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

ABSTRACT 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.