Leng, Daniel J. (2019) Chemical probing of nonribosomal peptide assemblies. PhD thesis, University of Warwick.

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Abstract

Nonribosomal peptides (NRPs) are secondary metabolites produced by nonribosomal peptide synthetases and comprise countless medicinally relevant compounds, such as the penicillin antibiotics and the anticancer agent bleomycin, as well as toxins such as microcystins. NRP biosynthesis involves the processing of enzyme-bound intermediates, which is challenging and constitutes a bottleneck in the stepwise elucidation of natural product maturation. Chemical probes mimicking natural biosynthetic extender units have been developed and used as effective tools to intercept and capture biosynthetic intermediates for identification and characterisation. This PhD work describes the use of chemical probes to investigate the NRP bio-assembly of echinomycin, colibactin, vancomycin, and scleric acid.

Echinomycin is a depsipeptide antibiotic produced by Streptomyces lasaliensis. Chemical probes containing a thioamide or N-methylated amino acid moieties inspected the substrate scope of the condensation domain, and the potential to generate unnatural peptides by NRPSs. To this end, in vivo editing of the gene cluster and in vivo reconstitution of the NRPS have also been pursued.

Colibactin is a PKS-NRPS derived genotoxin produced by E. coli. The biosynthetic pathway features several rare enzymatic processes, including those catalysed by novel amidase ClbL and the use of two aminomalonate extender units. Chemical probes designed to mimic aminomalonate and cysteine were synthesised and tested to explore the formation of thiazole rings and the incorporation of aminomalonate by the PKS ClbO in colibactin biosynthesis.

Vancomycin is a glycopeptide antibiotic of last resort produced by Amycolatopsis orientalis. A number of biosynthetic questions remain unanswered, particularly the timing of the chlorination by the halogenase VhaA. Chemical probes were used to examine timing of chlorine incorporation, with a likely timing proposed, and the substrate specificity of the pathways C domains.

Scleric acid is the product of an NRPS-containing, silent, cryptic gene cluster. Chemical synthesis has been used to unequivocally assign the structure, and the participation of a key biosynthetic intermediate. A number of analogues have also been synthesized to develop modest antitubercular activity exhibited by scleric acid.

Overall, in this work the chemical probing of NRPSs has provided insights into the substrate tolerance of the condensation domains, offering insights which can be used in the engineering of the NRPS pathways. Intermediates captured from the vancomycin pathway support the chlorination of an enzyme-bound intermediate, and the structure of scleric acid has been identified and key parts of its biosynthesis revealed.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QD Chemistry Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Peptides -- Synthesis, Molecular probes, Biosynthesis
Official Date:	September 2019
Dates:	Date Event September 2019 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Tosin, Manuela
Format of File:	pdf
Extent:	350 leaves : illustrations (some color)
Language:	eng

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