ORCID Profile
0000-0002-4668-7245
Current Organisations
The University of Tennessee Knoxville
,
University of Sydney
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Publisher: Elsevier BV
Date: 06-2017
Publisher: Oxford University Press (OUP)
Date: 11-10-2017
DOI: 10.1093/NAR/GKX893
Publisher: American Chemical Society (ACS)
Date: 15-05-2020
DOI: 10.1021/JACS.0C02549
Publisher: Elsevier BV
Date: 09-2017
Publisher: Elsevier BV
Date: 07-2017
Publisher: Elsevier BV
Date: 10-2023
Publisher: American Chemical Society (ACS)
Date: 17-10-2017
DOI: 10.1021/ACSCHEMBIO.7B00714
Abstract: Streptomyces genomes have a high G + C content and typically use an ATG or GTG codon to initiate protein synthesis. Although gene-finding tools perform well in low GC genomes, it is known that the accuracy in predicting a translational start site (TSS) is much less for high GC genomes. LipPks1 is a Streptomyces-derived, well-characterized modular polyketide synthase (PKS). Using this enzyme as a model, we experimentally investigated the effects of alternative TSSs using a heterologous host, Streptomyces venezuelae. One of the TSSs employed boosted the protein level by 59-fold and the product yield by 23-fold compared to the originally annotated start codon. Interestingly, a structural model of the PKS indicated the presence of a structural motif in the N-terminus, which may explain the observed different protein levels together with a proline and arginine-rich sequence that may inhibit translational initiation. This structure was also found in six other modular PKSs that utilize noncarboxylated starter substrates, which may guide the selection of optimal TSSs in conjunction with start-codon prediction software.
Publisher: Elsevier BV
Date: 07-2023
Publisher: American Chemical Society (ACS)
Date: 11-04-2023
Publisher: Wiley
Date: 22-12-2020
Abstract: Bacterial modular type I polyketide synthases (PKSs) are complex multidomain assembly line proteins that produce a range of pharmaceutically relevant molecules with a high degree of stereochemical control. Due to their colinear properties, they have been considerable targets for rational biosynthetic pathway engineering. Among the domains harbored within these complex assembly lines, ketoreductase (KR) domains have been extensively studied with the goal of altering their stereoselectivity by site‐directed mutagenesis, as they confer much of the stereochemical complexity present in pharmaceutically active reduced polyketide scaffolds. Here we review all efforts to date to perform site‐directed mutagenesis on PKS KRs, most of which have been done in the context of excised KR domains on model diffusible substrates such as β‐keto N‐acetyl cysteamine thioesters. We also discuss the challenges around translating the findings of these studies to alter stereocontrol in the context of a complex multidomain enzymatic assembly line.
Publisher: Wiley
Date: 05-09-2012
Abstract: PKS biocatalysis: The terminal module of erythromycin synthase was used for the in vitro production of chiral triketide lactones. Combining cofactor regeneration, substrate truncation, and enzymatic promiscuity afforded a scalable strategy to generate these molecules from abundant racemic and achiral precursors. The described biocatalytic platform thus facilitates the application and study of enzymes within PKS modules.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5CC07315D
Abstract: Structure–activity relationship studies guided stereocontrol engineering within a modular polyketide synthase ketoreductase to yield a more active enzyme whose reactivity can be explained through the Felkin–Anh model.
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3PY00001J
Publisher: Springer Science and Business Media LLC
Date: 25-01-2018
DOI: 10.1038/S41598-018-20016-W
Abstract: pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli . As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cell in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. These pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.
Publisher: Wiley
Date: 08-04-2014
Abstract: Mechanically sensitive biocomposites comprised of fluorescent proteins report stress through distinct pathways. Whereas a composite containing an enhanced yellow fluorescent protein (eYFP) exhibited hypsochromic shifts in its fluorescence emission maxima following compression, a composite containing a modified green fluorescent protein (GFPuv) exhibited fluorescence quenching under the action of mechanical force. These ratio‐ and intensiometric sensors demonstrate that insights garnered from disparate fields (that is, polymer mechanochemistry and biophysics) can be harnessed to guide the rational design of new classes of biomechanophore‐containing materials.
Publisher: Elsevier BV
Date: 06-2020
Location: United States of America
Location: United States of America
Start Date: 2018
End Date: 2018
Funder: National Institute of General Medical Sciences
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