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In order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Young's modulus values of the P(LA-co-3HB)s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB)s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] was developed in Escherichia coli and Corynebacterium glutamicum by an acetoacetyl-coenzyme A (CoA) synthase (AACS) recently isolated from terpenoid-producing Streptomyces sp. strain CL190. Expression of AACS led to significant productions of P(3HB) in E. coli (10.5 wt %) and C. glutamicum (19.7 wt %).

Lactate (LA)-polymerizing enzyme (LPE) is a newly established class of polyhydroxyalkanoate (PHA) synthase, which can incorporate LA units into a polymer chain. We previously synthesized P(LA-co-3-hydroxybutyrate)s [P(LA-co-3HB)s] in recombinant Escherichia coli using the first LPE, which is the Ser325Thr/Glu481Lys mutant of PHA synthase from Pseudomonas sp. 61-3 [PhaC1(Ps)ST/QK]. In this study, we finely regulated LA fraction in the copolymer by saturated mutations at position 392 (F392X), which corresponds to the activity-enhancing mutations at position 420 of PHA synthase from Ralstonia eutropha. Among the 19 saturated mutants of LPE at position 392, 17 mutants produced P(LA-co-3HB)s with various LA fractions (16-45 mol %), whereas PhaC1(Ps)ST/QK produced P(LA-co-3HB) with 26 mol % LA under the same culture condition. In particular, the F392S mutation exhibited the highest LA fraction of 45 mol %, and also increased polymer content (62 wt %) compared with PhaC1(Ps)ST/QK (44 wt %). Combination of the F392S mutant and anaerobic culture conditions, which promote LA production, led to a further increase in LA fraction up to 62 mol %. The P(LA-co-3HB)s with various LA fractions exhibited altered melting temperatures and melting enthalpy depending on their monomer composition. Accordingly, the mutations at position 392 in LPE greatly contributed to fine-tuning of the LA fraction in the copolymers that is useful for regulating LA fraction-dependent thermal properties.

Novel lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate(3HB)-co-3-hydroxyvalerate(3HV)]s (PLBVs), were produced in LA-overproducing mutant, Escherichia coli JW0885, which was found to be a superior host for the efficient production of LA-based polyesters. Recombinant E. coli JW0885 harboring the genes encoding LA-polymerizing enzyme (Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3) and three monomer supplying enzymes [propionyl-CoA transferase, beta-ketothiolase, and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-dependent acetoacetyl-CoA reductase] was aerobically grown on glucose with feeding of propionate as a precursor of 3-hydroxyvaleryl-CoA (3HV-CoA). Gas chromatography and nuclear magnetic resonance (NMR) analyses revealed that polymers accumulated in the cells were composed of LA, 3HB, and 3HV units, thus being identified as terpolymers, PLBVs. In addition, (1)H-NMR analysis suggested the existence of LA-3HV sequence in the terpolymer. When 100 mg/l of sodium propionate was added into the medium, 3HV fraction in the terpolymer linearly reached up to 7.2 mol%, while LA fraction was inversely decreased. This phenomenon could be due to the change in metabolic fluxes of lactyl-CoA (LA-CoA) and 3HV-CoA depending on the concentration of propionate fed into the medium.

Recombinant strains of Ralstonia eutropha and Pseudomonas putida harboring a chimeric polyhydroxyalkanoate (PHA) synthase, which consisted of PHA synthases of Aeromonas caviae and R. eutropha, produced 3-hydroxybutyrate (3HB)-based PHA copolymers comprised of 3-hydroxyhexanoate and 3-hydroxyoctanoate units from dodecanoate (87-97 mol % 3HB), indicating that the chimeric PHA synthase possesses desirable substrate specificity leading to the production of 3HB-rich copolymers.

Considerable enrichment of the lactate (LA) fraction in Poly[(R)-LA-co-(R)-3-hydroxybutyrate (3HB)] has been achieved, reaching up to 47 mol % from the previous 6 mol % [S. Taguchi et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (45), 17323-17327], when recombinant Escherichia coli W3110, harboring the LA-polymerizing enzyme gene together with three genes for supplying monomers, lactyl-coenzyme A (CoA), and 3-hydroxybutyryl-CoA, were grown on glucose under anaerobic conditions. The molecular weights of the copolymer were M(n) = 1.5 x 10(4) g/mol and M(w) = 2.0 x 10(4) g/mol, respectively. Notably, alkaline-hydrolyzed product analysis revealed that only the (R)-enantiomer of LA was incorporated in the copolymer. Furthermore, the triad sequence of LA-LA-LA was clearly detected in the polymer chain based on NMR analysis. A remarkably contrasting melting temperature was observed between the copolymer obtained here P[(R)-LA-co-(R)-3HB] (157 degrees C) and the chemically synthesized P[(S)-LA-co-(R)-3HB] (105 degrees C) with similar monomer composition.

PhaP is a major poly[(R)-3-hydroxybutyrate] [P(3HB)]-granule-associated protein. Its gene expression is controlled by an autoregulated repressor, PhaR, in Paracoccus denitrificans. The packing force of the P(3HB) granule by PhaP is greatly influenced by the number of PhaP molecules. In this study, the effects of DNA-binding-ability-reduced mutations of PhaR on morphological change in the cellular granule formation of P(3HB) were examined under a transmission electron microscope using an Escherichia coli recombinant system. Microscopic observation indicated that stronger packing of P(3HB) granules took place when the number of PhaP molecules was increased by reduction in the DNA-binding ability of PhaR.

PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).

The gene expression for phasins (PhaP), which are predominantly polyhydroxyalkanoates (PHAs) granule-associated proteins, is regulated by a repressor protein of PhaR through the dual binding abilities of PhaR to the target DNAs and the granules. In this study, the binding functions of PhaR to poly[(R)-3-hydroxybutyrate] (P(3HB)) were investigated quantitatively by using a quartz crystal microbalance (QCM) technique. Adsorption of PhaR onto a melt-crystallized film of P(3HB) (cr-P(3HB)) was detected as a negative frequency shift of the QCM. The time course of the frequency changes observed for PhaR adsorption was composed of a quick frequency decrease at an initial stage and a subsequent slower frequency decrease for several hours, indicating multilayered adsorption of PhaR molecules onto cr-P(3HB). The initial rapid adsorption, which corresponds to direct adsorption of PhaR molecules onto a bare surface of cr-P(3HB), was a diffusion-controlled process. Strong interactions between PhaR and cr-P(3HB) were also observed as apparently irreversible adsorption. The comparative QCM measurement of PhaR adsorption onto various types of polymers with different aliphatic chemical structures revealed that PhaR was adsorbed onto the surfaces of polymers, including cr-P(3HB), mainly by nonspecific hydrophobic interactions. These results illustrate the high affinity and low specificity for adsorption of PhaR to P(3HB).

Abstract: New lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate (3HB)-co-3-hydroxyhexananoate (3HHx)]s, were produced in recombinant Escherichia coli LS5218 harboring three genes encoding LA-polymerizing enzyme (LPE), propionyl-coenzyme A (CoA) transferase (PCT) and (R)-specific enoyl-CoA hydratase (PhaJ4). When the recombinant LS5218 was grown on glucose with the feeding of butyrate, 3HB-CoA and 3HHx-CoA were supplied, probably via reverse reactions of the β-oxidation pathway and PhaJ4. LPE copolymerized the two monomers 3HB-CoA and 3HHx-CoA with LA-CoA, which was generated by PCT, to yield the terpolymers. Gas chromatography analysis revealed that the terpolymers consisted of 2.7–34 mol% LA, 38–81 mol% 3HB and 17–33 mol% 3HHx units, which can be varied depending on the butyrate concentration fed in the medium. In addition, 1H-13C COSY NMR analysis provided evidence for a linkage between LA and 3HHx units in the polymer. [Copyright &y& Elsevier]