Abstract
• Characterization of PhaC from Ideonella sakaiensis 201-F6, a bacterium that is capable of degrading and assimilating PET. • PhaC Is polymerizes five types of monomers: 3HB, 3HV, 4HB, 5HV and 3H4MV. • Provides insights into the substrate specificity of PhaC Is , which is important for the metabolic engineering of I. sakaiensis that produces various types of PHA from PET. Ideonella sakaiensis 201-F6 is a bacterium capable of degrading and assimilating poly(ethylene terephthalate) (PET). A recent study revealed its capability in accumulating intracellular polyhydroxyalkanoate (PHA) at high levels using PET as a carbon source. The finding demonstrated that I. sakaiensis can mediate the direct conversion of non-biodegradable PET into environment-friendly PHA, providing a new approach for PET recycling. The PHA synthase gene of I. sakaiensis ( phaC Is ) was characterized in this study. Phylogenetic analysis of the PhaC protein sequences indicated PhaC Is belong to class I PhaC. To carry out in vivo characterization of PhaC Is , phaC Is was cloned and heterologously expressed in a PHA-negative mutant strain Cupriavidus necator PHB⁻4. The heterologous expression of the PhaC Is in PHB⁻4/pBBR1- C Is resulted in poly(3-hydroxybutyrate) [P(3HB)] accumulation of up to 71 wt% of the cell dry weight. In addition, the PhaC Is was shown to be able to polymerize 5 types of monomers, 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV), and 3-hydroxy-4-methylvalerate (3H4MV). In vitro enzymatic assay showed that PhaC Is was capable of catalyzing ( R )-3HB-CoA with a specific activity of 11.6 ± 0.6 nmol/min/mg and displayed an initial lag-phase. This study provides a basic information of PhaC Is that allows us to create variants applied for the metabolic engineering of I. sakaiensis that produces various types of PHA from PET.
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