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Applied and Environmental Microbiology, August 2005, p. 4297-4306, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4297-4306.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Expression of 3-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Genes Enhances Production of Polyhydroxyalkanoate Copolymer from Glucose in Recombinant Escherichia coli JM109

Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan,¹ CAS Key Laboratory of Polymer Science and Materials, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China²

Received 21 December 2004/ Accepted 21 February 2005

	ABSTRACT

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Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.

	INTRODUCTION

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Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that are produced by some bacteria grown under nutrient limitation in the presence of excess carbon and have attracted research interest because they can be used as biodegradable plastics that can be produced from renewable resources (11, 23). PHAs can be divided into three main types based on the number of carbons in the monomer units incorporated into the polymer chain. Short-chain-length (SCL) PHA consists of monomers from C3 to C5 in length, medium-chain-length (MCL) PHA consists of monomers from C6 to C14 in length, and SCL-MCL PHA copolymer consists of both SCL and MCL monomer units. Polymers composed of SCL monomers such as poly-3-hydroxybutyrate [P(3HB)] have thermoplastic properties but are generally brittle and must be processed in a special manner to improve their properties (2, 7), while PHA polymers composed of MCL subunits have elastomeric properties that may be improved with the addition of nanocomposite materials (5, 6). SCL-MCL PHA copolymers have qualities between the SCL and MCL PHA polymers, depending on the ratio of SCL and MCL monomers, and because of superior physical and thermal properties have a wide array of uses (15). It has been shown that an SCL-MCL PHA copolymer with a high mol% SCL monomer composition and a low mol% MCL monomer composition has properties similar to polyethylene (1). Because of the many possible applications of SCL-MCL PHA, it is important to examine and define the enzymes and metabolic pathways for SCL-MCL PHA production.

In our previous study, we demonstrated that coexpression of mutant 3-ketoacyl-acyl carrier protein (ACP) synthase III genes (fabH) with PHA synthase genes (phaC) leads to the production of SCL-MCL PHA in recombinant Escherichia coli grown in the presence of excess glucose (16). That study proposes that substrates for PHA production are derived from the fatty acid biosynthesis pathway (Fig. 1A) and also proposes that the 3-ketoacyl-ACP reductase (FabG) would be necessary to provide monomers for PHA production from the fatty acid biosynthesis pathway in recombinant E. coli (16).

View larger version (29K): [in this window] [in a new window]   FIG. 1. Metabolic pathways for monomers derived from nonrelated and related carbon sources. (A) Production of SCL and MCL monomers from fatty acid biosynthesis via the overproduction of genetically engineered FabH proteins and PHA synthase. 1. Fatty acid biosynthesis pathway. 2. FabH catalyzes the condensation of malonyl-ACP and acetyl-CoA to form acetoacetyl-ACP. 3 and 4. FabH mediates the transacylase reaction. 5. PhaC catalyzes the PHA polymerization reaction. (B) The production of SCL and MCL monomers from the ß-oxidation pathway. 1. The ß-oxidation pathway of E. coli. 2. PhaJ catalyzes the conversion of enoyl-CoA to (R)-3-hydroxyacyl-CoA. 3. FabG catalyzes the reduction of 3-ketoacyl-CoA to (R)-3-hydroxyacyl-CoA. 4. PhaC catalyzes the PHA polymerization reaction.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and cultivation conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. All transformations and DNA manipulations and PHA production were carried out using E. coli JM109 (Takara, Tokyo, Japan). For protein production, E. coli BL21(DE3) was used. For PHA production from nonrelated carbon sources, the strains were grown at 30°C in either Luria-Bertani (LB) medium supplemented with glucose to a final concentration of 2 mg ml^–1 or M9 medium supplemented with 0.001% thiamine and glucose to a final concentration of 2 mg ml^–1. For plasmid selection in recombinant E. coli strains, 100 µg of ampicillin and 50 µg of kanamycin were used. Pseudomonas sp. 61-3 was grown in LB medium at 30°C.

Construction of expression plasmids.
In order to evaluate PHA production in various E. coli strains, both wild-type Pseudomonas sp. 61-3 PHA synthase genes were cloned into the broad-host-range plasmid pBBR1-MCS2 (8). The plasmid pGEMC1AB (Table 1) or one of its derivatives and the pBBR1-MCS2 plasmids were digested with BamHI and SacII, and a DNA fragment harboring the phaC1 gene or one of its mutant derivatives was ligated into the same sites of the pBBR1-MCS2 plasmid to create pBBR1-phaC1, pBBR1SCQM, or pBBRSTQK. The E. coli fabG gene was amplified from E. coli JM109 genomic DNA by PCR with the following primers: for the N terminus, 5'-GCT CTA GAG AGG AAA ATC ATG AAT TTT GAA GG-3', and for the C terminus, 5'-TCA GAC CAT GTA CAT CCC GCC GTT CAC-3'. The subsequent PCR product was cloned into the TA-cloning vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA). The insertion, overall correctness, and orientation of the gene were determined by DNA sequencing and restriction digestion. The Pseudomonas sp. 61-3 fabG gene was amplified from Pseudomonas sp. 61-3 genomic DNA by PCR with the following primers: for the N terminus, 5'-CCT CTA ACC CTC AAT ACC CCA G-3', and for the C terminus, 5'-TTG AAG GGA TCC GTC ACA T-3'. As with the E. coli fabG gene PCR product, the Pseudomonas sp. 61-3 fabG PCR product was cloned into pCR2.1-TOPO to produce pCEFG+ and pCPSFG+, respectively, and the insertion and orientation of the gene were determined by DNA sequencing and restriction digestion. The fabG genes were isolated from the pCEFG+ and pCPSFG+ plasmids with HincII, and the DNA fragments were subcloned into the SmaI site of the pBBRC1, pBBRSCQM, and pBBRSTQK plasmids. The orientation of the insert was determined by sequence analysis.

The following DNA primers were used to amplify the fabG genes from E. coli and Pseudomonas sp. 61-3 in order to construct the expression vectors pETGE and pETGP for the production of His-tagged FabG_Ec and FabG_Ps: 5'-GAA ACA TAT GAA TTT TGA AG GAA AAA TCG CAC TGG-3' for the N terminus of fabG_Ec, where bold lettering indicates the introduction of an NdeI restriction site; 5'-GCG GAT CCC GGT CAG ACC ATG TAC ATC CCG CCG TTC ACA TG-3' for the C terminus of fabG_Ec, where bold lettering indicates the introduction of a BamHI site into the primer; 5'-GAA GCT CAT ATG AGT CTG CAA GGT AAA GTT GCA C-3' for the N terminus of fabG_Ps; and 5'-GAA GGG ATC CGT CAC ATT TAA CTC ATG TAC ATC CCG C-3' for the C terminus of fabG_Ps. The PCR products were purified and digested with NdeI and BamHI and inserted into the same sites of the pET15b plasmid (Novagen, Madison, WI) to make either the E. coli fabG expression plasmid pETGE or the Pseudomonas sp. 61-3 fabG expression plasmid pETGP (Table 1).

Overexpression of fabG genes and purification of His-tagged recombinant FabG proteins.
E. coli BL21(DE3) (Novagen, Madison, WI) was transformed with either the pETGE plasmid harboring the E. coli fabG gene or pETGP harboring the Pseudomonas sp. 61-3 fabG gene. Individual colonies were selected based on their resistance to ampicillin on LB ampicillin plates grown overnight at 37°C and used to inoculate starter cultures of 1.75 ml of LB medium that were grown overnight in a rotary shaker (200 rpm). Each starter culture was used to inoculate 100 ml of LB medium in 500-ml Sakaguchi flasks supplemented with 100 µg ml^–1 ampicillin. The cultures were grown for 3 h at 37°C, and the expression of the fabG genes was induced by the addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG), after which the cultures were incubated for an additional 2 h with constant shaking at 37°C before harvesting by centrifugation.

The harvested cells were washed with cold 50 mM phosphate buffer and resuspended in 4 ml of the same buffer. The cells were broken on ice by sonication with a model UD-200 ultrasonic disruptor (TOMY, Tokyo, Japan) by pulsing four times for 5 s each and centrifuged to purify the cell lysates. The cell lysates were subjected to purification via Ni column chromatography with the His-Bind purification kit (Novagen). The purified, His-tagged FabG proteins were eluted according to the manufacturer's protocol, and the purity of the final product was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single bands with molecular masses approximately equivalent to predicted molecular masses of the FabG proteins on the gels after staining with Rapid CBB stain (Kanto Chemical, Tokyo, Japan) indicated that the proteins were purified to electrophoretic homogeneity (data not shown).

Assay for substrate specificity of FabG proteins.
Purified His-tagged E. coli FabG and Pseudomonas sp. 61-3 FabG proteins were assayed for substrate specificity using a modified form of a previously developed method to evaluate the stereospecificity of enoyl-CoA hydratases (29). Reaction mixtures consisted of 0.25 mM trans-2-enoyl-CoA (C4, C6, C8, C10, or C12), 0.5 mM NADP⁺, and 1.0 U of hydratase (either PhaJ1 or PhaJ4) in 400 µl of 50 mM Tris-HCl (pH 8.0). Reactions were initiated by the addition of 1 U of either His-tagged E. coli FabG or His-tagged Pseudomonas sp. 61-3 FabG. Reactions were carried out at room temperature for up to 2 min, and substrate specificity was monitored by the increase in absorbance at 340 nm, which is due to the formation of NADPH linked with the oxidation of 3-hydroxyacyl-CoA substrates produced by PhaJ proteins to 3-ketoacyl-CoA.

PHA production from glucose in recombinant E. coli.
Plasmids harboring either the E. coli fabG gene or the Pseudomonas sp. 61-3 fabG gene with the wild-type phaC1 gene, phaC1(STQK) gene, or phaC1(SCQM) gene were cotransformed with pTrcFabH(F87T) into E. coli JM109. Transformants were isolated, and the presence of the pTrcFabH plasmid and a plasmid harboring phaC1 and fabG was confirmed by restriction digestion and PCR. Single colonies of confirmed transformants were cultured overnight in 1.75 ml of LB medium and used to inoculate 500-ml culture flasks with either 100 ml LB medium or 100 ml of M9 medium supplemented with glucose. Cultures were incubated at 30°C and constantly shaken at 150 rpm for 5 h, at which time expression of the fabH(F87T) gene was induced by the addition of 1 mM IPTG. Cultures grown in LB medium were incubated with constant shaking at 30°C for an additional 3 h, at which time glucose was added to a final concentration of 2 g ml^–1. The cells were grown for a total of 96 h before harvesting by centrifugation. PHA contents were determined by gas chromatography (GC) analysis of lyophilized cells as previously described (16).

GPC analysis of PHA polymers.
Cell materials for gel permeation chromatography (GPC) analysis were prepared and lyophilized as described for GC analysis except that a total of 2 liters of liquid culture per sample was harvested by centrifugation. The lyophilized cells were added to 100 ml of chloroform and were stirred in a covered beaker at room temperature for 48 h to extract the polymers. The chloroform-polymer solution was filtered first through filter paper to remove cell debris and then through a 0.45-µm polytetrafluoroethylene membrane to remove any residual solid materials. The chloroform was evaporated using a rotary vacuum evaporator (Eyela, Tokyo, Japan), and the isolated polymer was washed with 20 ml of methanol. The polymer was allowed to dry at room temperature and was redissolved in 20 ml of chloroform. The polymer was precipitated by the addition of 10 times the volume of methanol and collected by filtering the solution through a 0.45-µm polytetrafluoroethylene membrane. The membrane and polymers were allowed to dry at room temperature, and the polymers were dissolved by the addition of chloroform and collected in a beaker. The chloroform was allowed to evaporate at room temperature in a fume hood for 48 h, and the polymer cast film was weighed and used for GPC analysis. Molecular mass data of polyesters were obtained by GPC analysis using the Shimadzu 10A system with a RID-10A refractive-index detector with serial columns of ShodexK802 and K806 M as described previously (9).

Determination of PHA polymer composition by NMR.
Twenty mg of polymer isolated from E. coli JM109 harboring pBBRSTQKGEC and pTrcFabH(F87T) was dissolved in 1 ml of CDCl₃ and subjected to both ¹H and ¹³C nuclear magnetic resonance (NMR) analysis. ¹H NMR spectra were recorded using a JEOL -400 spectrometer with a 5.0-µs pulse width (45^o pulse angle), 3-s pulse repetition, 7,500-Hz spectra width, and 16K data points. For ¹³C NMR analysis, data were collected using a JEOL ECP-500 spectrometer with a 7.0-µs pulse width (45^o pulse angle), 5-s pulse repetition, 25,000-Hz spectra width, and 64K data points. Tetramethylsilane (Me₄Si) was used as an internal chemical shift standard.

Determination of thermal properties of SCL-MCL PHA polymers produced by recombinant E. coli.
The thermal data were recorded on a PerkinElmer Pyris 1 differential scanning calorimeter equipped with a liquid nitrogen cooling accessory. Data was collected under a nitrogen flow of 20 ml min^–1. Melt-quenched polyester samples (ca. 3 mg) encapsulated in aluminum pans were heated from –30°C to 200°C at a rate of 20°C min^–1, and the heat flow curves were recorded. The observed melting temperatures were determined from the positions of the endothermic peaks.

	RESULTS

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Cloning and identification of a novel 3-ketoacyl-ACP reductase (fabG) gene from Pseudomonas sp. 61-3.
Pseudomonas sp. 61-3 is a nonpathogenic bacterium isolated from soil that can produce a blend of P(3HB) homopolymer and random SCL-MCL PHA copolymers from both sugars and alkanoic acids (14). Previous studies have shown that 3-ketoacyl reductases can be used for monomer supply in PHA biosynthesis (21, 24), so we set out to investigate the potential role of reductase enzymes from Pseudomonas sp. 61-3. Southern blot analysis was performed using genomic DNA fragments from digestions with several restriction enzymes, and a 1.2-kbp EcoRI fragment that cross-hybridized with a heterologous E. coli fabG (EcfabG) PCR probe was cloned into pBluescript SK+ at the same restriction sites. The insert was sequenced and found to contain the entire open reading frame corresponding to the Pseudomonas sp. 61-3 fabG gene (DDBJ, AB193436). The predicted amino acid sequence of the Pseudomonas sp. 61-3 FabG was found to be 83% identical and 91% similar to the Pseudomonas aeruginosa FabG and 66% identical and 79% similar to the E. coli FabG protein via ClustalW alignment. The Pseudomonas sp. 61-3 fabG (PsfabG) gene product was further characterized and used to assess its potential role in the production of SCL-MCL PHA from glucose.

Substrate specificity of FabG proteins.
Although the natural reaction of the FabG protein is the reduction of 3-ketoacyl-ACP to form (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis (Fig. 1A), it was shown previously that FabG is capable of recognizing both (R)-3-ketoacyl-CoA and (R)-3-hydroxyacyl-CoA as substrates (Fig. 1B) (25). In this study, we used an assay previously developed to characterize stereoselectivity in P. aeruginosa enoyl-CoA hydratase (PhaJ) proteins (29) to assay the substrate specificity of the FabG proteins. PhaJ proteins can convert enoyl-CoAs into (R)-3-hydroxyacyl-CoAs (29). For example, PhaJ1 can convert crotonyl-CoA to (R)-3-hydroxybutyrl-CoA and PhaJ4 can convert hexenyl-CoA to (R)-3-hydroxyhexanoyl-CoA. In turn, these 3-hydroxyacyl-CoA forms can be converted to (R)-3-ketoacyl-CoA with the subsequent reduction of NADP⁺ to NADPH via FabG. In order to assess the substrate specificities of the FabG proteins, recombinant FabG proteins from Pseudomonas sp. 61-3 and E. coli were produced and purified and their substrate specificities were determined for C4 to C12 substrates. The results indicated that the E. coli FabG protein has high substrate specificity toward C8 to C12 substrates, with the highest substrate specificity toward C10 compounds (Fig. 2). In contrast to the E. coli FabG protein, the Pseudomonas sp. 61-3 FabG protein displayed higher substrate specificity toward C6 to C10 compounds, with the highest specificity for the C6 substrate (Fig. 2). In addition to their preferred substrate specificities, both proteins were able to react with a broad (C4 to C12) range of substrates, with the Pseudomonas sp. 61-3 FabG protein having a stronger preference for C4 substrates than the E. coli FabG protein (Fig. 2).

View larger version (17K): [in this window] [in a new window]   FIG. 2. FabG substrate specificity. Data represent results for three independent experiments ± standard deviations. Results are presented as relative reactivities toward a specific substrate. FabGEc, E. coli FabG protein-relative activities for substrates; FabGPs, Pseudomonas sp. 61-3 FabG protein-relative activities for substrates. C4, 3-hydroxybutyryl-CoA; C6, 3-hydroxyhexanyl-CoA; C8, 3-hydroxyoctonoyl-CoA; C10, 3-hydroxydecanoyl-CoA; C12, 3-hydroxydodecanoyl-CoA.

PHA production from glucose is further enhanced by the coexpression of fabG genes with the genetically engineered PHA synthase genes and fabH(F87T) in E. coli JM109.
Previously, our lab developed several highly active mutant type II PHA synthases via in vitro evolutionary engineering and saturation point mutagenesis (27). Two mutants obtained from that study were used to further characterize the involvement of the fatty acid biosynthesis genes in SCL-MCL PHA copolymer production from glucose in E. coli. The first mutant PHA synthase [PhaC1(STQK)] contains two point mutations. One of the point mutations changes amino acid 325 of the Pseudomonas sp. 61-3 PhaC1 enzyme from Ser to Thr, and a secondary point mutation changes the Gln at position 481 to Lys. The mutations at S325T/Q481K led to an 8.7-fold increase in activity toward 3-hydroxybutyrate-CoA and a 2.8-fold increase in activity toward 3-hydroxydecanoate-CoA in an in vitro assay (26). The second mutant PHA synthase [PhaC1(SCQM)] also had two point mutations, one changing the Ser at 325 to Cys and the second changing Gln at 481 to Met. These two mutants were found to be highly active for the production of P(3HB) homopolymer compared to the wild-type enzyme (27). Furthermore, these mutants also possessed the capacity to produce SCL-MCL PHA copolymer when grown in the presence of dodecanoate (26).

It was predicted that coexpression of highly active mutant forms of PHA synthases with the fabH(F87T) and fabG genes would further enhance SCL-MCL PHA production in E. coli JM109 from glucose. To test this, plasmids harboring the genetically engineered phaC1 genes alone (pBBRSTQK and pBBRSCQM), plasmids harboring the genetically engineered phaC1 genes with the EcfabG genes (pBBRSTQKGEC and pBBRSCQMGEC), and plasmids harboring the genetically engineered phaC1 genes with the PsfabG genes (pBBRSTQKGPS and pBBRSCQMGPS) were transformed with or without thepTrcFabH(F87T) plasmid into E. coli JM109. The relevant genotypes and phenotypes of the strains are described in Table 3. The ability of the transformed E. coli JM109 strains to accumulate SCL-MCL PHA copolymer from glucose was assessed by GC as described in Materials and Methods, and the results are shown in Table 3. Control strains harboring plasmids expressing either phaC1(STQK) or phaC1(SCQM) alone were unable to accumulate detectable levels of PHA in E. coli JM109. Strains harboring either EcfabG or PsfabG and phaC1(STQK) or phaC1(SCQM) were able to accumulate a small amount of P(3HB) homopolymer. A strain coexpressing E. coli fabH(F87T) with phaC1(STQK) was able to produce 0.96% of the cellular dry weight of SCL-MCL PHA copolymer consisting of 90.7 mol% C4, 6.0 mol% C6, 2.0 mol% C8, 1.3 mol% C10, and 1.2 mol% C12 (Table 3). This result indicates that the PhaC1(STQK) enzyme was able to broaden the number and type of substrates that could be incorporated into the SCL-MCL PHA copolymer and increase the yield compared to SCL-MCL PHA copolymer produced by the wild-type PHA synthase. The additional coexpression of the EcfabG gene with the E. coli fabH(F87T) and phaC1(STQK) genes resulted in a marked increase in PHA content (up to 4.5% of cellular dry weight) in addition to a change in the mol% composition of the SCL-MCL copolymer produced in this strain, with a shift toward the shorter-chain-length monomers compared to the strain that harbored only the fabH(F87T) and phaC1(STQK) genes (Table 3). These results contrast with those obtained by coexpressing the PsfabG gene with both the E. coli fabH(F87T) and the phaC1(STQK) genes, an event which produced an SCL-MCL copolymer with a dramatic shift toward C4 and C6 monomer incorporation compared to strains expressing the E. coli fabG gene. This indicates that the monomer-supplying enzymes are important factors in determining the composition of SCL-MCL PHA copolymers. Similar results were obtained when coexpression of the phaC1(SCQM) gene was performed with the fatty acid biosynthesis genes (Table 3). Overall, these results indicate that the coexpression of fabG genes with the fabH(F87T) and PHA synthase genes results in the enhanced production of SCL-MCL PHA copolymer in E. coli JM109 grown in the presence of excess glucose.

Physical characterization of SCL-MCL PHA copolymer isolated from recombinant E. coli.
Polymer was isolated from a JM109 strain harboring the pTrcFabH(F87T) and pBBRSTQKGEC plasmids as described in Materials and Methods. This polymer was chosen as a representative polymer produced by fatty acid biosynthetic enzymes and a mutant PHA synthase and was characterized by NMR spectroscopy, GPC, and differential scanning calorimetry. The results are shown in Table 5.

The weight-average molecular weight (M_w) of the polymer was determined by GPC and revealed that the M_w of the SCL-MCL PHA copolymer was slightly lower than that of P(3HB) homopolymer but that the relative distributions of the polydispersity indices (M_w/M_n) were similar for the SCL-MCL PHA copolymer and P(3HB) (Table 5). This result indicates that the relative distributions of the polymers isolated from each strain were similar despite the difference in the overall molecular weights.

Thermal properties of the SCL-MCL PHA copolymer isolated from recombinant E. coli harboring the pTrcFabH(F87T) and pBBRSTQKGEC plasmids were determined by differential scanning calorimetryDSC analysis and compared with the thermal properties of the P(3HB) homopolymer (Table 5). For the SCL-MCL PHA copolymer, two melting temperature peaks were observed as opposed to the single melting temperature peak observed for the P(3HB) homopolymer. The lower temperature melting peaks observed for the SCL-MCL PHA copolymers are from their respective original crystals, while the higher temperature melting peaks arose from the recrystallization of the copolymer during the heating process. The thermal properties for each polymer are summarized in Table 5. As determined by NMR analysis, the SCL-MCL PHA copolymer isolated from the recombinant E. coli strain harboring the pTrcFabH(F87T) and pBBRSTQKGEC plasmids consisted of polymers composed of 95.1 mol% 3HB, 3.6 mol% 3HHx, and 1.3 mol% 3HO monomer units as determined by NMR analysis. The addition of 4.9 mol% 3HHx and 3HO MCL-monomer units to the PHA copolymer lowered the melting temperature to 150°C from 170°C compared to the P(3HB) homopolymer sample (Table 5). In addition, the enthalpy of fusion was lowered to 32 J/g compared to 52 J/g for the P(3HB) homopolymer. These results indicate that the polymer is an SCL-MCL PHA copolymer and that the addition of MCL monomers within the copolyester dramatically alters the thermal properties of the polymer. Based on previous studies, these changes in thermal properties correspond to improved physical properties (1).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two important factors must be taken into consideration for the use of PHAs as bulk commodity plastics: the properties of the polymer and the cost of production. Polymer properties are greatly influenced by their monomer composition. P(3HB) homopolymer made of SCL monomer units is a stiff and brittle thermoplastic. On the other hand, SCL-MCL PHA copolymers with a high mol% of SCL monomers along with a low mol% of MCL monomers have properties similar to polypropylene and polyethylene and many potential uses as bulk commodity plastics (1, 15). Therefore, it is of special interest to develop pathways and enzymes for the production of SCL-MCL PHA copolymers with various MCL monomer compositions.

There are many factors that influence the cost of production of SCL-MCL PHA copolymers, but one of the key components is the cost of the carbon feedstock. One way to reduce the cost is by using inexpensive substrates such as sugars or molasses (11, 23). Another way is to bypass these carbon sources by using photosynthetic organisms and to use CO₂ as a carbon source for the production of SCL-MCL PHA copolymers. However, the lack of defined and usable pathways has limited the application of these methods for the economical production of PHAs. The current study represents a preliminary step to addressing these issues by defining a pathway for SCL-MCL PHA copolymer production from nonrelated carbon sources via the fatty acid biosynthesis pathway.

As shown in previous studies, FabG enzymes can convert 3-ketoacyl-CoA to (R)-3-hydroxyacyl-CoA for PHA production in recombinant bacteria (18, 21, 24). However, the enzymes used in these studies produced copolymers of similar MCL content when coexpressed with type II PHA synthases and the polymers produced had little or no SCL content. Polymers with low mol% SCL composition fall into the class of elastomers, which have limited applications. It is much more desirable to produce an SCL-MCL PHA copolymer with a relatively high mol% (ca. 90 to 95%) of SCL monomer with a low mol% (ca. 5 to 10%) of MCL monomers randomly distributed throughout the copolymer because the properties are most similar to those necessary to produce bulk commodity plastics (1, 15). In addition, production of MCL monomers from the related carbon sources is dependent on the flux through the ß-oxidation pathway and requires the use of organisms with fully functional ß-oxidation pathways.

The use of the fatty acid biosynthesis pathway for the supply of MCL monomers from nonrelated carbon sources represents an alternative to MCL monomer production from the ß-oxidation pathway (Fig. 1B). FabG and FabH enzymes are important components in the type II fatty acid biosynthetic (FAS II) pathway that is inherent in bacteria and plants. In our previous study (16), it was suggested that the FabG enzyme was involved in PHA monomer supply from nonrelated carbon sources via the fatty acid biosynthesis pathway (Fig. 1A). The current study demonstrated for the first time the enhancement of SCL-MCL PHA copolymer production from glucose by coexpression of either the E. coli fabG gene or the Pseudomonas sp. 61-3 fabG gene with the fabH(F87T) and PHA synthase genes. Furthermore, an SCL-MCL PHA copolymer isolated from recombinant E. coli coexpressing the E. coli fabG, fabH(F87T), and phaC1(STQK) genes had improved properties compared to the P(3HB) homopolymer. The yields of PHA from nonrelated carbon sources attained in this study were rather low (between 0.5 and 5.0% cellular dry weight). This is likely due to the low transacylase activity associated with FabH (28). Currently, techniques such as in vitro evolution, which was used to develop the highly active PHA synthases used in this study (13, 26), and rational design, which was used to develop the FabH(F87T) protein used in this study (16), are being applied to the PHA monomer-supplying enzymes in our laboratory and may lead to improved production of SCL-MCL PHA copolymers. In addition, we have shown that coexpression of fabH(F87T) and genetically modified PHA synthase genes with other monomer-supplying enzyme genes can increase the yield of SCL-MCL PHA produced in recombinant E. coli (17), strategies that are currently under investigation in our lab.

Interestingly, coexpression of fabG with fabH(F87T) and PHA synthase genes led to an increase in the mol% of C4 monomer incorporated into the copolymer. This is in marked contrast to the in vitro substrate specificity data. This phenomenon likely occurs because, although the FabH(F87T) protein is capable of enhancing MCL monomer supply for PHA production in recombinant E. coli, the majority of monomers produced by this protein are still SCL (Tables 2 through 4), since the ratio of SCL to MCL monomers produced by the mutant FabH protein is still skewed toward SCL monomer production. This study also showed that the medium used (LB or M9) could dramatically influence the composition and yields of the polymers produced (Tables 2 through 4). Based on these results, changing the medium would allow for the production of polymers with specific monomer compositions, dependent upon the desired properties.

Although E. coli may not represent the best organism to produce SCL-MCL PHA, it is an excellent organism to look at potential metabolic pathways for use in PHA production because it is easy to manipulate genetically, it grows rapidly, and it has a defined metabolic background. These qualities have allowed us to establish a defined system to produce SCL-MCL PHA copolymers from the fatty acid biosynthesis pathway in E. coli. The importance of establishing a route of monomer supply from nonrelated carbon sources must not be overlooked. Because of the ubiquity of the fatty acid biosynthesis pathway in all organisms, the defined use of both FabH and FabG enzymes as SCL-MCL PHA monomer-supplying enzymes provides a model that may be used in many different types of recombinant organisms. This is important because it may be possible to transfer these genes into chloroplasts, which harbor the proteins for fatty acid biosynthesis in photosynthetic organisms, thus facilitating the production of SCL-MCL PHA copolymers from carbon dioxide. In addition, it may be possible to overexpress these genes in native PHA-producing bacteria (Aeromonas sp., Ralstonia sp., and Pseudomonas sp.) to enhance PHA production from nonrelated carbon sources. These studies are currently under way in our laboratory and may lead to the more economical production of SCL-MCL PHA copolymers.

The current study has shown that E. coli FabG and Pseudomonas sp. 61-3 FabG have different substrates in vitro (Fig. 2) and that expression of these enzymes clearly influences the type of monomers incorporated in vivo into an SCL-MCL PHA copolymer in recombinant E. coli (Table 2 through 4). The FabG proteins share high sequence homology, so the current findings indicate that subtle differences in the amino acid sequences of FabG proteins can dramatically alter the substrate specificities of the enzymes. It is known that FabG enzymes from different organisms have different substrate specificities (12), and it may be possible to design new metabolic pathways capable of producing SCL-MCL PHA copolymers with specific compositions by incorporating FabG proteins with different substrate specificities. The crystal structures of the E. coli FabG (19, 20) and Mycobacterium tuberculosis FabG (4) enzymes are known and may allow for the use of a rational design strategy for changing substrate specificity in a manner similar to that used with FabH (16). Examination of the primary amino acid sequence of the M. tuberculosis FabG protein reveals a unique sequence at the C terminus from amino acids 243 through247 (MGMGH). This is a conserved sequence unique to ketoacyl reductases from Mycobacterium sp. and may correlate with the specificity toward larger substrates by the FabG enzymes from Mycobacterium species (12). The use of such an enzyme or genetically modified FabG enzymes based on the crystal structures of the proteins may result in the production of the specific monomers necessary to control the composition and thus the properties of SCL-MCL PHA copolymers.

Our conclusion is that FabG plays an important role in enhancing the yield and determining the monomer composition of the SCL-MCL PHA copolymers produced from nonrelated carbon sources. Because previous studies have established that FabG enzymes are capable of supplying monomers for SCL-MCL PHA copolymers from related carbon sources (18, 21, 24), it may be possible to look at different biochemical pathways in the cell to supply monomers for SCL-MCL PHA copolymer production based on carbon source and growth conditions of the bacteria. This flexibility in carbon source usage could allow for the production of polymers with specific material properties by changing the carbon source and/or enzyme combinations for PHA synthesis and represents another step toward the improved production of useful biodegradable polyesters.

	ACKNOWLEDGMENTS

 
We thank H. Abe and K. Matsumoto for helpful discussions regarding this work.

This work was supported by a grant from Ecomolecular Science Research (RIKEN). C. T. Nomura is supported by a JSPS Fellowship for Foreign Researchers (Japan Society for the Promotion of Science).

	FOOTNOTES

 
* Corresponding author. Mailing address: Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Phone: 81-48-467-9403. Fax: 81-48-462-4667. E-mail: cnomura{at}riken.jp.

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