Biosynthesis, characterization, and biodegradation of elastomeric polyhydroxyalkanoates consisting of α-dimethylated monomer units

Mierzati, M.; Sakurai, T.; Ishii-Hyakutake, M.; Miyahara, Y.; Nomura, C.T.; Taguchi, S.; Abe, H.; Tsuge, T.

doi:10.1016/j.mtsust.2023.100577

Cited by 4 publications

(3 citation statements)

References 62 publications

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“…Host strains used for PHA biosynthesis, plasmids, and cultivation strategies were similar to those of previous studies. ,− A β-oxidation deficient mutant Escherichia coli LS5218 [ fadR601 , atoC ( Con )] strain with a fadB fadJ double-deletion, named E. coli LSBJ, was used as the host strain to efficiently accumulate PHA. , Plasmids pBBR1P(D4N)CJ Ac AB Re NSDG and pTTQ19-PCT were used to produce the 3HB-rich P(3HB- co -3H2MP) copolymer. pBBR1P(D4N)CJ Ac AB Re NSDG contains a PHA granule-associated protein gene ( phaP Ac ) with D4N point mutation from Aeromonas caviae , which ensures high PHA yield after expression of the phaPCJ operon, a PHA synthase gene ( phaC Ac NSDG ) with N149S and D171G point mutations that further enhances the incorporation of the comonomer unit into the 3HB-based copolymer, along with the ( R )-specific enoyl-CoA hydratase gene ( phaJ Ac ) and genes involved in the 3HB-CoA biosynthesis pathway, 3-ketothiolase ( phaA Re ) and acetoacetyl-CoA reductase ( phaB Re ) from Ralstonia eutropha ( Cupriavidus necator ) H16.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…pBBR1P(D4N)CJ Ac AB Re NSDG contains a PHA granule-associated protein gene ( phaP Ac ) with D4N point mutation from Aeromonas caviae , which ensures high PHA yield after expression of the phaPCJ operon, a PHA synthase gene ( phaC Ac NSDG ) with N149S and D171G point mutations that further enhances the incorporation of the comonomer unit into the 3HB-based copolymer, along with the ( R )-specific enoyl-CoA hydratase gene ( phaJ Ac ) and genes involved in the 3HB-CoA biosynthesis pathway, 3-ketothiolase ( phaA Re ) and acetoacetyl-CoA reductase ( phaB Re ) from Ralstonia eutropha ( Cupriavidus necator ) H16. pTTQ19-PCT contains a propionyl-CoA transferase gene ( pct ) from Megasphaera elsdenii for supplying 3H2MP-CoA as a substrate for PhaC. , Plasmids pBBR1P(D4N)C Ac NSDG and pTTQ19-PCT were used to produce the 3H2MP-rich P(3HB- co -3H2MP) copolymer and the P(3H2MP) homopolymer.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…pTTQ19-PCT contains a propionyl-CoA transferase gene (pct) from Megasphaera elsdenii for supplying 3H2MP-CoA as a substrate for PhaC. 26,31 Plasmids pBBR1P(D4N)C Ac NSDG and pTTQ19-PCT were used to produce the 3H2MP-rich P(3HB-co-3H2MP) copolymer and the P(3H2MP) homopolymer.…”

Section: Bacterial Strain and Plasmidsmentioning

confidence: 99%

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Tacticity Characterization of Biosynthesized Polyhydroxyalkanoates Containing (S)- and (R)-3-Hydroxy-2-Methylpropionate Units

Mierzati,

Miyahara,

Curial

et al. 2023

Biomacromolecules

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Polyhydroxyalkanoates (PHAs), aliphatic polyesters synthesized by microorganisms, have gained considerable attention as biodegradable plastics. Recently, α-carbon-methylated PHAs have been shown to exhibit several interesting properties that differ from those of conventional PHAs, such as their crystallization behavior and material properties. This study investigated α-carbon methylated (S)-and (R)-3hydroxy-2-methylpropionate (3H2MP) as new repeating units. 3H2MP units were homopolymerized or copolymerized with (R)-3-hydroxybutyrate (3HB) by manipulating the culture conditions of recombinant Escherichia coli LSBJ. Consequently, PHAs with 3H2MP units ranging from 5 to 100 mol % were synthesized by external addition of (R)-and (S)-enantiomers or the racemic form of 3H2MPNa. The (S)-3H2MP precursor supplemented into the culture medium was almost directly polymerized into PHA while maintaining its chirality. Therefore, a highly isotactic P(3H2MP) (R:S = 1:99) was synthesized, which displayed a melting temperature of 114−119 °C and a relatively high enthalpy of fusion (68 J/g). In contrast, in cultures supplemented with (R)-3H2MP, the precursor was racemized and polymerized into PHA, resulting in the synthesis of the amorphous polymer atactic P(3H2MP) (R:S = 40:60). However, racemization was not observed at a low concentration of the (R)-3H2MP precursor, thereby synthesizing P(3HB-co-8 mol % 3H2MP) with 100% (R)-3H2MP units. The thermogravimetric analysis revealed that the thermal degradation temperatures at 5% weight loss of P(3H2MP)s occurred at approximately 313 °C, independent of tacticity, which is substantially higher than that of P(3HB) (257 °C). This study demonstrates a new concept for controlling the physical properties of biosynthesized PHA by manipulating the polymers' tacticity using 3H2MP units.

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Section: Materials and Methodsmentioning

confidence: 99%

Section: Materials and Methodsmentioning

confidence: 99%

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Tacticity Characterization of Biosynthesized Polyhydroxyalkanoates Containing (S)- and (R)-3-Hydroxy-2-Methylpropionate Units

Mierzati,

Miyahara,

Curial

et al. 2023

Biomacromolecules

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Biosynthesis of High Toughness Poly(3‐Hydroxypropionate)‐Based Block Copolymers With Poly(D‐2‐Hydroxybutyrate) and Poly(D‐Lactate) Segments Using Evolved Monomer Sequence‐Regulating Polyester Synthase

Kawakami,

Tomita,

Hien

et al. 2024

Biopolymers

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This study synthesized poly(3‐hydroxypropionate) [P(3HP)]‐containing polyhydroxyalkanoate (PHA) block copolymers, P(3HP)‐b‐P[2‐hydroxybutyrate (2HB)] and P(3HP)‐b‐P(D‐lactate) (PDLA), using Escherichia coli. The cells expressing an evolved sequence‐regulating PHA synthase, PhaCARNDFH, and propionyl‐CoA transferase were cultured with the supplementation of the corresponding monomer precursors in the medium. The block structure of P(3HP)‐b‐PDLA was confirmed by proton nuclear magnetic resonance analysis and solvent fractionation. The molecular weights of the polymers were in the range of 0.8–2.8 × 105. The solvent‐cast polymer films were subjected to isothermal treatment to promote phase separation and crystallization and were subsequently melt‐quenched to produce an amorphous phase. The melt‐quenched P(3HP)‐b‐P(2HB) film exhibited a high elongation at break (1153%), resulting in a toughness of 181 MJ/m3. The solvent‐cast film of P(3HP)‐b‐65 mol% PDLA exhibited partial elastic deformation, in which the P(3HP) phase functioned as a soft segment. The melt‐quenching of the polymer resulted in embrittlement presumably due to the high lactate fraction. Overall, the P(3HP)‐based block copolymers exhibited several mechanical properties depending on the higher‐order structure of the polymer and the properties of the P(2‐hydroxyalkanoate) segments. This study findings show that P(3HP)‐b‐P(2HB) and P(3HP)‐b‐PDLA can function excellently if their microstructures are properly controlled.

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Biosynthesis of polyhydroxyalkanoate copolymers consisting of α-methylated monomer units from glucose and propionate: thermal properties and chiral configuration

Miyahara,

Ishino,

Nomura

et al. 2024

Biotechnol Sustain Mater

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Background Expression of the Ascaris suum ketothiolase (Acat3) in recombinant Escherichia coli enables the production of α-methylated monomers such as 3-hydroxy-2-methylvalerate (3H2MV) and 3-hydroxy-2-methylbutyrate (3H2MB) for polyhydroxyalkanoate (PHA) biosynthesis from glucose and propionate as carbon sources. However, the chiral configurations and thermal properties of biosynthesized PHAs remain poorly understood. Results In this study, 3-hydroxybutyrate (3HB)-based PHA copolymers containing 3H2MV and 3H2MB units were synthesized from glucose and propionate using Acat3-expressing Escherichia coli LSBJ. The 3H2MV fraction of the synthesized PHA reached 15.7 mol%, while the 3H2MB fraction remained at approximately 0.2 mol%. Chirality analysis revealed that (2S,3R)- and (2R,3R)-3H2MV units were both detected; however, (2R,3R)-3H2MV units were dominant in the PHA copolymer produced by the strain expressing the (R)-specific enoyl-CoA hydratase (PhaJAc). To evaluate the effect of α-methylated monomers on the crystallization behavior of PHA copolymers, cold crystallization was compared for PHA polymers with different mol% 3-hydroxyvalerate (3HV) monomer units. The cold crystallization of the copolymer containing 11 mol% 3H2MV and 30 mol% 3HV was detected at 68 °C, while the non-α-methylated copolymer containing 24 mol% 3HV did not exhibit cold crystallization, indicating that α-methylated PHA had a greater tendency to crystallize. Conclusions This study conclusively demonstrated that (2S,3R)- and (2R,3R)-3H2MV units were both incorporated into PHA by expressing Acat3; however, the (2R,3R)-isomer became the dominant 3H2MV unit in the PHA copolymers by additionally expressing PhaJAc. The 3H2MV repeating unit facilitated the crystallization of PHA copolymers despite the high fraction of the 3HV unit. Graphical Abstract

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Biosynthesis, characterization, and biodegradation of elastomeric polyhydroxyalkanoates consisting of α-dimethylated monomer units

Cited by 4 publications

References 62 publications

Tacticity Characterization of Biosynthesized Polyhydroxyalkanoates Containing (S)- and (R)-3-Hydroxy-2-Methylpropionate Units

Tacticity Characterization of Biosynthesized Polyhydroxyalkanoates Containing (S)- and (R)-3-Hydroxy-2-Methylpropionate Units

Biosynthesis of High Toughness Poly(3‐Hydroxypropionate)‐Based Block Copolymers With Poly(D‐2‐Hydroxybutyrate) and Poly(D‐Lactate) Segments Using Evolved Monomer Sequence‐Regulating Polyester Synthase

Biosynthesis of polyhydroxyalkanoate copolymers consisting of α-methylated monomer units from glucose and propionate: thermal properties and chiral configuration

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