Comparative Analysis of phaC1 and phaC2 Genes Encoding Polyhydroxyalkanoate Synthase in Cupriavidus malaysiensis USMAA1020T

Two polyhydroxyalkanoate synthase genes, phaC1 and phaC2, were identified in three strains of Cupriavidus malaysiensis (C. malaysiensis): C. malaysiensis USMAA1020T, C. malaysiensis USMAHM13, and C. malaysiensis USMAA2-4. Interestingly, the genome of C. malaysiensis USMAA1020T 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 USMAA1020T and individually expressed in Cupriavidus necator (C. necator) PHB-4, which serves as a benchmark of functionality for other strains. Using gamma-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.