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Abstract: Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.

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Abstract: In this thesis, the bioconversion of palladium and ruthenium solutions into valuable monometallic and bimetallic nanoparticles with highly catalytic activity is described. Bacteria can enzymatically reduce Pd(II) at the expense of an exogenous electron donor to Pd(0). The resulting nanoparticles (NPs) are immobilised in the cytoplasm, membrane and periplasm of the cells resulting in small NPs with a homogeneous size and high catalytic properties. The catalytic activity of the biogenic Pd NPs is sometimes higher than the commercial catalysts with the additional advantage of being synthesized using more economic and environmentally friendly methodologies. Previous studies have shown the ability of some bacteria to recover gold and palladium from wastes using cells of Escherichia coli and Desulfovibrio desulfuricans and have elucidated some of the main enzymes involve in the initial nucleation and formation of Pd NPs. Other studies proved the ability of bacteria to synthesize bimetallic NPs of gold and palladium and palladium and ruthenium. In this study, cells of E. coli were used for the synthesis of bimetallic NPs of Pd/Ru. A detailed information of the structure of the bimetallic NPs was provided using a combination of spectroscopic and microscopic techniques; a “core/shell” (Ru core, Pd shell) structure was reported with a similar mechanism to that reported previously by Deplanche et al., (2012) with E. coli Pd/Au NPs. In addition, the catalytic properties of the bio-bimetallic NPs were reported for the conversion of 5-hydroxymethylfurfural (5-HMF) (a waste compound derived from the hydrolysis of starch and cellulose for obtaining biofuel) into 2,5-dimethylfuran (2,5-DMF), a valuable compound with similar properties to ethanol, with onward application as biodiesel. The ability to synthesize Pd/Ru NPs was tested using a consortium of acidophilic sulfidogenic (CAS) waste culture recovered from an unrelated biotechnology process together with a traditional sulfidogenic bacterium (D. desulfuricans). D. desulfuricans and CAS culture showed high activity for the conversion of 5-HMF into 2,5-DMF. Finally, an external source of radio-frequency (RF) (microwave energy (MW)) was applied to resting cells (i.e. before being exposed to Pd(II) solution) of E. coli and D. desulfuricans reporting changes in the dispersity of the resulted Pd NPs compared with untreated cells. The catalytic activity of Pd-NPs on MW-pretreated cells of D. desulfuricans was tested for the hydrogenation of 2-pentyne, showing increased catalytic properties as compared to Pd-NPs on untreated cells.

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Abstract: Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. Here, we report that this inhibition is dependent on incubation time, the solution conditions and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which our simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis, and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.

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Abstract: Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

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Abstract: Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.