Evolution-inspired engineering of non-ribosomal peptide synthetase assembly lines to create novel bioactive scaffolds
Lead Research Organisation:
University of Warwick
Department Name: Chemistry
Abstract
Microorganisms and plants supply the world with a considerable number of bioactive compounds used in medicine and agriculture. Small changes in the structure of these compounds can influence their efficacy as antibiotics and anti-cancer agents (used in hospitals to treat serious diseases), and pesticides (used in agriculture to protect crops).
Many of these biologically active compounds are produced in bacteria by large multi-enzyme systems known as non-ribosomal peptide synthetases (NRPSs), that work as an assembly line to create the final product. These assembly lines are encoded in bacterial genomes by sets of genes known as biosynthetic gene clusters (BGCs). By removing, duplicating, or substituting different sections of the BGCs encoding these assembly lines, bacteria can modify the final product.
My project aims to exploit publicly available bacterial genome sequence data to identify natural examples of these BGC variations in Pseudomonas bacteria using bioinformatics techniques and catalogue these variants. We can use this data to see the exact positions employed by Nature to modify the BGCs and apply these rules to make novel alterations while not disrupting the overall function of the assembly line. The BGC responsible for making the natural product called orfamide A will be captured and subsequently modified through the removal, duplication, or substitution of different sections. Initially, I will re-create examples of known BGCs to trial the modification strategy, but I will later apply this technique to create novel BGC variants and new natural products. This synthetic biology approach to creating BGC variants will bypass the need to acquire bacterial strains from difficult to access sources and lead to the creation of novel scaffolds. The natural product variants resulting from this approach will be tested for antimicrobial, anti-cancer, and biopesticidal properties.
The outcomes of this project will accelerate the rational design and bioengineering of NRPS systems enabling novel compounds with beneficial properties for clinical, industrial, and agricultural to be created.
Many of these biologically active compounds are produced in bacteria by large multi-enzyme systems known as non-ribosomal peptide synthetases (NRPSs), that work as an assembly line to create the final product. These assembly lines are encoded in bacterial genomes by sets of genes known as biosynthetic gene clusters (BGCs). By removing, duplicating, or substituting different sections of the BGCs encoding these assembly lines, bacteria can modify the final product.
My project aims to exploit publicly available bacterial genome sequence data to identify natural examples of these BGC variations in Pseudomonas bacteria using bioinformatics techniques and catalogue these variants. We can use this data to see the exact positions employed by Nature to modify the BGCs and apply these rules to make novel alterations while not disrupting the overall function of the assembly line. The BGC responsible for making the natural product called orfamide A will be captured and subsequently modified through the removal, duplication, or substitution of different sections. Initially, I will re-create examples of known BGCs to trial the modification strategy, but I will later apply this technique to create novel BGC variants and new natural products. This synthetic biology approach to creating BGC variants will bypass the need to acquire bacterial strains from difficult to access sources and lead to the creation of novel scaffolds. The natural product variants resulting from this approach will be tested for antimicrobial, anti-cancer, and biopesticidal properties.
The outcomes of this project will accelerate the rational design and bioengineering of NRPS systems enabling novel compounds with beneficial properties for clinical, industrial, and agricultural to be created.
Technical Summary
Many bacterial specialised metabolites are assembled by large multi-enzyme systems known as non-ribosomal peptide synthetases (NRPSs). These NRPS systems usually function in a linear fashion, moving the growing peptide chain through successive modules to create the final product. Pseudomonas bacteria are talented producers of natural products and carry multiple NRPS biosynthetic gene clusters (BGCs). Many of the NRPSs responsible for lipopeptide assembly are evolutionarily related and have diversified through a series of module duplication, deletion, or substitution events. These lipopeptides show differences in their efficacy as antibiotic, antifungal, insecticidal, and anti-cancer agents.
This project is focused on understanding the natural recombination events that underpin the evolution of NRPS BGCs and using this knowledge to create novel non-ribosomal peptides via biosynthetic engineering. A bioinformatics-driven genome mining approach of publicly available genome sequencing data will identify naturally occurring NRPS BGC variants and the specific recombination events responsible for creating them. Using a yeast-based recombination platform, the orfamide A BGC will be captured in an expression vector. This BGC will be engineered via module deletions and substitutions at evolutionarily amenable recombination sites to create NRPS variants. The engineered BGCs encoding these NRPS variants will be expressed in a heterologous host, and production of the corresponding metabolites investigated using UHPLC-ESI-Q-TOF-MS. Novel compounds will be purified by semi-preparative HPLC, and structurally elucidated using NMR spectroscopy. Screening novel compounds for antimicrobial, anticancer and biopesticidal activity will assist in the development of more efficacious derivatives with clinical, industrial, and agricultural applications. Understanding natural recombination sites will improve our ability to rationally design and bioengineer bacterial NRPS systems.
This project is focused on understanding the natural recombination events that underpin the evolution of NRPS BGCs and using this knowledge to create novel non-ribosomal peptides via biosynthetic engineering. A bioinformatics-driven genome mining approach of publicly available genome sequencing data will identify naturally occurring NRPS BGC variants and the specific recombination events responsible for creating them. Using a yeast-based recombination platform, the orfamide A BGC will be captured in an expression vector. This BGC will be engineered via module deletions and substitutions at evolutionarily amenable recombination sites to create NRPS variants. The engineered BGCs encoding these NRPS variants will be expressed in a heterologous host, and production of the corresponding metabolites investigated using UHPLC-ESI-Q-TOF-MS. Novel compounds will be purified by semi-preparative HPLC, and structurally elucidated using NMR spectroscopy. Screening novel compounds for antimicrobial, anticancer and biopesticidal activity will assist in the development of more efficacious derivatives with clinical, industrial, and agricultural applications. Understanding natural recombination sites will improve our ability to rationally design and bioengineer bacterial NRPS systems.
