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.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by high-cell-density cultivation of Aeromonas hydrophila

The newly screened Aeromonas hydrophila produces copolymer consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). The characteristics of cell growth and polymer accumulation were examined using various carbon sources. P(3HB-co-3HHx) was produced from lauric acid and oleic acid only. P(3HB-co-3HHx) content can be increased by limitation of phosphorus. A maximal P(3HB-co-3HHx) content of 28.8 wt% could be obtained in flask culture. By applying the optimally designed nutrient feeding strategy, cell dry weight, P(3HB-co-3HHx) content, and 3HHx fraction obtained over the course of 43 h were 95.7 g/L, 45.2 wt%, and 17 mol%, respectively, resulting in a productivity of 1.01 g polyhydroxyalkanoate (PHA)/L. h.     

Sequencing microbial copolymers of 3-hydroxybutyric and 3-mercaptoalkanoic acids by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR

Copolymers of 3-hydroxybutyrate (3HB) and 3-mercaptopropionate (3MP) or 3-mercaptobutyrate (3MB) units and minor amounts of 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), or 3-mercaptovalerate (3MV) were investigated regarding their microstructure by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR. These copolymers were produced by Ralstonia eutropha strain H16 when cells were cultivated in a mineral salts medium with gluconate as a carbon source for growth and 3MP or 3MB as precursor substrates for incorporation of 3-mercaptoalkanoates. Mass spectrometry analysis of partially methanolyzed or pyrolyzed samples proved the presence of true copolymers or terpolymers. (13)C NMR spectroscopy of intact polymer samples, with values of average block length and degree of randomness deviating from a random sequence model, suggested microblock structures; however, composition analysis by (1)H NMR of fractions obtained by size exclusion chromatography showed significant variations with molecular weight, revealing the presence of blends of poly(3HB-co-3MP-co-3HP) or poly(3HB-co-3MB) with poly(3HB). The experimental NMR carbonyl dyad signal intensities were satisfactorily matched by a random sequence model when the presence of poly(3HB) was taken into account.     

Lamellar thickening and cocrystallization of poly(hydroxyalkanoate)s on annealing.

Lamellar thickening behavior of microbial polyesters, poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] annealed at various temperatures was investigated to make sure of the occurrence of cocrystallization of both components. All the copolymers showed steep increases in melting points accompanied by partial melting as the annealing temperature increased up to just below the melting points. In contrast, long periods of P(3HB-co-7mol% 3HV) increased to twice, similar to those of P(3HB), with increasing annealing temperature up to just below the melting point, while long periods of P(3HB-co-7mol% 4HB) and P(3HB-co-92mol% 3HV) only increased up to one and a half times. Lattice indices of unit cell of the former crystal were increased slightly, while those of the latter crystal remained unchanged. These results imply that the P(3HB) crystal can occlude the 3HV component to some extent, but hardly includes the 4HB component, and P(3HV) crystal also excludes the 3HB component.     

Biosynthesis and local sequence specific degradation of poly(3-hydroxyvalerate-co-4-hydroxybutyrate) in Hydrogenophaga pseudoflava

A novel copolymer that consisted of 3-hydroxyvalerate and 4-hydroxybutyrate, P(3HV-co-4HB), was synthesized in Hydrogenophaga pseudoflava by growing it in media containing gamma-valerolactone and gamma-butyrolactone as a carbon source. The monomer ratio in the copolymer was changed by altering the feed ratio of the two lactones. The cultivation technique was composed of three steps: the first-step for high cell production in Luria-Bertani medium, the second-step for intracellular degrading removal of poly(3-hydroxybutyrate) (P(3HB)), which was formed in the first step, by culturing the cells in carbon-source-free medium, and the final step for accumulation of P(3HV-co-4HB) in a mixed lactone medium. All the P(3HV-co-4HB) copolymers contained less than 1 mol % of 3HB unit. These copolymers were characterized by NMR spectroscopy, differential scanning calorimetry, wide-angle X-ray diffraction, and first-order kinetic analysis of intracellular degradation. The copolymer with an approximately equal ratio of the comonomers was found amorphous. The NMR microstructural analysis showed that the copolymers contained appreciable amounts of 3HV-rich or 4HB-rich chains. The (13)C NMR splitting patterns associated with the four carbons in the 4HB unit of P(3HV-co-4HB) bear close resemblance to those observed in the 4HB unit of P(3HB-co-4HB). The signals arising from the carbons in the 3HV unit of P(3HV-co-4HB) split in a manner similar to those in the 3HB unit of P(3HB-co-4HB). Thus the sequences were assigned by comparing the NMR splittings for P(3HV-co-4HB) with those for P(3HB-co-4HB) and P(3HB-co-3HV). The sequence assignment was further checked by comparing the signal intensities before and after degradation of the copolymers. This was considered reasonable because the H. pseudoflava intracellular PHA depolymerase is more specific to the 3HV unit than to the 4HB unit, which was also confirmed by the higher degradation rate constant for the 3HV unit in the first-order kinetic analysis.     

Side-chain effect of second monomer units on crystalline morphology, thermal properties, and enzymatic degradability for random copolyesters of (R)-3-hydroxybutyric acid with (R)-3-hydroxyalkanoic acids

Three types of random copolymers with 94 mol % (R)-3-hydroxybutyric acid (3HB) and 6 mol % (R)-3-hydroxyalkanoic acids with different side-chain lengths, (R)-3-hydroxypentanoic acid (3HV), (R)-3-hydroxyhexanoic acid (3HHx), and medium-chain-length (R)-3-hydroxyalkanoic acids (mcl-3HA, C8-C12), were prepared by biological synthetic techniques. The solid-state structure and thermal properties of melt-crystallized films for copolymers were characterized by means of wide-angle X-ray diffraction, small-angle X-ray scattering, differential scanning calorimetry, and optical microscopy. The randomly distributed second monomer units, except for 3HV in copolyesters, act as defects of the P(3HB) crystal and are excluded from the P(3HB) crystalline lamellae. The lamellar thickness of copolymers decreased with an increase in the side-chain length of second monomer units. In addition, the growth rate of spherulites decreased with an increase in the carbon numbers of second monomer units at an identical crystallization temperature. These results indicate that a steric bulkiness of the second monomer unit affects the crystallization of (R)-3HB segments in random copolyesters. An enzymatic degradation test of melt-crystallized copolymer films was carried out in the presence of PHB depolymerase from Alcaligenes faecalis T1. Erosion rate of copolyesters was dependent on both the crystallinity and the lamellar thickness of samples. As the result, the rate of enzymatic degradation for copolymer films increased with an increase in the carbon numbers of second monomer units.     

Biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer by Cupriavidus sp. USMAA1020 isolated from Lake Kulim, Malaysia

Cupriavidus sp. USMAA1020 was isolated from Malaysian environment and able to synthesize poly(3-hydroxybutyrate-co-4-hydroxybutyrate), [P(3HB-co-4HB)] when grown on gamma-butyrolactone as the sole carbon source. The polyester was purified from freeze-dried cells and analyzed by nuclear magnetic resonance (NMR) spectroscopy. 1H and 13C NMR results confirmed the presence of 3HB and 4HB monomers. In a one-step cultivation process, P(3HB-co-4HB) accumulation by Cupriavidus sp. USMAA1020 was affected by carbon to nitrogen ratio (C/N). A two-step cultivation process accumulated P(3HB-co-4HB) copolyester with a higher 4HB fraction (53 mol%) in nitrogen-free mineral medium containing gamma-butyrolactone. The biosynthesis of P(3HB-co-4HB) was also achieved by using 4-hydroxybutyric acid and alkanediol as 1,4-butanediol. The composition of copolyesters varied from 32 to 51 mol% 4HB, depending on the carbon sources supplied. The copolyester produced by Cupriavidus sp. USMAA1020 has a random sequence distribution of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) units when analyzed by nuclear magnetic resonance (NMR) spectroscopy. When gamma-butyrolactone was used as the sole carbon source, the 4HB fraction in copolyester increased from 25 to 60 mol% as the concentration of gamma-butyrolactone in the culture medium increased from 2.5 g/L to 20.0 g/L.     

Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126

A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.     

Formation of blends of various poly(3-hydroxyalkanoic acids) by a recombinant strain of Pseudomonas oleovorans

Summary Recombinant strains of Pseudomonas oleovorans, which harbour the poly(3-hydroxybutyrate)-biosynthetic genes of Alcaligenes eutrophus, accumulated poly(hydroxyalkanoates), composed of 3-hydroxybutyrate(3HB), 3-hydroxyhexanoate (3HHx) and 3-hydroxyactanoate (3HO), up to 70% of the cell dry weight if the cells were cultivated with sodium octanoate as the carbon source. Physiological and chemical analysis revealed multiple evidence that this polymer is a blend of the homopolyester poly(3HB) and of the copolyester poly(3HHx-co-3HO) rather than a random or a block copolyester of 3HB, 3HHx and 3HO. The molar ratio between poly(3HHx-co-3HO) and poly(3HB) varied drastically during the process of fermentation. Whereas synthesis of poly(3HHx-co-3HO) started immediately after ammonium was exhausted in the medium, synthesis of poly(3HB) occurred only after a lag-phase. From freeze-dried cells poly(3HHx-co-3HO) was much more readily extracted with chloroform than was poly(3HB). The blend was fractionated into petrol-ether-insoluble poly(3HB) and petrol-ether-soluble poly(3HHx-co-3HO). The molecular weight values of these polyesters measured by gel permeation chromatography were 2.96 × 10⁶ and 0.35 × 10⁶ and were similar of those polymers accumulated by A. eutrophus or by wild-type P. oleovorans, respectively. Offprint requests to: A. Steinbüchel     

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.     

Enzymatic hydrolysis of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4

Enzymatic degradability has been investigated for a series of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s (P(3HB-co-3HP)s) with 3-hydroxypropionate (3HP) unit contents from 11 to 86 mol % as well as poly(3-hydroxybutyrate) (P(3HB)) and chemosynthesized poly(3-hydroxypropionate) (P(3HP)). The behavior of degradation by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pikettii T1 and Acidovorax Sp. TP4, defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were compared in relation to the thermal properties and crystalline structures of the PHA samples elucidated by differential scanning calorimetry and wide-angle X-ray diffraction. The degradation products were characterized by high-performance liquid chromatography and one- (1D) and two-dimension (2D) (1)H NMR spectroscopy. It was found that the PHA depolymerase of Acidovorax Sp. TP4 showed degradation behavior different from that shown by depolymerase of R. pikettii T1. PHA depolymerase from Acidovorax Sp. TP4 degraded the P(3HB-co-3HP) films with lower crystallinity in higher rates than those with higher crystallinity, no matter what kinds of crystalline structures they formed. In contrast, PHA depolymerase from R. pikettii T1 degraded P(3HB-co-3HP) films forming P(3HB) crystalline structure in higher rates than those forming P(3HP)s. The increase in amorphous nature of the P(3HB-co-3HP) films with P(3HB)-homopolymer-like crystalline structure increases and then decreases the rate of degradation by depolymerase from R. pikettii T1. The 3-hydroxybutyrate (3HB) monomer was produced as a major product by the hydrolysis of P(3HB) film by PHA depolymerase from Acidovorax Sp. TP4. The P(3HB-co-3HP) films could be degraded into 3HB and 3-hydroxypropionate (3HP) monomer at last, indicating that the catalytic domain of the enzyme recognized at least two monomeric units as substrates. While the PHA depolymerase from R. pikettii T1 hydrolyzed P(3HB) film into 3HB dimer as a major product, and the catalytic domain recognized at least three monomeric units. The degradation behavior of P(3HB-co-3HP) films by the PHA depolymerase of Acidovorax Sp. TP4 could be distinguished from that by the depolymerase of R. pikettii T1.     

Acid catalyzed transesterification as a route to poly(3-hydroxybutyrate-co-epsilon-caprolactone) copolymers from their homopolymers

Copolymers of (R)-3-hydroxybutyric acid (HB) and epsilon-caprolactone (CL) with a composition ranging from 28 to 81 mol % of HB were synthesized by transesterification of the corresponding homopolymers in solution in the presence of 4-toluenesulfonic acid. The copolyesters were characterized with regard to their molecular weights, thermal properties, molar compositions, and average block length of repeating units by gel permeation chromatography (GPC), differential scanning calorimetry, (1)H NMR, and (13)C NMR, respectively. Random and microblock copolymers could be obtained depending on experimental conditions, with weight-average molecular weights of up to 20,000. The glass transition temperature decreased from 2 to -42 degrees C as the CL content was increased from 0 to 72 mol %. The melting temperature (T(m)) of the PCL phase decreased from 70 to 46 degrees C as the HB content changed from 0 to 47 mol %, while the T(m) of the PHB phase decreased from 177 degrees C to 163 degrees C as the CL content changed from 0 to 72 mol %. Matrix-assisted laser desorption ionization time-of-flight mass spectra of GPC fractionated samples allowed us to ascertain that copolymers rich in HB units have mostly hydroxyl and carboxyl end groups, while copolymers rich in CL units have mostly tosyl and carboxyl end groups.     

Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei.

Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.     

Expression of Aeromonas caviae polyhydroxyalkanoate synthase gene in Burkholderia sp. USM (JCM15050) enables the biosynthesis of SCL-MCL PHA from palm oil products

AIMS: Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaC(Ac)) was transformed into this strain. Methods and Results: The resulting transformant incorporated approximately 1 ± 0·3 mol% of 3HHx in the polymer when crude palm kernel oil (CPKO) or palm kernel acid oil was used as the sole carbon source. In addition, when the transformed strain was cultivated in the mixtures of CPKO and sodium valerate, PHA containing 69 mol% 3HB, 30 mol% 3-hydroxyvalerate and 1 mol% 3HHx monomers was produced. Batch feeding of carbon sources with 0·5% (v/v) CPKO at 0 h and 0·25% (w/v) sodium valerate at 36 h yielded 6 mol% of 3HHx monomer by controlled-feeding strategies. CONCLUSIONS: Burkholderia sp. USM (JCM15050) has the metabolic pathways to supply both the short-chain length (SCL) and medium-chain length (MCL) PHA monomers. By transforming the strain with the Aer. caviae PHA synthase with broader substrate specificity, SCL-MCL PHA was produced. Significance and Impact of the Study: This is the first study demonstrating the ability of transformant Burkholderia to produce P(3HB-co-3HHx) from a single carbon source.     

Sequence distribution in microbial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polyesters determined by NMR and MS

The microstructure of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters (PHBV) as well as a mixture of two PHBV copolyesters of different comonomer composition and sequence distribution was studied by 13C NMR based on dyad and triad analysis and multistage electrospray ionization mass spectrometry (ESI-MSn). Both techniques gave results that were in good agreement for all investigated samples. The effect of microstructure on PHBV thermal properties was investigated from the melting behavior of samples. A PHBV copolyester with randomly distributed hydroxyvalerate units (12.0 mol % HV) showed a single melting peak, whereas samples with nonrandom composition distribution showed multiple melting peaks in their thermograms. Such complex melting behavior suggested that the 12.9 and 27.1 mol % PHBV copolyesters were actually blends of several copolymers with widely different comonomer-unit composition.     

Production and characterization of poly-beta-hydroxyalkanoate copolymers from Burkholderia cepacia utilizing xylose and levulinic acid

Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (P(3HB-co-3HV)) copolymers were prepared via shake-flask fermentations of Burkholderia cepacia (formerly Pseudomonas cepacia) containing 2.2% (w/v) xylose and concentrations of levulinic acid ranging from 0.07% to 0.67% (w/v). Periodic harvest of shake-flask cultures from 48 to 92 h post-inoculation yielded 4.4-5.3 g/L of dry cell biomass, containing 42-56% (w/w) P(3HB-co-3HV), with optimal product yield occurring between 66 and 74 h. Growth and PHA accumulation enhancement were observed with concentrations of levulinic acid from 0.07 to 0.52% (w/v), producing dry cell biomass and P(3HB-co-3HV) yields of 9.5 and 4.2 g/L, respectively, at the 0.52% (w/v) concentration of levulinic acid. Representative samples were subjected to compositional analysis by 300 MHz 1H and 150 MHz 13C NMR, indicating that these random copolymers contained between 0.8 and 61 mol % 3-hydroxyvalerate (3HV). Solvent-cast film samples were characterized by differential scanning calorimetry, which demonstrated melting temperatures (Tm) to decrease in a pseudoeutectic fashion from 174.3 degrees C (0.8 mol % 3HV) to a minimum of 154.2 degrees C (25 mol % 3HV) and the glass transition temperatures (Tg) to decrease linearly from 2.1 to -11.9 degrees C as a function of increasing mol % 3HV. Thermogravimetric analysis of the copolymer series showed the temperature for onset of thermal decomposition (T(decomp)) to vary as a function of mol % 3HV from 273.4 to 225.5 degrees C. Intrinsic viscosities (eta) varied from 3.2 to 5.4 dL/g, as determined by dilute solution viscometry. Viscosity average molecular weights (Mv) of the copolymers were determined to range from 469 to 919 kDa, indicating that these P(3HB-co-3HV) copolymers are of sufficient molecular mass for commercial application.     

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.     

Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M _n) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 × 10⁶ and 2.5 × 10⁶ g mol⁻¹, respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.     

Biosynthesis of polyhydroxyalkanoates (PHA) by recombinant Ralstonia eutropha and effects of PHA synthase activity on in vivo PHA biosynthesis.

Recombinant strains of Ralstonia eutropha PHB 4, which harbored Aeromonas caviae polyhydroxyalkanoates (PHA) biosynthesis genes under the control of a promoter for R. eutropha phb operon, were examined for PHA production from various alkanoic acids. The recombinants produced poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from hexanoate and octanoate, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxypentano ate) [P(3HB-co-3HV-co-3HHp)] from pentanoate and nonanoate. One of the recombinant strains, R. eutropha PHB 4/pJRDBB39d3 harboring ORF1 and PHA synthase gene of A. caviae (phaC(Ac)) accumulated copolyesters with much more 3HHx or 3HHp fraction than the other recombinant strains. To investigate the relationship between PHA synthase activity and in vivo PHA biosynthesis in R. eutropha, the PHB- 4 strains harboring pJRDBB39d13 or pJRDEE32d13 were used, in which the heterologous expression of phaC(Ac) was controlled by promoters for R. eutropha phb operon and A. caviae pha operon, respectively. The PHA contents and PHA accumulation rates were similar between the two recombinant strains in spite of the quite different levels of PHA synthase activity, indicating that the polymerization step is not the rate-determining one in PHA biosynthesis by R. eutropha. The molecular weights of poly(3-hydroxybutyrate) produced by the recombinant strains were also independent of the levels of PHA synthase activity. It has been suggested that a chain-transfer agent is generated in R. eutopha cells to regulate the chain length of polymers.     

Viscoelastic relaxations and thermal properties of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

3-Hydroxybutyrate-3-hydroxyvalerate (3HB-3HV) as well as 3-hydroxybutyrate-4-hydroxybutyrate (3HB-4HB) copolyesters have been investigated by differential scanning calorimetry, thermogravimetric analysis and dynamic mechanical spectroscopy, over a wide range of compositions (0-95 mol% 3HV; 0-82 mol% 4HB). Both series of isolated copolyesters are partially crystalline at all compositions. Quenched samples show a glass transition that decreases linearly with increasing co-monomer molar fraction, more markedly when the co-monomer is 4HB. Above Tg, all copolyesters, rich in 3HB units, show a cold crystallization phenomenon followed by melting, while at the other end crystallization on heating is observed only in 3HB-3HV copolymers. The viscoelastic spectrum, strongly affected by thermal history, shows two relaxation regions: the glass transition, whose location depends on copolymer type and composition, and a secondary dispersion region at low temperatures (-130/-80 degrees C). The latter results from a water-related relaxation analogous to that of P(3HB) and, in 3HB-4HB copolymers, from another overlapping absorption peak centered at -130 degrees C, attributed to local motion of the methylene groups in the linear 4HB units.