The polyhydroxyalkanoate (PHA) synthase 1 of Pseudomonas sp. H9 synthesized a 3-hydroxybutyrate-dominant hybrid of short- and medium-chain-length PHA

Abstract

Pseudomonas sp. H9 (H9), an environmental strain isolated from a riverbank soil sample collected in southern Taiwan, is Gram-negative and shares a 99 % sequence identity to Pseudomonas putida KT2440 based on 16S rRNA gene analysis. H9 produced novel polyhydroxyalkanoates (PHA) including a hybrid PHA, comprised of 3-hydroxyvalerate (37 mol%) and medium-chain-length (MCL) monomers from valerate, as well as a 3-hydroxyhexanoate-dominant (93 mol%) MCL-PHA from hexanoate. Next-generation sequencing analysis showed H9 had a typical class II PHA operon, consisted of phaC1H9-phaZH9-phaC2H9, in which phaC1H9 was the sole active PHA synthase in H9. Deletion of phaC1H9 gene led to a complete loss of its PHA accumulation capability. Knockout of phaC2H9 gene, in contrast, affected neither bacterial growth nor PHA accumulation. When co-expressed with the phaAB genes of Ralstonia eutropha H16 in the PHA mutant strain Pseudomonas sp. H9ΔC1, phaC1H9 synthesized a hybrid PHA consisted of 3-hydroxybutyrate (3HB) (75 mol%) and MCL-monomers, confirmed in analyses using hot-acetone fractionation and 13C-NMR spectroscopy. As a novel PHA synthase, PhaC1H9 possesses a broad substrate specificity to synthesize a hybrid of SCL- and MCL-PHA, known to have many mechanical properties for potential applications.