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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.

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are key enzymes in PHA polymerization. PhaCs with broad substrate specificity are attractive for synthesizing structurally diverse PHAs. In the PHA family, 3-hydroxybutyrate (3HB)-based copolymers are industrially produced using Class I PhaCs and can be used as practical biodegradable thermoplastics. However, Class I PhaCs with broad substrate specificities are scarce, prompting our search for novel PhaCs. In this study, four new PhaCs from the bacteria Ferrimonas marina, Plesiomonas shigelloides, Shewanella pealeana, and Vibrio metschnikovii were selected via a homology search against the GenBank database, using the amino acid sequence of Aeromonas caviae PHA synthase (PhaCAc), a Class I enzyme with a wide range of substrate specificities, as a template. The four PhaCs were characterized in terms of their polymerization ability and substrate specificity, using Escherichia coli as a host for PHA production. All the new PhaCs were able to synthesize P(3HB) in E. coli with a high molecular weight, surpassing PhaCAc. The substrate specificity of PhaCs was evaluated by synthesizing 3HB-based copolymers with 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate, 3-hydroxy-2-methylbutyrate, and 3-hydroxypivalate monomers. Interestingly, PhaC from P. shigelloides (PhaCPs) exhibited relatively broad substrate specificity. PhaCPs was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.

The use of stable isotope-labeled polymers in in situ biodegradation tests provides detailed information on the degradation process. As isotope-labeled raw chemicals are generally expensive, it is desirable to prepare polymer samples with high production yields and high isotope-labeling ratios. The biodegradable plastic poly[(R)-3-hydroxybutyrate)] (P(3HB)) is produced by microorganisms. In this study, to produce carbon 13 (13C)-labeled P(3HB) from [U-13C6]D-glucose (13C-glucose), the culture conditions needed for high production yields and high 13C-labeling ratios were investigated using Ralstonia eutropha NCIMB 11599 and recombinant Escherichia coli JM109. We found that over 10 g/L of P(3HB) could be obtained when these microorganisms were cultured in Luria-Bertani (LB3) medium containing 3 g/L NaCl and 40 g/L 13C-glucose, while 1.4–4.7 g/L of P(3HB) was obtained when a mineral salt (MS) medium containing 20 g/L 13C-glucose was used. The 13C-labeling ratio of P(3HB) was determined by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry (GC-MS), and both analytical methods yielded nearly identical results. High 13C-labeling ratios (97.6 atom% by GC-MS) were observed in the MS medium, whereas low 13C-labeling ratios (88.8–94.4 atom% by GC-MS) were observed in the LB3 medium. Isotope effects were observed for the P(3HB) content in cells cultured in the LB3 medium and the polydispersity of P(3HB).