Open Access Te Herenga Waka-Victoria University of Wellington
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Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

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posted on 2021-12-09, 11:54 authored by Kane, Alex

Manufacturing of high-grade plastics from petroleum-based feedstocks is a high-cost, unsustainable process resulting in expensive products. My overall goal was to engineer the pathway of bacterial bio-polyester formation, in order to produce high-grade bioplastics. More specifically, the aim was to introduce aromatic rings into the main-chain of the polyhydroxyalkanoate (PHA) polymer currently produced by specialist bacteria. This research aimed to create these bio-plastics from renewable resources, rather than relying on petroleum-based sources.  A key enzyme for this process is the polyhydroxyalkanoate synthase, PhaC. This enzyme is capable of polymerizing activated hydroxybutyrate-CoA monomers. I began with the establishment of a system that allowed the use of directed evolution. I constructed a minimal plasmid for the expression of PhaC and a second plasmid with the CoA ligase genes required for substrate activation. I generated error-prone PCR libraries of the Cupriavidus necator phaCa, Chromobacterium sp. USM2 phaCb and an ancestrally reconstructed phaCb-LCA that contained differing spectra of mutations. A life-or-death selection was employed to select for PhaC variants able to polymerise aromatic substrates based upon the toxicity of the un-polymerized aromatic hydroxyacid monomers. I determined the minimum inhibitory concentrations (MICs) for six of these monomers in Escherichia coli for downstream selection. Lastly, I adapted a Nile red screening method to test wild-type PHA accumulation of PhaC enzymes.  Selections for mutants capable of polymerizing aromatic monomers were implemented on the libraries generated from phaCa and phaCb. Whereas, the library generated from phaCb-LCA was screened for variants with increased wild-type activity. Selections yielded no candidates for further testing. However, the screen isolated several variants with increased wild-type activity. These variants may serve as a new scaffold for further mutagenesis experiments to achieve the overall goal; to produce a high-grade bioplastic.


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Date of Award



Te Herenga Waka—Victoria University of Wellington

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Author Retains Copyright

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Degree Grantor

Te Herenga Waka—Victoria University of Wellington

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Degree Name

Master of Science

ANZSRC Type Of Activity code

970106 Expanding Knowledge in the Biological Sciences

Victoria University of Wellington Item Type

Awarded Research Masters Thesis



Victoria University of Wellington School

School of Biological Sciences


Patrick, Wayne