Location of functional region at N-terminus of polyhydroxyalkanoate (PHA) synthase by N-terminal mutation and its effects on PHA synthesis

Polyhydroxyalkanoate (PHA) synthase PhaC plays a very important role in biosynthesis of microbial polyesters PHA. Compared to the extensively analyzed C-terminus of PhaC, N-terminus of PhaC was less studied. In this paper, the N-terminus of two class I PHA synthases PhaC_Re and PhaC_Ah from Ralstonia eutropha and Aeromonas hydrophila, respectively, and one class II synthase PhaC2_Ps of Pseudomonas stutzeri strain 1317, were investigated for their effect on PHA synthesis. For PhaC_Re, deletion of 2–65 amino acid residues on the N-terminus led to enhanced PHB production with high PHB molecular weight of 2.50 × 10⁶ Da. For PhaC_Ah, the deletion of the N-terminal residues resulted in increasing molecular weights and widening polydispersity accompanied by a decreased PHA production. It was found that 3-hydroxybutyrate (3HB) monomer content in copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (3HHx) increased when the first 2–9 and 2–13 amino acid residues in the N-terminus of PhaC2_Ps were deleted. However, deletion up to the 40th amino acid disrupted the PHA synthesis. This study confirmed that N-terminus in different types of PHA synthases showed significant roles in the PHA productivity and elongation activity. It was also indicated that N-terminal mutation was very effective for the location of functional regions at N-terminus.     

Polyhydroxyalkanoates production from carbohydrates by a genetic recombinant Aeromonas sp.

Aims: To develop an Aeromonas strain able to utilize inexpensive carbon sources such as starch for the synthesis of polyhydroxyalkanoates (PHA). Methods and Results: A recombinant Aeromonas sp. (strain KC007‐1) was constructed by introducing the PHB synthesis genes (phaCAB) into the bacterium. Strain KC001‐R1 can not only use carbohydrate (including starch) for growth but also accumulate significant amounts of polyhydroxybutyrate (PHB) in the cells. Conclusions: One of the present focuses on PHA production has been on lowering the production costs. Starch is an example of an inexpensive carbohydrate for use in industrial production of PHA. We have demonstrated that by introducing the phaCAB operon into Aeromonas sp. allowed the bacterium able to accumulated PHB using this substrate. Significance and Impact of the Study: Aeromonas spp. are able to synthesize PHA using fatty acids as carbon source. Although good robust growth results with use of starch as sole carbon source for Aeromonas, PHA synthesis does not occur. Strain KC007‐R1 showed the ability to accumulate PHA in relative high amount with both carbohydrates and fatty acids as carbon source, and can be cultivated to a significant amount of cell mass and hence is a potential strain for further development for industrial applications.     

Immobilization of glycolate oxidase from Medicago falcata on magnetic nanoparticles for application in biosynthesis of glyoxylic acid

Glycolate oxidase was isolated from Medicago falcata Linn. after a screening from 13 kinds of C₃ plant leaves, with higher specific activity than the enzyme from spinach. The M. falcata glycolate oxidase (MFGO) was partially purified and then immobilized onto hydrothermally synthesized magnetic nanoparticles via physical adsorption. The magnetic nanoparticles were characterized with scanning electron microscope (SEM), transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectroscopy. The maximum load of MFGO was 56 mg/g support and the activity recovery was 45%. Immobilization of MFGO onto magnetic nanoparticles enhanced the enzyme stability, and the optimum temperature was significantly increased from 15 °C to 30 °C. The immobilized biocatalyst was successfully used in a batch reactor for repeated oxidization of glycolic acid to synthesize glyoxylic acid, retaining ca. 70% of its initial activity after 4 cycles of reaction at 30 °C for nearly 70 h, and its half-life was calculated to be 117 h.     

Polyhydroxyalkanoate (PHA) accumulating bacteria from the gut of higher termite <Emphasis Type="Italic">Macrotermes carbonarius</Emphasis> (Blattodea: Termitidae)

The continuous quest for bacterial strains capable of accumulating polyhydroxyalkanoate (PHA) utilizing cheaper and renewable carbon source prompted us to explore newer and diverse environments like the gut of termites. Among the bacterial strains isolated from the gut of higher termite Macrotermes carbonarius, three strains were found to accumulate PHA, as observed by microscopic studies and PHA production experiments. Among them, strain MC1 with rapid growth and higher PHA accumulation was selected for further studies. API kit-50 CHB and 16S rRNA gene sequence analysis results indicated the strain to have 99% homology with Bacillus megaterium and Bacillus flexus. Bacillus sp. MC1 was able to accumulate PHA during the growth phase utilizing different carbon sources like glucose, fructose, sodium acetate, sodium valerate and 1,4-butanediol. Gas chromatography analysis of the polymer has shown it to be basically composed of poly (3-hydroxybutyrate) (PHB). Growth associated PHB biosynthesis was best in the presence of sodium acetate with 39 wt% after 16 h of cultivation. Though previous studies provided evidence confirming the presence of PHA producing bacteria in termite gut, isolation and characterization of these strains in pure culture has not been documented yet. Presence of other morphotypes in the termite gut with PHA like granular inclusions was evident from the transmission electron microscopy studies. This is a novel report and shows the feasibility of using potent strains capable of utilizing lignocellulosic degradation products as a renewable carbon source for the production of PHA in the future.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

In silico prospection of microorganisms to produce polyhydroxyalkanoate from whey: Caulobacter segnis DSM 29236 as a suitable industrial strain

Polyhydroxyalkanoates (PHAs) are polyesters of microbial origin that can be synthesized by prokaryotes from noble sugars or lipids and from complex renewable substrates. They are an attractive alternative to conventional plastics because they are biodegradable and can be produced from renewable resources, such as the surplus of whey from dairy companies. After an in silico screening to search for ß-galactosidase and PHA polymerase genes, several bacteria were identified as potential PHA producers from whey based on their ability to hydrolyse lactose. Among them, Caulobacter segnis DSM 29236 was selected as a suitable strain to develop a process for whey surplus valorization. This microorganism accumulated 31.5% of cell dry weight (CDW) of poly(3-hydroxybutyrate) (PHB) with a titre of 1.5 g l⁻¹ in batch assays. Moreover, the strain accumulated 37% of CDW of PHB and 9.3 g l⁻¹ in fed-batch mode of operation. This study reveals this species as a PHA producer and experimentally validates the in silico bioprospecting strategy for selecting microorganisms for waste re-valorization.     

An extracellular polyhydroxybutyrate depolymerase in <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8

The thermophilic bacterium Thermus thermophilus HB8 has been characterized as a polyhydroxybutyrate (PHB)-degrading microorganism since it grows efficiently and forms clear zones on agar plates containing PHB as sole carbon source. T. thermophilus extracellular PHB depolymerase was purified to homogeneity using an affinity chromatography protocol. The purified enzyme was estimated to have an apparent molecular mass of 42 kDa. The extracellular PHB depolymerase gene was identified as the TTHA0199 gene product of T. thermophilus HB8. The amino acid sequence of the TTHA0199 gene product shared significant homologies to other carboxylesterases. A catalytic triad was identified consisting of S₁₈₃, E₃₁₀, and H₄₀₅. A pentapeptide sequence (GX₁SX₂G) exists within the molecule, characteristic for PHB depolymerases (lipase box) and for other serine hydrolases. Purified extracellular PHB depolymerase was stable at high temperatures with an optimum activity at pH 8.0. The apparent Km value of the purified enzyme for PHB was 53 μg/ml. As the main product of the enzymic hydrolysis of PHB, the monomer 3-hydroxybutyrate was identified, suggesting that the enzyme acts principally as an exo-type hydrolase.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

Continuous production of poly([<Emphasis Type="BoldItalic">R</Emphasis>]-3-hydroxybutyrate) by <Emphasis Type="BoldItalic">Cupriavidus necator</Emphasis> in a multistage bioreactor cascade

Poly(hydroxyalkanoates) (PHAs) constitute biodegradable polyesters and are considered among the most promising candidates to replace common petrochemical plastics in various applications. To date, all commercial processes for PHA production employ microbial discontinuous fed-batch fermentations. These processes feature drawbacks such as varying product quality and the inevitable periods of downtime for preparation and post-treatment of the bioreactor equipment. An unprecedented approach to PHA production was chosen in the presented work using a multistage system consisting of five continuous stirred tank reactors in series (5-SCR), which can be considered as a process engineering substitute of a continuous tubular plug flow reactor. The first stage of the reactor cascade is the site of balanced bacterial growth; thereafter, the fermentation broth is continuously fed from the first into the subsequent reactors, where PHA accumulation takes place under nitrogen-limiting conditions. Cupriavidus necator was used as production strain. The focus of the experimental work was devoted to the development of a PHA production process characterized by high productivity and high intracellular polymer content. The results of the experimental work with the reactor cascade demonstrated its potential in terms of volumetric and specific productivity (1.85 g L⁻¹ h⁻¹ and 0.100 g g⁻¹ h⁻¹, respectively), polymer content (77%, w/w) and polymer properties (M _w = 665 kg/mol, PDI = 2.6). Thus, implementing the technology for 5-SCR production of PHB results in an economically viable process. The study compares the outcome of the work with literature data from continuous two-stage PHA production and industrial PHA production in fed-batch mode.     

Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules

A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3 _Rru ) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3 _Rru turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3 _Rru with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3 _Rru was specific for short-chain-length polyhydroxyalkanoates (PHA_SCL) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3 _Rru . Low concentrations of calcium or magnesium ions (1–5 mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3 _Rru is the representative of a new type of the growing number of intracellular PHB depolymerases.     

Peroxisomal polyhydroxyalkanoate biosynthesis is a promising strategy for bioplastic production in high biomass crops

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers with diverse plastic‐like properties. PHA biosynthesis in transgenic plants is being developed as a way to reduce the cost and increase the sustainability of industrial PHA production. The homopolymer polyhydroxybutyrate (PHB) is the simplest form of these biodegradable polyesters. Plant peroxisomes contain the substrate molecules and necessary reducing power for PHB biosynthesis, but peroxisomal PHB production has not been explored in whole soil‐grown transgenic plants to date. We generated transgenic sugarcane (Saccharum sp.) with the three‐enzyme Ralstonia eutropha PHA biosynthetic pathway targeted to peroxisomes. We also introduced the pathway into Arabidopsis thaliana, as a model system for studying and manipulating peroxisomal PHB production. PHB, at levels up to 1.6%–1.8% dry weight, accumulated in sugarcane leaves and A. thaliana seedlings, respectively. In sugarcane, PHB accumulated throughout most leaf cell types in both peroxisomes and vacuoles. A small percentage of total polymer was also identified as the copolymer poly (3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) in both plant species. No obvious deleterious effect was observed on plant growth because of peroxisomal PHA biosynthesis at these levels. This study highlights how using peroxisomal metabolism for PHA biosynthesis could significantly contribute to reaching commercial production levels of PHAs in crop plants.     

Polyhydroxybutyrate production in halophilic marine bacteria Vibrio proteolyticus isolated from the Korean peninsula

Polyhydroxybutyrates (PHB) are biodegradable polymers that are produced by various microbes, including Ralstonia, Pseudomonas, and Bacillus species. In this study, a Vibrio proteolyticus strain, which produces a high level of polyhydroxyalkanoate (PHA), was isolated from the Korean marine environment. To determine optimal growth and production conditions, environments with different salinity, carbon sources, and nitrogen sources were evaluated. We found that the use of a medium containing 2% (w/v) fructose, 0.3% (w/v) yeast extract, and 5% (w/v) sodium chloride (NaCl) in M9 minimal medium resulted in high PHA content (54.7%) and biomass (4.94 g/L) over 48 h. Addition of propionate resulted in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(HB-co-HV)) copolymer as propionate acts as a precursor for the HV unit. In these conditions, the bacteria produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) containing a 15.8% 3HV fraction with 0.3% propionate added as the substrate. To examine the possibility of using unsterilized media with high NaCl content for PHB production, V. proteolyticus was cultured in sterilized and unsterilized conditions. Our results indicated a higher growth, leading to a dominant population in unsterilized conditions and higher PHB production. This study showed the conditions for halophilic PHA producers to be later implemented at a larger scale.

     

Production of polyhydroxyalkanoate from starch by the native isolate <Emphasis Type="Italic">Bacillus cereus</Emphasis> CFR06

A bacterial strain that produces amylase and polyhydroxyalkanoate (PHA) was isolated, identified, and classified under the Bacillus cereus group based on 16S rRNA gene sequences and specific reaction in poly-myxin egg yolk Mannitol bromothymol blue agar (PEMBA) medium and in combination with microbiological and biochemical tests. The complete ORF of phaC gene was cloned by PCR technique and nucleotide sequences were determined. Results indicated that the phaC gene had 99% homology with phaC of B. cereus (AE016877.1), 98% with B. thuringiensis (AY331151.1), and 94% with several strains of B. anthracis and B. cereus group including Bacillus sp. INT005. However, only 90% sequence homology with phaC of B. megaterium (AF109909.2) was observed. The PHA production using different fermentable sugars was tested and it was found that the CFR06 was able to accumulate 36–60% of PHA in cell dry weight (CDW). Zymogram of amylase indicated that native strain produces an extracellular enzyme of ∼80 kDa. The potency of the organism to hydrolyze starch due to the intrinsic amylase activity was considered, and starch was used as the sole carbon source for growth and PHA production. GC, FTIR, and ¹H NMR analysis of the polymer indicated that the strain was a potent polyhydroxybutyrate (PHB) producer. The bacterium accumulated about 48% PHA in CDW in a starch containing medium.     

Enhanced Accumulation and Changed Monomer Composition in Polyhydroxyalkanoate (PHA) Copolyester by In Vitro Evolution of Aeromonas caviae PHA Synthase

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC_Ac, was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC_Ac gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) {by LS5218 [fadR601 atoC(Con)] strain}. The expression vector for the phaC_Ac gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).     

A transferable heterogeneous two-hybrid system in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on polyhydroxyalkanoates synthesis regulatory protein PhaR

Background  

Polyhydroxyalkanoate (PHA) synthesis regulatory protein PhaR contains a DNA binding domain (DBD) and a PHA granule binding domain (GBD), it anchors to the promoter region of PHA granule-associated protein (PhaP) to repress phaP expression. However, PhaR will bind to PHB granules and be released from phaP promoter region when PHA granules are formed in vivo, initiating expression of phaP gene. Based on this regulatory mechanism, a bacterial two-hybrid system was developed: PhaR was separated into two parts: DBD was used to fuse with the bait, GBD with the prey, and phaP was replaced by a reporter gene lacZ. However, GBD protein expressed in vivo formed inclusion bodies. Thus, PhaP with strong binding ability to PHB granules was employed to replace GBD.     

Overexpression of NAD kinase in recombinant <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> harboring the <Emphasis Type="BoldItalic">phbCAB</Emphasis> operon improves poly(3-hydroxybutyrate) production

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y _PHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y _PHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

A lower specificity PhaC2 synthase from Pseudomonas stutzeri catalyses the production of copolyesters consisting of short-chain-length and medium-chain-length 3-hydroxyalkanoates

A polyhydroxyalkanoate (PHA) synthase gene phaC2 _Ps from Pseudomonas stutzeri strain 1317 was introduced into a PHA synthase gene phbC _Re negative mutant, Ralstonia eutropha PHB⁻4. It conferred on the host strain the ability to synthesize PHA, the monomer compositions of which varied widely when grown on different carbon sources. During cultivation on gluconate, the presence of phaC2 _Ps in R. eutropha PHB⁻4 led to the accumulation of polyhydroxybutyrate (PHB) homopolymer in an amount of 40.9 wt% in dry cells. With fatty acids, the recombinant successfully produced PHA copolyesters containing both short-chain-length and medium-chain-length 3-hydroxyalkanoate (3HA) of 4–12 carbon atoms in length. When cultivated on a mixture of gluconate and fatty acid, the monomer composition of accumulated PHA was greatly affected and the monomer content was easily regulated by the addition of fatty acids in the cultivation medium. After the (R)-3-hydroxydecanol-ACP:CoA transacylase gene phaG _Pp from Pseudomonas putida was introduced into phaC2 _Ps-containing R. eutropha PHB⁻4, poly(3HB-co-3HA) copolyester with a very high 3-hydroxybutyrate (3HB) fraction (97.3 mol%) was produced from gluconate and the monomer compositions of PHA synthesized from fatty acids were also altered. This study clearly demonstrated that PhaC2_Ps cloned from P. stutzeri 1317 has extraordinarily low substrate specificity in vivo, though it has only 54% identity in comparison to a previously described low-substrate-specificity PHA synthase PhaC1_Ps from Pseudomonas sp. 61–3. This study also indicated that the monomer composition and content of the synthesized PHA can be effectively modulated by controlling the addition of carbon sources or by modifying metabolic pathways in the hosts.