Tailor-Made Polyhydroxyalkanoates by Reconstructing Pseudomonas Entomophila

Abstract

Microbial polyhydroxyalkanoates (PHA) containing short- and medium/long-chain-length monomers, abbreviated as SCL-co-MCL/LCL PHAs, generate suitable thermal and mechanical properties. However, SCL-co-MCL/LCL PHAs with carbon chain longer than nine are difficult to synthesize due to the low specificity of PHA synthase PhaC and the lack of either SCL- or MCL/LCL monomer precursor fluxes. This study succeeds in reprogramming a β-oxidation weakened Pseudomonas entomophila containing synthesis pathways of SCL 3-hydroxybutyryl-CoA (3HB) from glucose and MCL/LCL 3-hydroxyalkanoyl-CoA from fatty acids with carbon chain lengths from 9 to 18, respectively, that are polymerized under a low specificity PhaC_61-3 to form P(3HB-co-MCL/LCL 3HA) copolymers. Through rational flux-tuning approaches, the optimized recombinant P. entomophila accumulates 55 wt% poly-3-hydroxybutyrate in 8.4 g L^-1 cell dry weight. Combined with weakened β-oxidation, a series of novel P(3HB-co-MCL/LCL 3HA) copolymers with over 60 wt% PHA in 9 g L^-1 cell dry weight have been synthesized for the first time. P. entomophila has become a high-performing platform to generate tailor-made new SCL-co-MCL/LCL PHAs.

Keywords: Pseudomonas entomophila; SCL-co-LCL PHA copolymer; SCL-co-MCL PHA copolymer; flux tuning; metabolic engineering; polyhydroxyalkanoates; synthetic biology.