Comparative Analysis of <i>phaC1</i> and <i>phaC2</i> Genes Encoding Polyhydroxyalkanoate Synthase in <i>Cupriavidus malaysiensis</i> USMAA1020^T

Two polyhydroxyalkanoate synthase genes, phaC1 and phaC2, were identified in three strains of Cupriavidus malaysiensis (C. malaysiensis): C. malaysiensis USMAA1020^T, C. malaysiensis USMAHM13, and C. malaysiensis USMAA2-4. Interestingly, the genome of C. malaysiensis USMAA1020^T revealed the presence of the polyhydroxyalkanoate granule-associated protein (phaF), which was not present in C. malaysiensis USMAHM13 and C. malaysiensis USMAA2-4. A Maximum Likelihood phylogenetic analysis shows that the phaC genes were classified into Class I synthases. The phaC1 and phaC2 genes in the three C. malaysiensis strains formed a separate, distinct cluster. To further examine the function of phaC, both phaC genes were cloned from C. malaysiensis USMAA1020^T and individually expressed in Cupriavidus necator (C. necator) PHB-4, which serves as a benchmark of functionality for other strains. Using γ-butyrolactone as the sole carbon source, the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) contains up to 83.00 mol% 4-hydroxybutyrate (4HB) and 26.50% PHA content. However, the transformant C. necator PHB-4 with phaC2 produced only 2.30% PHA content and no 4HB monomer. The phaC2 transformant produces up to 100 mol% 3HB monomer and 41.90% PHA content, while the phaC1 transformant produces only 25.80% PHA content when using oleic acid as the sole carbon source. When provided with a mixed substrate of oleic acid and 1-pentanol, the transconjugants accumulated up to 20% PHA content but produced a low 3HV content of only 4%-5%. These findings significantly contribute to the scientific literature by improving the understanding of the genetic and biochemical diversity of the two PHA synthases, phaC1 and phaC2, in Cupriavidus species.