Biosynthesis and Characterization of Medium-Chain-Length Polyhydroxyalkanoate with an Enriched 3-Hydroxydodecanoate Monomer from a <i>Pseudomonas chlororaphis</i> Cell Factory

Abstract

Polyhydroxyalkanoates (PHAs) have been reported with agricultural and medical applications in virtue of their biodegradable and biocompatible properties. Here, we systematically engineered three modules for the enhanced biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA) in <i>Pseudomonas chlororaphis</i> HT66. The <i>phzE</i>, <i>fadA</i>, and <i>fadB</i> genes were deleted to block the native phenazine pathway and weaken the fatty acid β-oxidation pathway. Additionally, a PHA depolymerase gene <i>phaZ</i> was knocked out to prevent the degradation of mcl-PHA. Three genes involved in the mcl-PHA biosynthesis pathway were co-overexpressed to increase carbon flux. The engineered strain HT4Δ::<i>C1C2J</i> exhibited an 18.2 g/L cell dry weight with 84.9 wt % of mcl-PHA in a shake-flask culture, and the 3-hydroxydodecanoate (3HDD) monomer was increased to 71.6 mol %. Thermophysical and mechanical properties of mcl-PHA were improved with an enriched ratio of 3HDD. This study demonstrated a rational metabolic engineering approach to enhance the production of mcl-PHA with the enriched dominant monomer and improved material properties.