Polyhydroxyalkanoate Copolymer Production by Recombinant Ralstonia eutropha Strain 1F2 from Fructose or Carbon Dioxide as Sole Carbon Source.

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 CO 2 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 CO 2. 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 CO 2 . 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 × 10 6 on fructose. Thus, it demonstrates the positive effects of hbdH deletion on the copolymer composition and molecular weight of PHA.