Novel Synthetic Biology for the Generation of 'Unnatural' Peptides

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Many bioactive natural products currently utilised as anticancer and antibiotic agents are biosynthesized in microorganisms by multifunctional enzymes known as nonribosomal peptide synthetases (NRPSs). These naturally occurring 'peptide factories' are seen as the promising starting point for the development of novel synthetic biology towards the generation of 'unnatural' peptides: these are molecules still produced via biosynthesis but are structurally different from the original peptide products, and may be of equal or superior value.
The aim of this PhD project is to develop a novel chemoenzymatic approach towards the in vivo generation of unnatural peptides: by utilising chemical building blocks and engineered NRPS enzymes/pathways it is hoped to generate a library of novel peptide compounds of pharmaceutical and industrial interest.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
1643091 Studentship BB/M01116X/1 05/10/2015 30/09/2019 Daniel Leng
 
Description As a result of this award, we have discovered the previous unknown timing of key steps in the assembly of the clinically used antibiotic vancomycin. The tools which were developed during the award enabled this, and enable new approaches to studying previously inaccessible steps in the biosynthesis of other medicinal compounds.
Exploitation Route Understanding of the timing and ordering of biosynthetic processes is key to rationally engineering biosynthetic pathways to create new products. The tools developed during this award allow a more detailed elucidation of the timing of biosynthetic processes, and the pathway intermediates involved. Identifying the substrates of these unknown steps is vital to understanding the possibility of enzyme engineering to create novel derivatives of natural products, many of which are medicinally relevant. This work provides a base knowledge from which to begin a redesign process.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Title Chemical probes for the study of nonribosomal peptide synthetases 
Description Chemical probes mimic the amino-acyl phosphopantetheinyl moiety used to extend the peptide chain during the biosynthesis of nonribosomal peptides. By acting as viable substrates for the formation of an amide bond by the enzymes condensation domain, the probes can form a non-hydrolysable bond to the intermediates formed and remove them from the enzyme. Once no longer enzyme bound, the intermediates can be extracted and studied using high-resolution mass spectrometry. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact This work has been published in Chemical Communications in 2017, and was also used to enhance the understanding of a number of other NRPS biosynthetic pathways, including that of the clinically relevant glycopeptide antibiotic vancomycin, for which a manuscript is in preparation. 
URL https://pubs.rsc.org/en/content/articlelanding/2017/cc/c7cc02427d#!divAbstract
 
Title Mass spectrometry methods for the study of nonribosomal peptide intermediates 
Description A mass spectrometry approach for the separation of nonribosomal peptide intermediates from a complex mixture from a whole cell extract, to allow their identification. The ion selection and fragmentation energy of the tandem mass spectrometry has also been optimised to improve the amount of information about the structure of the intermediates that can be derived. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact The development of this method enabled a publication in Chemical Communications in 2017, and is an integral part of the manuscript in preparation on the biosynthetic timing of halogenation in vancomycin biosynthesis. 
 
Description Cost action CM1407: Challenging organic syntheses inspired by nature from natural products chemistry to drug discovery 
Organisation European Cooperation in Science and Technology (COST)
Country Belgium 
Sector Public 
PI Contribution Contribution of a number of synthetic compounds for in silico and in vitro screening for a range of activities, including anti-viral, anti-cancer, and anti-TB activities.
Collaborator Contribution In silico and in vitro screening for a range of activities, including anti-viral, anti-cancer, and anti-TB activities.
Impact No compounds were discovered to possess sufficient activity to pursue further.
Start Year 2015
 
Description Glycopeptide collaboration 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution As a part of this collaboration, I synthesized a wide range of chemical probes based upon the cognate amino acids involved in the biosynthesis of vancomycin, in addition to chemical probes based upon non-cognate amino acids of similar structure. I carried out a large number of experiments supplementing glycopeptide antibiotic producing bacterial strains with chemical probes in order to intercept biosynthetic intermediates from the pathway. I analysed the organic extracts obtained from the supplemented cultures by LC-MS/MS to characterise the intermediate species offloaded by the chemical probes.
Collaborator Contribution Our partners contributed rare amino acid precursors, notably dihydroxyphenylglycine, for the development of chemical probes for glycopeptide antibiotic producing organisms. They also conducted a small number of experiments supplementing the vancomycin producer A. Orientalsis with chemical probes, and carried out a preliminary analysis of the data produced from the LC-MS of the culture extracts.
Impact There is a manuscript in preparation, detailing the first elucidation of the timing of the first chlorination during the biosynthesis of vancomycin.
Start Year 2017