Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.polymdegradstab.2024.110975

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Language：English   Publishing type：Research paper (scientific journal)  

Ralstonia eutropha strain H16 is a chemoautotrophic bacterium that oxidizes hydrogen and accumulates poly[(R)-3-hydroxybutyrate] [P(3HB)], a prominent polyhydroxyalkanoate (PHA), within its cell. R. eutropha utilizes fructose or CO2 as its sole carbon source for this process. A PHA-negative mutant of strain H16, known as R. eutropha strain PHB-4, cannot produce PHA. Strain 1F2, derived from strain PHB-4, is a leucine analog-resistant mutant. Remarkably, the recombinant 1F2 strain exhibits the capacity to synthesize 3HB-based PHA copolymers containing 3-hydroxyvalerate (3HV) and 3-hydroxy-4-methyvalerate (3H4MV) comonomer units from fructose or CO2. This ability is conferred by the expression of a broad substrate-specific PHA synthase and tolerance to feedback inhibition of branched amino acids. However, the total amount of comonomer units incorporated into PHA was up to around 5 mol%. In this study, strain 1F2 underwent genetic engineering to augment the comonomer supply incorporated into PHA. This enhancement involved several modifications, including the additional expression of the broad substrate-specific 3-ketothiolase gene (bktB), the heterologous expression of the 2-ketoacid decarboxylase gene (kivd), and the phenylacetaldehyde dehydrogenase gene (padA). Furthermore, the genome of strain 1F2 was altered through the deletion of the 3-hydroxyacyl-CoA dehydrogenase gene (hbdH). The introduction of bktB-kivd-padA resulted in increased 3HV incorporation, reaching 13.9 mol% from fructose and 6.4 mol% from CO2. Additionally, the hbdH deletion resulted in the production of PHA copolymers containing (S)-3-hydroxy-2-methylpropionate (3H2MP). Interestingly, hbdH deletion increased the weight-average molecular weight of the PHA to over 3.0 × 106 on fructose. Thus, it demonstrates the positive effects of hbdH deletion on the copolymer composition and molecular weight of PHA.

DOI： 10.3390/bioengineering11050455

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

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)-3-hydroxy-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.

DOI： 10.1021/acs.biomac.3c01069

PubMed

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