Biosynthesis and characterization of novel poly(3-hydroxybutyrate-co-3-hydroxy-2-methylbutyrate): thermal behavior associated with α-carbon methylation

Abstract

3-Hydroxy-2-methylbutyrate (3H2MB) has been identified as a minor component of polyhydroxyalkanoates (PHAs) synthesized by bacteria living in activated sludge. In this study, we found that PHA synthase derived from Aeromonas caviae (PhaC Ac ) polymerizes 3H2MB. By expressing PhaC Ac in recombinant Escherichia coli LS5218 and growing cells in the presence of tiglic acid, a PHA copolymer [P(3HB-co-3H2MB)], mainly consisting of 3-hydroxybutyrate (3HB) and up to 37 mol% 3H2MB, was obtained. Analysis of the thermal properties of this novel copolymer indicates that incorporation of 3H2MB into P(3HB) sequence reduced the glass transition temperature (T g ), melting temperature (T m ), and melting enthalpy (DH m ). The cold crystallization temperature (T cc ) was also lowered by incorporating 7 or 23 mol% 3H2MB, in contrast to the findings for other PHA copolymers. This result suggests that P(3HB-co-3H2MB) copolymers are easier to crystallize than P(3HB) and other PHAs. Thus, 3H2MB provides promising new opportunities to generate 3HB-based polymers with novel thermal properties.

Plasticized poly(3‐hydroxybutyrate) with improved melt processing and balanced properties

“…The 1 H‐NMR spectra of the two samples showed several differences. Both types of PHBs showed three large signals at 2.47–2.61, 5.26, and 1.28 ppm; these signals were assigned to the three types of protons: (1) methylene, (2) methine, and (3) methyl . However, the PHB granules from Goodfellow also showed additional resonances corresponding to the characteristic protons coming from the plasticizer.…”

Engineering in Vivo Production of α-Branched Polyesters

“…The PHA expression system was assembled with synthetic genes encoding the thiolase (AsAcat3 or CapPhaA) and ketoreductase (CapPhaB0-3, RePhaB, and AsHadH2) as a single operon on the pTrc99a backbone (Figure A and Table S1). The three different PHA polymerases (CapPhaC1, CapPhaEC, and AcPhaC* which is a mutant PHA polymerase from Aeromonas caviae used to produce branched PHA from tiglic acid) were then placed under the control of the T7 lac promoter on the pTrc33a backbone. Plasmids for 18 different ketoreductase–PHA polymerase were constructed and transformed into Escherichia coli BAP1, a modified BL21­(DE3) strain engineered to increase intracellular concentration of propionyl-CoA .…”

“…Initial studies have shown that a variety of longer chain lengths as well as monomers that contain distal branch sites can be incorporated with wild-type PHA polymerases. , Additional work has shown that non-natural poly­(2-, 4-, or 5-hydroxyalkanoates) can also be biosynthesized with mutant and some nonengineered polymerases. − Of note, the biosynthesis of aromatic polyesters, similar to poly­(ethylene terephthalate) (PET), was recently achieved . These studies, as well as others, have shown that structural characteristics such as chain length and branching can be key to improving relevant mechanical properties such as toughness, flexibility, and other properties related to manufacturing, processing, and applications. ,, In particular, PHA materials extracted from activated sludge containing an α-branch site have been shown to display lower degrees of crystallinity and broader melting temperature ranges than the most common bacterial PHA, poly­(hydroxybutyrate) (PHB) . Furthermore, Tisma et al have demonstrated that substitution of α-hydrogen atoms by alkyl groups in the polyester, poly­(α-dimethylpropiolactone), reduced the rate of thermal degradation when compared to the unsubstituted poly­(β-propiolactone) .…”

Chemical and Biochemical Engineering Approaches in Manufacturing Polyhydroxyalkanoate (PHA) Biopolyesters of Tailored Structure with Focus on the Diversity of Building Blocks

“…Especially the decreased T c indicated that poly(3HBco-3H2MB) showed higher affinity towards crystallization than previously described scl-PHA copolyesters. 130 Further pathway engineering of this strain carried out by Furutate and colleagues, by weakening its β-oxidation metabolism and inserting a propionyl-CoA transferase gene, enabled the production of another new class of PHA copolyesters: cultivation in tiglic acid and hexanoic acid resulted in accumulation of poly(3H2MB-co-3HHx); these copolyesters, consisting of both branched scl-PHA and linear mcl-PHA building blocks, revealed low T g values and totally lacking T m , hence, these products show no crystallinity in contrast to poly(3HBco-3H2MB). 131 In addition, the production of PHA harboring 3HB, 3HV, 3H2MV and 3-hydroxy-2methylbutyrate (3H2MB) was reported by Dai and colleagues, who cultivated a mixed culture rich in glycogen-accumulating organisms with high phylogenetic similarity with Defluviicoccus vanus under anaerobic conditions in not nutrient-limited media; propionic acid and acetate were used as carbon sources, with higher propionic acid fractions favoring incorporation of the branched monomers.…”