Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

<p>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.</p>

Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

<p>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.</p>

Biosynthesis of poly(glycolate-co-3-hydroxybutyrate-co-3-hydroxyhexanoate) in Escherichia coli expressing sequence-regulating polyhydroxyalkanoate synthase and medium-chain-length 3-hydroxyalkanoic acid coenzyme A ligase

Chimeric polyhydroxyalkanoate synthase PhaCAR is characterized by the capacity to incorporate unusual glycolate (GL) units and spontaneously synthesize block copolymers. The GL and 3-hydroxybutyrate (3HB) copolymer synthesized by PhaCAR is a random-homo block copolymer, poly(GL-ran-3HB)-b-poly(3HB). In the present study, medium-chain-length 3-hydroxyhexanoate (3HHx) units were incorporated into this copolymer using PhaCAR for the first time. The coenzyme A (CoA) ligase from Pseudomonas oleovorans (AlkK) serves as a simple 3HHx-CoA supplying route in Escherichia coli from exogenously supplemented 3HHx. NMR analyses of the obtained polymers revealed that 3HHx units were randomly connected to 3HB units, whereas GL units were heterogeneously distributed. Therefore, the polymer is composed of two segments: P(3HB-co-3HHx) and P(GL-co-3HB-co-3HHx). The thermal and mechanical properties of the terpolymer indicate no contiguous P(3HB) segments in the material, consistent with the NMR results. Therefore, PhaCAR synthesized the novel block copolymer P(3HB-co-3HHx)-b-P(GL-co-3HB-co-3HHx), which is the first block PHA copolymer comprising two copolymer segments.

PLAN-M; Mycobacteriophage Endolysins Fused to Biodegradable Nanobeads Mitigate Mycobacterial Growth in Liquid and on Surfaces

The Mycobacteria are a genus of Actinobacteria that include human pathogens such as Mycobacterium tuberculosis (TB). Active TB disease can spread by airborne transmission to healthcare workers and to their community. The HHMI SEA-PHAGES program has contributed to discovering bacteriophages that are able to infect M. smegmatis MC2 155, a close relative of M. tuberculosis. This collection of diverse Mycobacteriophages is an excellent resource for trialling bacteriophage-sourced enzymes in novel applications. Herein we measured the ability Mycobacteriophage endolysins to lyse their host strain when functionally fused to biodegradable polyhydroxyalkanoate (PHA) nanobeads. PHA nanobeads facilitate both the expression and the application of enzymes to surfaces and have been demonstrated to stabilize a wide array of proteins for practical applications whilst eliminating the challenges of traditional protein purification. We selected two Lysin A and six Lysin B homologs to be functionally fused to the polyhydroxyalkanoate synthase C (PhaC). Expression of these constructs resulted in functional lysins displayed on the surface of PHA nanobeads. The lysins thus directionally displayed on nanobeads lysed up to 79% of the M. smegmatis MC2 155 population using 80 mg/mL of nanobeads in pure culture. In order to determine whether the nanobeads would be effective as a protective layer in PPE we adapted a fabric-based test and observed a maximum of 1 log loss of the cell population after 5 h of exposure on a textile (91% cell lysis). Lysin B enzymes performed better than the Lysin A enzymes as a protective barrier on textiles surface assays. These results suggest that bacterial endolysins are efficient in their action when displayed on PHA nanobeads and can cause significant population mortality in as little as 45 min. Our results provide the proof-of-principle that Mycobacteriophage endolysins can be used on functionalized nanobeads where they can protect surfaces such as personal protective equipment (PPE) that routinely come into contact with aerosolised bacteria.

Biosynthesis of Random-Homo Block Copolymer Poly[Glycolate-ran-3-Hydroxybutyrate (3HB)]-b-Poly(3HB) Using Sequence-Regulating Chimeric Polyhydroxyalkanoate Synthase in Escherichia coli

Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in Escherichia coli expressing the engineered chimeric PHA synthase PhaCAR and coenzyme A transferase. The cells produced poly[GL-co-3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaCAR is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using 1H nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL-ran-3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1PsSTQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaCAR synthesized a novel random-homo block copolymer, P(GL-ran-3HB)-b-P(3HB).

The structural insight of class III of polyhydroxyalkanoate synthase from Bacillus sp. PSA10 as revealed by in silico analysis

PhaC synthase is an enzyme responsible for PHA polymerization. In this work, the catalytic mechanism class III of PhaC synthase from Bacillus sp. PSA10 (BacPhaCSynt) was reported through in silico modelling approach based on the primary sequence of the PhaC synthase. The open reading frame BacPhaCSynt has been successfully isolated, cloned and overexpressed the recombinant protein in Escherichia coli BL21(DE3). To know the global architecture and catalytic mechanism, the structural prediction of BacPhaCSynt has been carried out by using MODELLER. The recombinant BacPhaCSynt exhibited monomeric molecular weight (MW) of 43.6 kDa, when it was analyzed on 12% SDS‐PAGE gel. Based on the structural prediction, BacPhaCSynt exhibited global architecture of α/β hydrolase fold, with the root mean square deviation (r.m.s.d) value of 0.94Å. The catalytic residues composition of BacPhaCSynt consists of C151, D307, and H336, but the H336 and D307 residues of the model have been distorted 62.8o and 175.2o from the corresponding residues of the template. Since the D307 is quite a distance from the H336, it might act as a general base for the activation of ‐OH group of the substrate. The results strongly suggested that the mode of action of BacPhaCSynt obeyed the covalent catalysis mechanism.