Production of medium-chain-length polyhydroxyalkanoates from engineered bacteria

Polyhydroxyalkanoates (PHAs) are sustainable microbial biopolymers that, due to their biodegradability and biocompatibility, could potentially replace petroleum-based plastic. Medium chain length polyhydroxyalkanoates (mcl-PHAs) are a type of PHA that consist of monomers that are between 6 and 14 carbons long. They are elastic and flexible and can be used for a range of applications where these properties are advantageous such as soft tissue implants, heart valves, nerve tissue scaffolds etc. In all microbial cells, mcl-PHA is synthesized by the PHA synthase enzyme (PhaC) from 3-hydroxyacyl-CoA which is an intermediate metabolite of the β-oxidation pathway. The main objective of this project was to optimize the β-oxidation pathway to channel more precursors into biosynthesis of a novel mcl-PHA with high hydroxydodecanoate (C12) and/or hydroxytetradecanoate (C14) monomer composition (≥ 30%) using sodium dodecanoate or tetradecanoate as substrates. Escherichia coli was chosen as a chassis to compare enzymes activity related to this pathway and to compare the biosynthesized PHAs. A method for digestion and quantification of PHA using gas chromatography (GC) was first validated and standard curves were successfully constructed for PHAs of different chain lengths and for identification of residual substrates in the samples. Subsequently, a metabolic engineering strategy was followed using techniques in molecular microbiology to biosynthesise PHAs in E. coli. Firstly, 3 different phaCs from different Pseudomonas ( Pseudomonas mendocina, NK-01, P. putida KT2440 and P. stutzeri 1317 ) were cloned in 2
different plasmid backbones each and the production of the target mcl-PHA inside Escherichia coli DH5α and inside the ΔfadA knockout E.coli- K-12 was compared using sodium dodecanoate (C12) and/or sodium tetradecanoate (C14) as substrates. The β oxidation inhibitor sodium acrylate was also tested for its ability to channel more intermediate metabolites towards PHA production. Secondly, 2 different FabG enzymes ( 3-ketoacyl-acyl carrier protein (ACP) reductases) from E. coli -K-12 (EcolifabG) and M. tuberculosis (MtubfabG) were expressed in E. coli and compared for their ability to channel more intermediate metabolites towards PHA production. Lastly, the gene encoding the FadR protein (β-oxidation repressor) was knocked out from the ΔfadA E. coli K-12 strain to test the constitutive expression of β-oxidation pathway for the same purpose. All the dry biomass collected from the recombinant strains cultures was digested and analyzed using GC-FID for PHA content. Results showed that further optimization needs to be undertaken for production to reach levels that are detectable and quantifiable.