Biosynthesis and Characterization of Poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from Palm Oil Products in a Wautersia eutropha Mutant

Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by ¹³C NMR analysis. The number average molecular weight (M_n) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6–3.9.     

Synthesis of biodegradable polyesters by Gram negative bacterium isolated from Malaysian environment

A locally isolated Gram negative bacterium, Cupriavidus sp. USMAA9-39 was able to produce various types of biodegradable polyesters through a two-step cultivation process. These are copolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)]. These polymers were synthesized by this bacterium when grown with a combination of some carbon sources. The biosynthesis of P(3HB-co-4HB) was achieved by using carbon sources such as γ-butyrolactone or 1,4-butanediol or by a combination of oleic acid with either γ-butyrolactone or 1,4-butanediol. Meanwhile, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was produced using 1-pentanol or valeric acid or by a combination of oleic acid with either 1-pentanol or valeric acid. When γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol were used as mixed carbon sources, P(3HB-co-3HV-co-4HB) terpolymer were produced. The presence of 3HB, 3HV or/and 4HB monomers were confirmed by gas chromatography and nuclear magnetic resonance (NMR) spectroscopy.     

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.     

Biosynthesis of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) copolyesters with a high molar fraction of 3-hydroxyvalerate by an insect-symbiotic <Emphasis Type="Italic">Burkholderia</Emphasis> sp. IS-01

Burkholderia sp. IS-01 capable of biosynthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] copolyesters with a high molar fraction of 3HV was isolated from the gut of the adult longicorn beetle, Moechotypa diphysis. The strain IS-01 was relatively tolerant to high concentrations of levulinic acid and accumulated a poly(13.5 mol% 3HB-co-86.5 mol% 3HV) copolyester when cultivated on a mixture of gluconate (20 g/L) and levulinic acid (12.5 g/L). In this case, the content of the copolyester in the cells was approximately 60.0%. The compositions of the copolyesters were easily regulated by altering the molar ratio of gluconate and levulinic acid in the medium. The organism was found to possess a class I PHA synthase (PhaC) gene (1,881 bp) that encodes a protein with a deduced molecular mass of 68,538 Da that consists of 626 amino acids. The PhaC of this organism was most similar to that of B. cenocepacia PC184 (92% similarity).     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with high molar fractions of 3-hydroxyvalerate by a threonine-overproducing mutant of Alcaligenes sp. SH-69

A threonine overproducing mutant of Alcaligenes sp. SH-69 was isolated and its ability to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), was investigated. The 3HV fraction in poly(3HB-co-3HV) produced from glucose as the sole carbon source exceeded 22 mol%, which is approximately six times higher than that achieved by the wild type under the same culture conditions. Furthermore, the addition of a relatively low concentration (10 mM) of propionic acid, valeric acid or levulinic acid to the glucose medium greatly increased the molar fraction of 3HV in the copolyester, to 38–77 mol%. The results suggest that metabolic engineering of the biosynthetic pathways supplying polyhydroxyalkanoate monomers, such as the threonine biosynthetic pathway, can lead to new poly(3HB-co-3HV)-producing strains.     

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.     

Isolation of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) producer from Malaysian environment using γ-butyrolactone as carbon source

Samples from various natural environments in Peninsular Malaysia were screened for microorganisms that are capable of producing poly(3-hydroxybutyrate-co-4-hydroxybutyrate). A total of 663 isolates were isolated and 119 out of these isolates were identified as possible PHA producers based on Nile red staining methods. All these potential producers emitted pink fluorescence when grown on solid mineral salts medium (MSM) containing Nile red and exposed to UV light. The isolates obtained in this study were cultivated in MSM containing γ-butyrolactone as the carbon source. Gas chromatography (GC) analysis confirmed that 95 out of the 119 isolates were PHA producers. Among the 95 positive isolates, 77 isolates produced only P(3HB) homopolymer and 18 isolates produced PHA containing 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) monomers. Of these 18 isolates, USMAA1020 was screened as the best P(3HB-co-4HB) producer based on GC analysis. For further confirmation, PHA was extracted from the isolate and analyzed by GC as well as nuclear magnetic resonance (NMR). Results from both analyses confirmed that this isolate was capable of producing PHA containing 3HB and 4HB. Based on, biochemical characterization, 16S rRNA sequencing, DNA base composition, cellular fatty acids analysis and DNA–DNA hybridization, it is clearly indicated that this isolate belongs to the genus Cupriavidus. Poly(3HB-co-4HB) was synthesized by this bacterium in one-stage, two-stage and three-stage cultivation using γ-butyrolactone as the carbon source. The highest 4HB composition of 82 mol% was obtained through three-stage cultivation.     

Biosynthesis of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) terpolymer by <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4 through two-step cultivation process

A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was found capable of producing terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] using γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol as the carbon source. The present of 3HB, 3HV and 4HB monomers were confirmed by gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. PHA concentration of 1.9 g/l was the highest value obtained using the combination of 1,4-butanediol and 1-pentanol through one-step cultivation process. PHA concentration obtained through two-step cultivation process was higher for all the combinations and the highest value achieved was 2.5 g/l using γ-butyrolactone and 1-pentanol as carbon source. Various molar fractions of 4HB and 3HV ranging from 6 to 14 mol% and 39 to 87 mol%, respectively were produced through two-step cultivation process by manipulating the concentration of γ-butyrolactone. As the culture aeration was reduced, the molar fraction of 3HV and 4HB increased from 40 to 67 mol% and 10 to 24 mol%, respectively while the dry cell weight and PHA content decreased. The terpolymer produced was characterized using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The number-average molecular weight (M _n) and the melting temperature (T _m)) of the terpolymer were in the range of 177–484 kDa and 160–164°C, respectively.     

Enhanced production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) copolymer with manipulated variables and its properties

Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M _n) of copolymers ranged from 260 × 10³ to 590 × 10³Da, and the polydispersities (M _w/M _n) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T _m), glass transition temperature (T _g), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.     

Characteristics of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production byRalstonia eutropha NCIMB 11599 and ATCC 17699

Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures ofR. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the γ-butyrolactone concentration, and the 4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures ofR. eutropha NCIMB 11599, glucose and γ-butyrolactone were fed as the carbon sources, under a phosphate limitation strategy. When glucose was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB) homopolymer was obtained from 201 g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased to 104 g/L, with glucose fed in the first step and constant feeding of γ-butyrolactone, at 6 g/h, in the second, final cell concentration at 67 h was 106 g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7 mol%. When the same feeding strategy was applied to the fedbatch culture ofR. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and γ-butyrolactone (1.5 g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74 h were 51 g/L, 35% and 32 mol%, respectively. In summary,R. eutropha ATCC 17699 was better thanR. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions.     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

Production of copolymer poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) through a one-step cultivation process

A one-step cultivation process for the production of biodegradable polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by Cupriavidus sp. USMAA2-4 was carried out using various carbon sources. It was found that Cupriavidus sp. USMAA2-4 could produce approximately 44 wt.% copolymer of P(3HB-co-4HB) with 27 mol% 4HB composition when the combination of oleic acid and 1,4-butanediol are used as carbon sources in 60 h cultivation. The manipulation of carbon-to-nitrogen ratio (C/N) resulted in the increase of dry cell weight, PHA content as well as 4HB composition. A new strategy of introducing oleic acid and 1,4-butanediol together and separately at different concentration demonstrated different yield in PHA content ranging from 47 to 58 wt.%. The molecular weight obtained was 234 kDa (by adding 1,4-butanediol and oleic acid together) and 212 kDa (by adding 1,4-butanediol separately). The copolymer of P(3HB-co-4HB) produced by Cupriavidus sp. USMAA2-4 was detected statistically as a random copolymer when analysed by nuclear magnetic resonance (NMR) spectroscopy.     

Heterologous expression of <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4 PHA synthase gene in PHB<Superscript>−</Superscript>4 mutant for the production of poly(3-hydroxybutyrate) and its copolymers

Ecological deterioration and human health concerns arising from the usage of non-biodegradable plastics have prompted mankind to search for greener alternatives which are biodegradable, biocompatible and easily produced from renewable sources. Polyhydroxyalkanoates (PHA), among other biopolymers, are emerging as a viable replacement for fossil fuel-based synthetic plastics. A PHA-producing strain, identified as Cupriavidus sp. (designated Cupriavidus sp. USMAA2-4) was isolated from a soil sample from western peninsular Malaysia. Heterologous expression of the PHA synthase gene (phaC _USMAA2-4) in mutant C. necator PHB⁻4 complemented its PHA-producing ability. More than 60 wt% of P(3HB) was synthesized from various plant oils. The highest P(3HB) production of 2.38 g/l at 68 wt% was attained when crude palm kernel oil was fed as the sole carbon source. The 3HV molar fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was significantly affected by the type of the precursor used and their respective feeding time. The 3HV molar fraction ranged from 4 to 31 mol% when sodium propionate/valerate was fed at different cultivation times. In addition, with the supplementation of 4HB-monomer precursors, approximately 67 wt% P(3HB-co-4HB) with 4–5 mol% of 4-hydroxybutyrate monomer was synthesized, regardless of the precursor feeding time used. Variation in the molar fraction of the second monomer along with its biodegradability and biocompatibility characteristics promotes the potential of these copolymers as replacements for traditional commodity plastics.     

Poly-β-hydroxyalkanoate production by halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> U7

Medium optimization for production of poly-β-hydroxyalkanoate (PHA) from Rhodobacter sphaeroides U7 cultivated in glutamate–acetate (GA) medium supplemented with 40 mM valeric acid as co-substrate under aerobic-dark condition was investigated. Studies on effect of nitrogen source and cultivation temperature by conventional and statistical methods illustrated that (NH₄)₂SO₄ (0.2 g/l) had no effect and the optimal temperature was at 30°C. The optimum environmental conditions were found to be anaerobic-light (3000 lux) cultivation with aeration rate of 1.0 vvm and agitation speed of 200 rpm for PHA production (2.5 g/l) with the highest PHA content (65.15%) at 0.5 vvm, and 200 rpm. Under this optimized medium and condition, PHA production from R. sphaeroides U7 increased 3.86-folds (from 0.69 to 2.66 g/l) (PHA content increased 1.5-folds). The biopolymer was purified and characterized by using ¹³C NMR, FTIR, DSC, X-ray diffraction and intrinsic viscosity techniques to be a copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) consisting of 84.8 mol% β-hydroxybutyric acid (HB) and 15.2 mol% β-hydroxyvaleric acid (HV).     

Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC. It was found that, with introduction of atoAD and with propionate as a co-substrate, 3HV fraction in PHA was increased up to 7.3-fold higher than a strain without atoAD expressed in trans (67.9 mol%). By the analysis of CoA pool concentrations in vivo and in vitro using HPLC and LC-MS, overexpression of AtoAD was shown to decrease the amount of acetyl-CoA and increase the propionyl-CoA/acetyl-CoA ratio, ultimately resulting in an increased 3HV fraction in PHA. Finally, synthesis of P(3HB-co-3HV) containing 57.9 mol% of 3HV was achieved by fed-batch fermentation of YJ101 with propionate.

     

Production of copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate by fed-batch culture of Alcaligenes SP. SH-69

Summary Production of copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate [poly(3HB-co-3HV)] by fed-batch culture of Alcaligenes sp. SH-69 was investigated using glucose as a sole carbon source. Synthesis of poly(3HB-co-3HV) during the polymer accumulation stage was favored under dissolved oxygen tension at 20% and C/N ratio (mol glucose/mol ammonium) of 23.1. When conditions were optimal, 36 g liter^-1 of poly(3HB-co-3HV) containing 3.0 mol% of 3HV was produced. Decreasing C/N ratio resulted in an increase of 3HV fraction in the copolymer to a maximum level of 6.3 mol%.     

Biosynthesis of diverse α,ω-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis

Polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible plastics with potential to replace petroleum based plastics. Diversity of PHA monomer structures provides flexibility in material properties to suit more applications. In this study, 5-hydroxyvalerate (5HV) synthesis pathway was established based on intrinsic alcohol/aldehyde dehydrogenases. The PHA polymerase cloned from Cupriavidus necator functions to polymerize 5HV into its copolymers in ratios ranging from 8% to 32%. Elastic copolymer P(85% 3HB-co-15% 5HV) was generated with an elongation at break and a Young's modulus of 1283% and 73.1 MPa, respectively. The recombinant H. bluephagenesis was able to convert various diols including 1, 3-propanediol, 1, 4-butanediol and 1, 5-pentanediol into PHA, leading to 13 PHA polymers including transparent P(53% 3HB-co-20% 4HB-co-27% 5HV) and sticky P(3HB-co-3HP-co-4HB-co-5HV). The engineered H. bluephagenesis was successfully grown in a 7-L bioreactor to produce the highly elastic P(85% 3HB-co-15% 5HV) and the sticky P(3HB-co-3HP-co-4HB-co-5HV), demonstrating their potential for industrial scale-up.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Corynebacterium glutamicum using propionate as a precursor

Lipopolysaccharides free P[3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)] production was achieved using recombinant Corynebacterium glutamicum harboring polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha. Cells grown on glucose with feeding of propionate as a precursor of 3HV unit accumulated 8-47 wt% of P(3HB-co-3HV). The 3HV fraction in the copolymer was varied from 0 to 28 mol% depending on the propionate concentrations.     

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.     

Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) P(3HB-<Emphasis Type="Italic">co</Emphasis>-3HV) with a broad range of 3HV content at high yields by <Emphasis Type="Italic">Burkholderia sacchari</Emphasis> IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y _3HV/prop) increasing from 1.10 to 1.34 g g⁻¹. Copolymer productivity of 1 g l⁻¹ h⁻¹ was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y _3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).