Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp.

The PHA-degrading isolate, strain P37C, was enriched from residential compost for its ability to hydrolyze the medium chain length PHA, poly(beta-hydroxyoctanoate) (PHO). It was subsequently found to grow on a wide range of PHAs, including both short chain length and medium chain length PHAs. The isolate was identified as belonging to the genus Comamonas. Strain P37C formed clear zones on poly(beta-hydroxybutyrate) (PHB), (PHO) and poly(beta-hydroxyphenylvalerate) (PHPV) overlay plates. PHA clear zone tubes were prepared using seven different kinds of PHAs, ranging from PHB with four-carbon repeating units, to poly(beta-hydroxyoctanoate-co-beta-hydroxyundecanoate) (PHOU) with 8- and 11-carbon repeating units. There was a direct correlation between PHA side chain length and rate of hydrolysis of the PHAs. A series of PHOUs containing varying percentages of unsaturated bonds were used to make a series of epoxidized PHOUs (PHOEs) with varying percentages of epoxy functions. Results of clear zone tube assays showed that these functionalized PHAs were all biodegradable by strain P37C, and there was no apparent correlation between rate of biodegradation and the proportion of functional groups in the PHAs. Biodegradability of these PHAs was verified using respirometry and enzyme assays. Cell-free supernatants containing activity toward PHAs were prepared, and strain P37C was shown to synthesize at least two distinct PHA depolymerases for the hydrolysis of SCL and MCL PHAs.     

Monohydroxylated poly(3-hydroxyoctanoate) oligomers and its functionalized derivatives used as macroinitiators in the synthesis of degradable diblock copolyesters

The presence of a hydroxyl group at the end of poly(3-hydroxyoctanoate) oligomers, noted PHO oligomers, is required to prepare diblock copolymers with improved properties by ring-opening polymerization of cyclic monomer as epsilon-caprolactone. Several chemical methods such as basic hydrolysis, acid-catalyzed reaction with APTS, and methanolysis were used to prepare well-defined low molar masses PHO oligomers. The methanolysis reaction was allowed to proceed for 10-60 min to produce PHO oligomers with Mn values ranging from 20,000 to 800 g mol-1 with low polydispersity index. Detailed analysis of the MALDI-TOF mass spectra of the obtained oligomers has revealed the presence of linear structures bearing methyl ester on one side and hydroxyl end group on the other side. The same procedure was applied to poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate), PHOU, a poly(3-hydroxyalkanoate) containing unsaturated units in its side chains. These oligomers were further used to initiate the polymerization of epsilon-caprolactone by varying the PHO (or PHOU) and PCL lengths. By copolymerization with epsilon-caprolactone, the properties of PHO or PHOU have been improved. The crystallinity of the obtained copolymers was modified by controlling the length of the two different blocks. The unsaturations in the side chains of the PHOU block were oxidized in acid carboxylic functions to obtain a novel artificial biopolyester. Moreover, degradation was followed to study the influence of carboxylic groups on the hydrolysis of the copolymers.     

Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.     

Exhibition of soft and tenacious characteristics based on liquid crystal formation by introduction of cholesterol groups on biodegradable lactide copolymer

Cholesterol side-functionalized poly(depsipeptide- co- dl-lactide) (PGD- dl-LA-(cholesterol) n ) and poly(depsipeptide) (PGD-(cholesterol) n) were prepared as novel biodegradable liquid crystalline (LC) polymers. These polymer films exhibited different LC phases depending on the cholesterol unit content in the polymers. The thermodynamic stability of these LC phases was quite high, and PGD-(cholesterol) n film exhibited continuous LC phases up to 202 degrees C. The resulting cholesterol LC phases were indicated to act as physical cross-linking points to form noncovalent network structures among the polymer chains. Therefore, PGD- dl-LA-(cholesterol) n film exhibited a rubbery and stretchy nature at 37 degrees C due to physical cross-linking points based on cholesterol LC phase well-dispersed in the film. The cholesterol side-group effects leading to rubbery character and hydrolytic resistance reported herein are rather unique. The biodegradable LC material exhibiting a soft and tenacious nature is a promising candidate for a new class of implant biomaterials used with dynamic organs of the body such as the heart and blood vessels.     

Mechanism of polymer-induced hemolysis: nanosized pore formation and osmotic lysis

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0-47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose-response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.     

Biosynthesis and biotechnological production of degradable polyhydroxyalkanoic acid

The use of biodegradable polymers is one of the key solution to environmental problems and the development of biocompatible material. The impact of such a large commercial opportunity is one of the primary reasons for much interest in the field of microbial polyester, polyhydroxyalkanoic acid (PHA). Its valuable properties of biodegradability, biocompatibility and thermoplasticity have attracted considerable commercial interest, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has been launched as the first market product. Recent advances in molecular genetics and microbial physiology of PHA biosynthesis have been uncovering the biosynthetic mechanics at molecular level, and extensive efforts for the developments of practical applications and cost-effective mass production of PHA will profell the commercialization of PHA towards the commodity market for biodegradable plastics. The biosynthesis of new members of PHA family with new monomer or unusual composition will also lead to the biotechnological production of tailor-made biopolymer for various applications.     

Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers

The Gram-negative, facultative chemolithoautotrophic bacterium Ralstonia eutropha has been intensively investigated for almost 50 years. Today it is the best studied 'Knallgas' bacterium and producer of poly(3-hydroxybutyric acid). This polyester provides the basis for renewable resource-based biodegradable plastic materials and has attracted much biotechnological interest. The polymer is accumulated in large amounts in the cell and can be used for various applications ranging from replacement of fossil resource-based bulk plastics to high-value special purpose polymers. To further enhance productivity and to allow tailormade poly(hydroxyalkanoic acids) (PHA) with different monomer compositions by metabolic engineering, the knowledge of metabolic pathways and of the biochemical properties of the enzymes involved is essential. Furthermore, proteins covering the PHA granule surface, which are referred to as phasins, and fusions of these phasins to other proteins are promising candidates for various protein technologies. The recently published genome sequence of strain H16 allows researchers to take a closer look at the genetic potential of this versatile bacterium. R. eutropha is, however, not limited to PHAs and to PHA-related polymers like poly(mercaptoalkanoic acids) as it can also be employed for production of a range of other interesting polymers including polyamides like cyanophycin.     

Poly(organo phosphazene) nanoparticles surface modified with poly(ethylene oxide)

The use of biodegradable derivatives of poly(organo phosphazenes) for the preparation of nanoparticles and their surface modification with the novel poly(ethylene oxide) derivative of poly(organo phosphazene) has been assessed using a range of in vitro characterization methods. The nanoparticles were produced by the precipitation solvent evaporation method from the derivative co-substituted with phenylalanine and glycine ethyl ester side groups. A reduction in particle size to less than 200 nm was achieved by an increase in pH of the preparation medium. The formation (and colloidal stability) of these nanoparticles seems to be controlled by two opposite effects: attractive hydrophobic interactions between phenylalanine ester groups and electrostatic repulsions arising from the carboxyl groups formed due to (partial) hydrolysis of the ester bond(s) at the high pH of the preparation medium. The poly[(glycine ethyl ester)phosphazene] derivative containing 5000-Da poly(ethylene oxide) as 5% of the side groups was used for the surface modification of nanoparticles. Adsorbed onto the particles, the polymer produced a thick coating layer of approximately 35 nm. The coated nanoparticles exhibited reduced surface negative potential and improved colloidal stability toward electrolyte-induced flocculation, relative to the uncoated system. However, the steric stabilization provided was less effective than that of a Poloxamine 908 coating. This difference in effectiveness of the steric stabilization might indicate that, although both the stabilizing polymers possess a 5000-Da poly(ethylene oxide) moiety, there is a difference in the arrangements of these poly(ethylene oxide) chains at the particle surface. (c) 1996 John Wiley & Sons, Inc.     

Effect of side group chemistry on the properties of biodegradable L-alanine cosubstituted polyphosphazenes

Biodegradable polyphosphazenes have been investigated for a variety of applications, such as controlled drug delivery matrixes, tissue-engineering scaffolds, membranes, and bone-type composites. In this study we have evaluated the effect of side group chemistry on the properties of biodegradable phosphazene polymers that contain ethyl alanato side groups together with ethyl glycinato, p-methylphenoxy, or p-phenylphenoxy side groups. The polymers were synthesized by a macromolecular substitution route. The molecular weights of aryloxy/amino acid ester cosubstituted polymers were much higher than the amino acid ester substituted polyphosphazenes described earlier. Polymer properties, such as glass transition temperature, hydrolytic degradation, surface wettability, tensile strength, and modulus of elasticity varied over a wide range following changes to the type of co-substituents on the polymer backbone. The glass transition temperatures varied from -10 to 35 degrees C and increased with the bulkiness of the side groups. Polymer films in phosphate buffer saline solution showed molecular weight declines ranging from 58% to >80% and mass loss ranging from 4% to 90% over a period of 7 weeks. Water contact angles for polymer films varied from 63 degrees to 107 degrees , with the highest angles for the alanine ethyl ester and p-phenylphenoxy cosubstituted polyphosphazene. The tensile strengths were in the range of 2.4-7.6 MPa and the modulus of elasticity was in the range of 31.4-455.9 MPa. Thus, in this study we have demonstrated the tunability of biodegradable polyphosphazenes to suit a range of biomedical applications.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate- co-3-hydroxy-heptanoate) terpolymers by recombinant Alcaligenes eutrophus

Recombinant strains of Alcaligenes eutrophusharboring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae synthesized poly(3-hydroxybutyrate- co-3-hydroxyvalerate- co-3-hydroxyheptanoate) terpolymers from alkanoic acids of odd carbon numbers. The results indicated the specificity of PHA synthase of A. caviae toward 3-hydrox-yalkanoate units from C to C . The composition of the polyesters formed varied as the carbon numbers of the alkanoic acids fed increased.     

The turnover of medium-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by a wide range of bacteria, including Pseudomonads. These polymers are accumulated in the cytoplasm as carbon and energy storage materials when culture conditions are unbalanced and hence, they have been classically considered to act as sinks for carbon and reducing equivalents when nutrients are limited. Bacteria facing carbon excess and nutrient limitation store the extra carbon as PHAs through the PHA polymerase (PhaC). Thereafter, under starvation conditions, PHA depolymerase (PhaZ) degrades PHA and releases R -hydroxyalkanoic acids, which can be used as carbon and energy sources. To study the influence of a deficient PHA metabolism in the growth of Pseudomonas putida KT2442 we have constructed two mutant strains defective in PHA polymerase ( phaC1 )- and PHA depolymerase ( phaZ )-coding genes respectively. By using these mutants we have demonstrated that PHAs play a fundamental role in balancing the stored carbon/biomass/number of cells as function of carbon availability, suggesting that PHA metabolism allows P. putida to adapt the carbon flux of hydroxyacyl-CoAs to cellular demand. Furthermore, we have established that the coordination of PHA synthesis and mobilization pathways configures a functional PHA turnover cycle in P. putida KT2442. Finally, a new strain able to secrete enantiomerically pure R -hydroxyalkanoic acids to the culture medium during cell growth has been engineering by redirecting the PHA cycle to biopolymer hydrolysis.     

Perspectives to produce positively or negatively charged polyhydroxyalkanoic acids

An overview is provided on the possibilities of producing positively and negatively charged poly(β-hydroxyalkanoates), PHAs. A large variety of bacterial polyesters with functionalized terminal side chains can be produced in microbial fermentation processes by a direct polymerization of respective carbon sources, that is, carbon sources that carry functional groups in their ω-position. However, charged PHAs are not accessible by a direct approach and must be synthesized via polymer-analogous reactions of functionalized bacterial polyesters. PHA polyanions are produced by converting the terminal functional groups into carboxylate groups, while PHA polycations are produced by introducing terminal amino groups. PHAs with terminal vinyl groups emerged as most suitable PHA precursors, as they can be produced in relatively high yields and the double bonds are sufficiently reactive. The oxidation of vinyl groups yields PHA polyanions. The conversion of terminal vinyl groups into epoxides with a subsequent ring-opening reaction with an amine yields PHA polycations. Other functionalized PHA that potentially lend themselves to polymer-analogous reactions are reviewed.     

Expression of 3-ketoacyl-acyl carrier protein reductase (fabG) genes enhances production of polyhydroxyalkanoate copolymer from glucose in recombinant Escherichia coli JM109

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

Biosynthesis of Poly[(R)-3-hydroxyalkanoate] Copolymers with Controlled Repeating Unit Compositions and Physical Properties

As applications for biodegradable and biologically produced poly[(R)-3-hydroxyalkanoates] (PHAs) grow into more specialized areas, the need to precisely control the repeating unit composition and consequently the physical properties of these polymers has become essential. A previous study reported our development of Escherichia coli LSBJ in order to produce PHA polymers composed of single repeating units ranging from 4 to 12 carbon atoms. This investigation expands the scope of our effort toward controlling the repeating unit composition of a variety of PHA copolymers. The sizes for the repeating units within the copolymers were modulated by feeding specific ratios of fatty acids with defined carbon lengths to E. coli LSBJ, which resulted in defined mole ratios for the repeating units. Various physical properties of the copolymers (including the Young's modulus, elongation to break, and glass-transition temperature) were shown to be strongly dependent upon the mole ratios of repeating units. This work demonstrates that copolymers of PHAs with repeating units from 4 to 12 carbons can be incorporated accurately to obtain any desired mole ratio within the PHA copolymers. Our methodology may thus be extended to generate tailor-made PHA copolymers with prescribed values for key sets of physical properties.     

Biosynthesis and native granule characteristics of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Delftia acidovorans

The ability of Delftia acidovorans to incorporate a broad range of 3-hydroxyvalerate (3HV) monomers into polyhydroxyalkanoate (PHA) copolymers was evaluated in this study. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] containing 0–90 mol% of 3HV was obtained when a mixture of sodium 3-hydroxybutyrate and sodium valerate was used as the carbon sources. Transmission electron microscopy analysis revealed an interesting aspect of the P(3HB-co-3HV) granules containing high molar ratios of 3HV whereby, the copolymer granules were generally larger than those of poly(3-hydroxybutyrate) [P(3HB)] granules, despite having almost the same cellular PHA contents. The large number of P(3HB-co-3HV) granules occupying almost the entire cell volume did not correspond to a higher amount of polymer by weight. This indicated that the granules of P(3HB-co-3HV) contain polymer chains that are loosely packed and therefore have lower density than P(3HB) granules. It was also interesting to note that a decrease in the length of the side chain from 3HV to 4-hydroxybutyrate (4HB) corresponded to an increase in the density of the respective PHA granules. The presence of longer side chain monomers (3HV) in the PHA structure seem to exhibit steric effects that prevent the polymer chains in the granules from being closely packed. The results reported here have important implications on the maximum ability of bacterial cells to accumulate PHA containing monomers with longer side chain length.     

Preparation of a class of versatile, chemoselective, and amorphous polyketoesters

A straightforward and versatile strategy for preparing a class of biodegradable and amorphous polyketoesters is reported. A series of ketone-containing diesters and diacids were combined with di(ethylene glycol) through condensation polymerization, achieving values of up to 10.1 x 10(3) g/mol. Glass transition temperatures ranged from -41 to -6 degrees C, rendering all of the materials liquid at room temperature. By including ketone groups in the repeat unit, facile postpolymerization modifications were possible by reaction with oxyamine-tethered ligands through the formation of an oxime linkage. Upon reaction with molecules that contain oxyamines, under mild conditions, these polymers can easily have a diverse set of side chains appended without coreagents or catalysts. The chemoselective oxime-forming coupling strategy is compatible with physiological conditions and can be done in the presence of a wide range of functional groups and biomolecules, including proteins and nucleic acids. We demonstrate the utility of this strategy by immobilizing a cell adhesive peptide (H2NO-RGD) to polyketoester films, creating cell adhesive elastomers. This immobilization strategy is synthetically flexible for designing and tailoring polymers for targeted biological applications.     

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.     

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA): its conformational and photodynamic properties

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA) polymers with 22 and 35 mol % of azo chromophores in the side chains have been synthesized by condensing 4-methoxy-4'-aminoazobenzene and poly(l-glutamic acid). These polymers have been characterized by NMR, FT-IR, and UV-visible spectroscopic techniques. The conformational features of the polymer backbone chains in the films that were cast from the polymer solutions prepared in different solvents have been investigated by circular dichroism spectroscopy. Experimental data suggested that the thermal cis-trans relaxation and photoinduced birefringence, which are related to the azo chromophores in the side chains of polymer, are not affected by the conformations of polymer backbones. However, the modulations of the surface relief gratings, the result of photoinduced mass transport process, recorded on these polymers are sensitive to polymer main chain conformation, as well as the degree of functionalization.     

Biosynthetic polyesters consisting of 2-hydroxyalkanoic acids: current challenges and unresolved questions

2-Hydroxyalkanoates (2HAs) have become the new monomeric constituents of bacterial polyhydroxyalkanoates (PHAs). PHAs containing 2HA monomers, lactate (LA), glycolate (GL), and 2-hydroxybutyrate (2HB) can be synthesized by engineered microbes in which the broad substrate specificities of PHA synthase and propionyl-CoA transferase are critical factors for the incorporation of the monomers into the polymer chain. LA-based polymers, such as P[LA-co-3-hydroxybutyrate (3HB)], have the properties of pliability and stretchiness which are distinctly different from those of the rigid poly(lactic acid) (PLA) and P(3HB) homopolymers. This versatile platform is also applicable to the biosynthesis of GL- and 2HB-based polymers. In the case of the synthesis of 2HB-based polymers, the enantiospecificity of PHA synthase enabled the production of isotactic (R)-2HB-based polymers, including P[(R)-2HB], from racemic precursors of 2HB. P(2HB) is a pliable material, in contrast to PLA. Furthermore, to obtain a new 2HA-polymerizing PHA synthase, the class I PHA synthase from Ralstonia eutropha was engineered so as to achieve the first incorporation of LA units. The analysis of the polymer synthesized using this new LA-polymerizing PHA synthase unexpectedly focused a spotlight on the studies on block copolymer biosynthesis.