Molecular structure of PCR cloned PHA synthase genes of Pseudomonas putida KT2440 and its utilization for medium-chain length polyhydroxyalkanoate production

A new phaC gene cluster encoding polyhydroxyalkanoate (PHA) synthase I PHA depolymerase, and PHA synthase 11 was cloned using the touchdown PCR method, from medium-chain length (mcl-) PHA-producing strain Pseudomonas putida KT2440. The molecular structure of the cloned phaC1 gene was analyzed, and the phylogenic relationship was compared with other phaC1 genes cloned from Pseudomonas species. The cloned phaC1 gene was expressed in a recombinant E. coli to the similar level of PHA synthase in the parent strain P putida KT2440, but no significant amount of mcl-PHA was accumulated. The isolated phaC1 gene was re-introduced into the parent strain P putida KT2440 to amplify the PHA synthase I activity, and the recombinant P putida accumulated mcl-PHA more effectively, increasing from 26.6 to 43.5%. The monomer compositions of 3-hydroxylalkanoates in mcl-PHA were also modified significantly in the recombinant P putida enforcing the cloned phaC1 gene.