway in Escherichia coli.

Polyhydroxyalkanoate biosynthesis genes of Aeromonas caviae were expressed in Escherichia coli LS5218 (fadR atoC(Con)), and the polyhydroxyalkanoate-producing ability of the recombinants was investigated. A LS5218 strain harboring only phaCAc (polyhydroxyalkanoate synthase gene) did not accumulate any polyhydroxyalkanoate from dodecanoate in spite of the existence of translated polyhydroxyalkanoate synthase protein, whereas co-expression phaCAc and phaJAc ((R)-specific enoyl-CoA hydratase gene) resulted in the accumulation of P(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer up to 7-11 wt% of dry cell weight from octanoate and dodecanoate. These results indicated that both phaCAc and phaJAc are essential for E. coli LS5218 to establish the polyhydroxyalkanoate biosynthesis pathway from alkanoic acids. The copolyester content in the strain expressing both the genes under the lac promoter control reached to 38 wt% from dodecanoate. Enzyme assays suggest that efficient monomer formation via beta-oxidation by a high level expression of phaJAc was important to achieve a high polyhydroxyalkanoate content in the recombinant E. coli.

Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis.

Two Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ2(Pa) were constructed and introduced into Escherichia coli DH5alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities, that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle.

Alteration of chain length substrate specificity of Aeromonas caviae R-enantiomer-specific enoyl-coenzyme A hydratase through site-directed mutagenesis.

Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ(Ac)) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid beta-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ(Ac) by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ(Ac) revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C(8)) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C(12)) was examined by using the mutated PhaJ(Ac) as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1(Ps)). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

Solubilization and partial purification of an enzyme involved in rat liver microsomal fatty acid chain elongation: beta-hydroxyacyl-CoA dehydrase.

The solubilization and partial purification of beta-hydroxyacyl-CoA dehydrase from rat liver microsomes has been accomplished through deoxycholate solubilization, ammonium sulfate fractionation, and ion exchange chromatography. A purification of about 90-fold based on total soluble activity was achieved, with an overall yield of 40%. However, the initial solubilization is accompanied by the loss of the secondary portion of the v/s curve observed with intact microsomes. The enzyme requires detergent during the purification procedure to remain "soluble," and is strongly activated by the inclusion of Triton-X-100 at concentrations above its critical micelle concentration in the assay mixture. In addition a preference for micelles has been inferred based on discontinuities in the v/s curves relative to the measured critical micelle concentration of the substrates in the absence of Triton X-100. Kinetic parameters calculated on the basis of micelle-specific activity indicated that beta-hydroxyacyl-CoA substrates possessing even-numbered alkyl chains from 14 to 20 carbon atoms differed little in Vm', but had progressively larger Km' as the chain length increased. The partially purified preparation was also active with beta-hydroxy-8,11-eicosadienoyl-CoA; and with 2-trans-enoyl-CoA substrates in a reverse (hydration) reaction.

Light microscopic immunocytochemical demonstration of peroxisomal enzymes in epon sections.

A procedure is described for light microscopic immunocytochemical localization of catalase and three enzymes of peroxisomal lipid beta-oxidation: acyl-CoA oxidase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase in semi-thin sections of rat liver processed for routine electron microscopy. Satisfactory immunostaining required the removal of the epoxy resin with sodium ethoxide, controlled digestion of deplasticized sections with proteases and, in case of osmiumfixed tissue, bleaching with oxidants. Resin removal was essential for successful immunostaining, and protease treatment enhanced markedly the intensity of the reaction. This study shows that tissues processed for conventional ultrastructural studies can be used for postembedding immunocytochemical demonstration of various peroxisomal enzymes.

Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer.

Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.

Fiber composition, fiber size and enzyme activities in vastus lateralis of elite athletes involved in high intensity exercise.

In order to determine the influence of an extensive history of participation in high intensity activity on muscle fiber type, fiber size, and metabolic profile, elite ice hockey players were selected for investigation from three different leagues. Biopsy samples from the vastus lateralis muscle were obtained from different groups of players prior to and following the season and compared with control subjects. No significant differences were found in the percentage (49.6 vs. 43.8%) or the size of the ST fibers between the elite athletes and the control group, nor was there any significant alteration following the season of play in these variables. For the FT fiber subgroups, a reduction in the FTb (12.2 vs. 3.9%) and an increase in FTa (38.0 to 45.2%) fiber populations occurred over the season. Similarly, increases in fiber area were observed for both FT subgroups pre to post season. Of the enzymes studied only 3-hydroxyacyl CoA dehydrogenase was elevated in the post season measures, while total phosphorylase and phosphofructokinase were significantly lower. The metabolic pattern exhibited does not appear to be substantially different from what would be expected from an untrained group of similar fiber distribution.

Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae.

Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in the pha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as phaJ(Ac). Escherichia coli BL21(DE3) carrying phaJ(Ac). under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of phaJ(Ac) except for the initial Met residue. The enoyl-CoA hydratase encoded by phaJ(Ac) exhibited (R)-specific hydration activity toward trans-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (R)-specific hydration of 2-enoyl-CoA catalyzed by the translated product of phaJ(Ac) is a channeling pathway for supplying (R)-3-hydroxyacyl-CoA monomer units from fatty acid beta-oxidation to poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis in A. caviae.

Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae.

A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains.

The recombinant Escherichia coli strain, equipped with the newly cloned Aeromonas PHA biosynthesis genes, could produce a terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from dodecanoic acid plus odd carbon number fatty acid. In addition, the orf1 gene of Aeromonas hydrophila was found to play a critical role in assimilating the 3HV monomer and in regulating the monomer fraction in the terpolymer.

4-Hydroxycinnamoyl-CoA hydratase/lyase, an enzyme of phenylpropanoid cleavage from Pseudomonas, causes formation of C(6)-C(1) acid and alcohol glucose conjugates when expressed in hairy roots of Datura stramonium L.

4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL), a crotonase homologue of phenylpropanoid catabolism from Pseudomonas fluorescens strain AN103, led to the formation of 4-hydroxybenzaldehyde metabolites when expressed in hairy root cultures of Datura stramonium L. established by transformation with Agrobacterium rhizogenes. The principal new compounds observed were the glucoside and glucose ester of 4-hydroxybenzoic acid, together with 4-hydroxybenzyl alcohol- O-beta- D-glucoside. In lines actively expressing HCHL, these together amounted to around 0.5% of tissue fresh mass. No protocatechuic derivatives were found, although a trace of vanillic acid-beta- D-glucoside was detected. There was no accumulation of 4-hydroxybenzaldehydes, whether free or in the form of their glucose conjugates. There was some evidence suggesting a diminished availability of feruloyl-CoA for the production of feruloyl putrescine and coniferyl alcohol. The findings are discussed in the context of a diversion of phenylpropanoid metabolism, and the ability of plants and plant cultures to conjugate phenolic compounds.

The role of the fatty acid beta-oxidation multienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: molecular characterization of the fadBA operon from P. oleovorans and of the enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida.

In order to investigate the role of the putative epimerase function of the beta-oxidation multienzyme complex (FadBA) in the provision of (R)-3-hydroxyacyl-CoA thioesters for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis, the fadBA(Po) operon of Pseudomonas oleovorans was cloned and characterized. The fadBA(Po) operon and a class-II PHA synthase gene of Pseudomonas aeruginosa were heterologously co-expressed in Escherichia coli to determine whether the putative epimerase function of FadBA(Po) has the ability to provide precursors for PHA accumulation in a non-PHA-accumulating bacterium. Cultivation studies with fatty acids as carbon source revealed that FadBA(Po) did not mediate PHA(MCL) biosynthesis in the E. coli wild-type strain harboring a PHA synthase gene. However, PHA accumulation was strongly impaired in a recombinant E. coli fadB mutant, which harbored a PHA synthase gene. These data indicate that in pseudomonads FadBA does not possess the inherent property, based on a putative epimerase function, to provide the ( R)-enantiomer of 3-hydroxyacyl-CoA efficiently and that other linking enzymes are required to efficiently channel intermediates of beta-oxidation towards PHA(MCL) biosynthesis. However, the phaJ gene from P. oleovorans and from Pseudomonas putida, both of which encoded a 3- Re enoyl-CoA hydratase, was identified. The co-expression of phaJ(Po/Pp) with either a class-II PHA synthase gene or the PHA synthase gene from Aeromonas punctata in E. coli revealed that PhaJ(Po/Pp) mediated biosynthesis of either PHA(MCL), contributing to about 1% of cellular dry mass, or of poly(3-hydroxybutyrate- co-3-hydroxyhexanoate), contributing to 3.6% of cellular dry mass, when grown on decanoate. These data indicate that FadBA(Po)does not mediate the provision of (R)-3-hydroxyacyl-CoA, which resembles FadBA of non-PHA-accumulating bacteria, and that 3- Re enoyl-CoA hydratases are required to divert intermediates of fatty acid beta-oxidation towards PHA biosynthesis in P. oleovorans.