Synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli

Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesters poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) was investigated with special regard to intermediate products, kinetics, and yields. During the degradation of PHBV acetate, propionate, n-butyrate, and n-valerate were detected. Additionally, 3-hydroxybutyrate and 3-hydroxyvalerate and four dimeric esters of these two molecules were identified by GC-MS measurements. Three different test systems for the anaerobic degradation of polyesters were studied. It was not possible to get reproducible results by means of the Anaerobic Sturm-test, a simple system based on carbon dioxide measurement. Secondly, a system based on the GC measurement of accumulated organic acids was investigated. A degradation of 90% in two days was calculated by a carbon balance. Best results were reached with the third test system based on the measurement of methane with a gas meter. A degradation of 99% was observed within 30 days.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesterspoly-3-hydroxybutyrate (PHB) andpoly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) wasinvestigated with special regard to intermediateproducts, kinetics, and yields. During the degradationof PHBV acetate, propionate, n-butyrate, andn-valerate were detected. Additionally,3-hydroxybutyrate and 3-hydroxyvalerate and fourdimeric esters of these two molecules were identifiedby GC-MS measurements. Three different test systemsfor the anaerobic degradation of polyesters werestudied. It was not possible to get reproducibleresults by means of the Anaerobic Sturm-test, a simplesystem based on carbon dioxide measurement. Secondly,a system based on the GC measurement of accumulatedorganic acids was investigated. A degradation of 90%in two days was calculated by a carbon balance. Bestresults were reached with the third test system basedon the measurement of methane with a gas meter. Adegradation of 99% was observed within 30 days.     

Rhodospirillum rubrum: utilization of condensed corn solubles for poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) production

Aims: This study sought to develop a less expensive medium for growth of the polyhydroxyalkanoate-producing bacterium Rhodospirillum rubrum from the ethanol production coproduct, condensed corn solubles (CCS). Methods and Results: Small-scale trials using R. rubrum were performed in aerated or anaerobic stoppered serum bottles filled with media. The CCS (240 g l⁻¹) achieved a maximum cell density and growth rate comparable with the defined supplemented malate-ammonium medium (mSMN) or tryptic soy broth. Microaerophilic solubles medium cultures exhibited significantly higher maximum cell densities and growth rates than did strictly anaerobic cultures; while illumination, nickel or biotin addition had no effect. Growth of R. rubrum in a pH controlled bioreactor was significantly better in CCS (240 g l⁻¹) than in mSMN medium and supported production of 0·36% (cell dry weight) poly-(3-hydroxybutyrate-Co-3-hydroxyvalerate) after 24 h. Conclusions: A CCS medium was devised that supported R. rubrum growth for biopolymer production as effective as the defined medium. Significance and Impact of the Study: This study demonstrates that a more economical medium can be developed for biopolymer production using a low value coproduct from ethanol production. The impact is that this inexpensive solubles medium may make it more economical to produce the biopolymer on a commercial scale.     

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch cultures of Pseudomonas sp EL-2

Pseudomonas sp EL-2 was cultivated to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from a structurally unrelated carbon source, glucose, by a fed-batch culture technique. Variation of the carbon to nitrogen (C/N) ratio of the medium produced optimal P(3HB-co-3HV) production at a C/N ratio of 95. Production of P(3HB-co-3HV) was favored by a dissolved oxygen tension of 40%. A maximum biomass concentration of 38 g L⁻¹ containing 53% P(3HB-co-3HV) was achieved after 45 h of cultivation. This corresponds to a volumetric productivity of 0.84 g L⁻¹ h⁻¹. The copolymer contained 7.5 mol% 3-hydroxyvalerate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 36–40. Received 28 January 1999/ Accepted in revised form 11 September 1999     

Polyphasic characterization of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (p(HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains

For the purpose of denitrification in small drinking water plants, a bacterial mixed population was isolated from a packed bed column bioreactor with poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) as a substrate for the denitrification of ground water (10 degrees C). Isolates 2nIII from the mixed culture, with the ability to denitrify and metabolize P(HB-co-HV), were used as starter cultures for the elimination of nitrate in ground water. The strains were characterized by diverse techniques. Classical phenotypic studies lead to rRNA group III of the genus Pseudomonas. Results obtained by molecular techniques demonstrated that the 2nIII strains are members of the Comamonadaceae and shows similarities to the genus Acidovorax. However, an integration of the 2nIII isolates within one of the known Acidovorax species is not possible for the moment. The 2nIII starter cultures clustered close to Av. temperans according to their whole cell proteins and fatty acids, whereas in DNA/DNA hybridization no significant DNA binding (< 25%) was found. In contrast a significant but low degree of DNA/DNA hybridization was found between the 2nIII strains and Av. facilis and Av. delafieldii. Our polyphasic results lead to the conclusion that the 2nIII strains may constitute a separate Acicdovorax species.     

Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Burkholderia cepacia D1.

Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced by Burkholderia cepacia D1 at 30°C in nitrogen-free culture solutions containing n-butyric acid and/or n-valeric acid. When n-valeric acid was used as the sole carbon source, the 3HV fraction in copolyester increased from 36 to 90 mol% as the concentration of n-valeric acid in the culture solution increased from 1 to 20 g/l. The addition of n-butyric acid to the culture solution resulted in a decrease in the 3HV fraction in copolyester. The copolymers biosynthesized by this method were mixtures of random copolymers having a wide variety of composition of the 3HV component. The melting points of the fractionated copolymers show a concave curve with the minimum at the 3HV content of ≈40 mol%. The a-parameter of lattice indices of the P(3HB) crystal for the fractionated copolymers largely increased as the 3HV composition increased. Biodegradability of the copolymer increased with the lower content of 3HV composition and/or the lower crystallinity.     

Oleic acid improves poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Ralstonia eutropha in inverted sugar and propionic acid

With the objective of verifying the influence of oleic acid as a nutritional supplement in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Ralstonia eutropha, cultures were established with 0.3 g oleic acid l^–1 and without this supplement, in 30 g inverted sugar l^–1 and 1 g propionic acid l^–1. The use of this supplement increased the accumulation of polymer from 18.3% to 28.3% (w/w) although the mass of 3-hydroxyvalerate in the polymer remained constant for both cultures.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

Economic considerations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacterial fermentation

The process for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB/V)] by bacterial fermentation and its recovery was analysed. The effects of various factors such as P(3HB/V) content, P(3HB/V) productivity, P(3HB/V) yield and 3-hydroxyvalerate (3HV) fraction in P(3HB/V) on the production cost of P(3HB/V) were examined. The increase in the 3HV yield on a carbon source did not significantly decrease the production cost when the 3HV fraction was 10 mol%, because the cost of the carbon substrate for 3HV was relatively small in terms of the total cost. However, at a 3HV fraction of 30 mol%, the 3HV yield on a carbon source had a significant effect on the total P(3HB/V) production cost. The production cost of P(3HB/V) increased linearly with the increase in the 3HV fraction in P(3HB/V). Received: 8 September 1999 / Received revision: 2 December 1999 / Accepted: 3 December 1999     

Regulation of poly-(R)-(3-hydroxybutyrate-co-3-hydroxyvalerate) biosynthesis by the AtoSCDAEB regulon in phaCAB + Escherichia coli

AtoSC two-component system (TCS) upregulates the high-molecular weight poly-(R)-3-hydroxybutyrate (PHB) biosynthesis in recombinant phaCAB ⁺ Escherichia coli strains, with the Cupriavidus necator phaCAB operon. We report here that AtoSC upregulates also the copolymer P(3HB-co-3HV) biosynthesis in phaCAB ⁺ E. coli. Acetoacetate-induced AtoSC maximized P(3HB-co-3HV) to 1.27 g/l with a 3HV fraction of 25.5 % wt. and biopolymer content of 75 % w/w in a time-dependent process. The atoSC locus deletion in the ?atoSC strains resulted in 4.5-fold P(3HB-co-3HV) reduction, while the 3HV fraction of the copolymer was restricted to only 6.4 % wt. The ?atoSC phenotype was restored by extrachromosomal introduction of AtoSC. Deletion of the atoDAEB operon triggered a significant decrease in P(3HB-co-3HV) synthesis and 3HV content in ?atoDAEB strains. However, the acetoacetate-induced AtoSC in those strains increased P(3HB-co-3HV) to 0.8 g/l with 21 % 3HV, while AtoC or AtoS expression increased P(3HB-co-3HV) synthesis 3.6- or 2.4-fold, respectively, upon acetoacetate. Complementation of the ?atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. Individual inhibition of β-oxidation and mainly fatty acid biosynthesis pathways by acrylic acid or cerulenin, respectively, reduced P(3HB-co-3HV) biosynthesis. Under those conditions, introduction of atoSC or atoSCDAEB regulon was capable of upregulating biopolymer accumulation. Concurrent inhibition of both the fatty acid metabolic pathways eliminated P(3HB-co-3HV) production. P(3HB-co-3HV) upregulation in phaCAB ⁺ E. coli by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards biotechnologically improved polyhydroxyalkanoates biosynthesis.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Aspergillus sp. NA-25

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading thermophilic fungus was isolated from soil sample collected from waste disposal site, Islamabad, Pakistan. It was able to grow efficiently on a medium containing PHBV as a sole source of carbon and has been identified as Aspergillus sp. NA-25 by 18S rRNA. Using 9% of inoculum maximum production of PHBV depolymerase was observed at 45°C, pH 7.0 in the presence of 0.2% lactose as an additional carbon source. PHBV depolymerase was purified by precipitation with 80% ammonium sulfate and gel filtration chromatography on Sephadex G-75. The four enzyme forms obtained after gel filtration were analyzed on SDS-PAGE and their molecular weights (36, 68, 72 and 90 kDa) were determined. They were characterized on the basis of effect of different temperatures, pH, metal ions and different reagents on the PHBV activity and stability. It is obvious that the fungal strain Aspergillus sp. NA-25 is capable of degrading PHBV with the help of different types of depolymerases.     

Biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters produced byAlcaligenes sp. MT-16

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), copolyesters, with 3-hydroxyvalerate (3HV) contents ranging from 17 to 60 mol%, were produced byAlcaligenes sp. MT-16, and their biocompatibility evaluated by the growth of Chinese hamster ovary (CHO) cells and the adsorption of blood proteins and platelets onto their film surfaces. The number of CHO cells that adhered to and grew on these films was higher with increasing 3HV content. In contrast, the tendency for blood proteins and platelets to adhere to the copolyester surfaces significantly decreased with increasing 3HV content. Examination of the surface morphology using atomic force microscopy revealed that the surface roughness was an important factor in determining the biocompatibility of theses copolyesters. The results obtained in this study suggest that poly(3HB-co-3HV) copolyesters, with >30 mol% 3HV, may be useful in biocompatible biomedical applications.     

n-Amyl alcohol as a substrate for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacteria

Abstract n -Amyl alcohol was examined as a source for the synthesis of the 3-hydroxyvalerate (3HV) unit of the biopolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), by Alcaligenes sp., Pseudomonas sp. and several methylotrophic bacteria. A. eutrophus and Ps. lemoignei synthesized P(3HB-co-3HV) from glucose and n -amyl alcohol under nitrogen-deficient conditions. Many of methylotrophic bacteria grown on methanol synthesized the copolyester from methanol and n -amyl alcohol under nitrogen-deficient conditions. The content and composition of the polyester varied from strain to strain. Paracoccus denitrificans differed from all others in having a higher content of 3-hydroxyvalerate units in the copolyester synthesized.     

High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli.

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1

Here, we report on the biodegradation of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. Preliminary screening and isolation of the bacterium was done using polyhydroxyalkanoate latex medium (PHALM). The isolate was cultured with P(3HB-co-3HV) as the only carbon source and by-products of degradation were derivatized with [N,O-bis(trimethylsilyl)trifluroacetamide] (BSTFA). These products were identified by gas chromatography/mass spectrometry (GC-MS) as silylated hydroxybutyric acid (3HB) and hydroxyvaleric acid, suggesting extracellular depolymerase activity by the isolate. The depolymerase was isolated by (NH₄)₂SO₄ fractionation, dialyzed and purified using fast protein liquid chromatography (FPLC), and confirmed using P(3HB-co-3HV) as a sole source of carbon. The molecular mass of the FPLC purified enzyme occurred between 45 and 66 kDa (SDS-PAGE), but was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to be 62 kDa. Enzyme activity was significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and Tween 80, but induced by azide (N³⁻). Sensitivity to PMSF, DTT, and Tween 80 suggests the involvement of serine as an active site amino acid with disulphide bonds contributing to the catalytic activity, as well as the presence of hydrophobic regions in the enzyme. Non-inhibition of activity by azide indicates that metal ions may not be required as cofactors for activity. This observation was further corroborated by the decrease in enzyme activity in the presence of metal ions such as Ca²⁺, Mg²⁺, Na⁺, and K⁺. The kinetic parameters, V_max and K_m, in the presence of p-nitrophenylbutyrate as substrate, were determined to be 5.06 × 10⁻¹ ??mol min⁻¹ and 6.73 × 10⁻¹ mM, respectively.     

Enzymatic and non-enzymatic degradation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters produced by Alcaligenes sp. MT-16

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), copolyesters with a variety of 3HV contents (ranging from 17 to 60 mol%) were produced by Alcaligenes sp. MT-16 grown on a medium containing glucose and levulinic acid in various ratios, and the effects of hydrophilicity and crystallinity on the degradability of the copolyesters were evaluated. Measurements of thermo-mechanical properties and Fourier-transform infrared spectroscopy in the attenuated total reflectance revealed that the hydrophilicity and crystallinity of poly(3HB-co-3HV) copolyesters decreased as 3HV content in the copolyester increased. When the prepared copolyester film samples were non-enzymatically hydrolysed in 0.01 N NaOH solution, the weights of all samples were found to have undergone no changes over a period of 20 weeks. In contrast, the copolyester film samples were degraded by the action of extracellular polyhydroxybutyrate depolymerase from Emericellopsis minima W2. The overall rate of weight loss was higher in the films containing higher amounts of 3HV, suggesting that the enzymatic degradation of the copolyester is more dependent on the crystallinity of the copolyester than on its hydrophilicity. Our results suggest that the degradability characteristics of poly(3HB-co-3HV) copolyesters, as well as their thermo-mechanical properties, are greatly influenced by the 3HV content in the copolyesters.