Design and engineering of antibiotics using systems and synthetic biology
Lead Research Organisation:
University of Manchester
Department Name: Chemistry
Abstract
Recent advances in molecular biology, in particular our improved ability to read and (most importantly) write genomic sequences, have led to renewed excitement in the area of genetic engineering. This ambitious new generation of genetic engineering, Synthetic Biology, has biotechnological potential. One of its most promising application areas is the generation of high-value bioactive natural products, including antibiotics and anticancer drugs.
The synthesis of bioactive natural products is usually encoded by large gene clusters in microbial genomes. A typical genome contains dozens of such clusters, but only a tiny fraction of these has a known end product; the others are silent or "cryptic". We will focus on one such gene cluster identified together with Demuris in a BBSRC/Innovate UK-funded project which ended in 2016. This project is part of an ambitious attempt to develop general strategies for awakening such cryptic clusters and characterizing their products.
1. Redesign and refactor the identified gene cluster for inducible overexpression ("awakening") and characterize the end product in detail, using for example, transcription studies, DNA assembly, CRISPR/Cas system, targeted and untargeted metabolite analysis.
2. Identify gene units that tend to be co-transferred horizontally between organisms or unique accessory enzymes, using a deep evolutionary analysis of thousands of sequenced gene clusters across hundreds of genomes. Each of these units is expected to carry out a well-defined type of chemical modification.
3. Develop a screening system in which the biochemical function of these units can be identified. This will be based on a metabolomics strategy that comprehensively characterizes changes in small molecule profiles of microbial indicator strains due to switching on of a specific gene unit.
4. Determine the identified gene units' functionality through enzymology in different background and on different chemical scaffolds, including that of gentamicin, to generate new biochemical diverse antimicrobials.
5. Conduct potency and activity spectrum testing against clinically relevant pathogens (MDR Grambacteria) and further scale-up fermentation (Demuris) for subsequent structural elucidation and the evaluation of cytotoxicity.
The track record of collaboration of the project partners and their world-leading expertise in the research area guarantee the feasibility of the project plan, which is based on a solid foundation of pilot studies.
The synthesis of bioactive natural products is usually encoded by large gene clusters in microbial genomes. A typical genome contains dozens of such clusters, but only a tiny fraction of these has a known end product; the others are silent or "cryptic". We will focus on one such gene cluster identified together with Demuris in a BBSRC/Innovate UK-funded project which ended in 2016. This project is part of an ambitious attempt to develop general strategies for awakening such cryptic clusters and characterizing their products.
1. Redesign and refactor the identified gene cluster for inducible overexpression ("awakening") and characterize the end product in detail, using for example, transcription studies, DNA assembly, CRISPR/Cas system, targeted and untargeted metabolite analysis.
2. Identify gene units that tend to be co-transferred horizontally between organisms or unique accessory enzymes, using a deep evolutionary analysis of thousands of sequenced gene clusters across hundreds of genomes. Each of these units is expected to carry out a well-defined type of chemical modification.
3. Develop a screening system in which the biochemical function of these units can be identified. This will be based on a metabolomics strategy that comprehensively characterizes changes in small molecule profiles of microbial indicator strains due to switching on of a specific gene unit.
4. Determine the identified gene units' functionality through enzymology in different background and on different chemical scaffolds, including that of gentamicin, to generate new biochemical diverse antimicrobials.
5. Conduct potency and activity spectrum testing against clinically relevant pathogens (MDR Grambacteria) and further scale-up fermentation (Demuris) for subsequent structural elucidation and the evaluation of cytotoxicity.
The track record of collaboration of the project partners and their world-leading expertise in the research area guarantee the feasibility of the project plan, which is based on a solid foundation of pilot studies.
People |
ORCID iD |
| Eriko Takano (Primary Supervisor) | |
| Katherine Baker (Student) |
| Description | A novel Micromonospora species was identified as being an interesting antibiotic producer; a gene cluster (a co-localised group of enzymes in the genome, hypothesised in this case to produce a specific aminoglycoside antibiotic) from this organism was engineered for heterologous expression in Streptomyces spp.. Production of antibiotic at low levels in the heterologous hosts were achieved by varying culture conditions. Further modification of the gene cluster through CRISPR-Cas9 is ongoing to determine whether one antibiotic can be isolated from its congeners, as well as whether production can be improved. The biosynthetic pathway to this antibiotic was also refactored for heterologous expression in previous work (outside of this funding period). Studies into the expression levels of the enzymes by strains carrying this refactored plasmid suggested that the enzymes were not being expressed properly, as these could not be visualised through Western blot. This plasmid was rebuilt to ensure that all enzymes would be expressed properly in the heterologous host. A method for DNA assembly, ligase cycling reaction, was tested for the combinatorial assembly of enzymes with a cognate promoter of random strength. This did not work well with the GC-rich DNA of the pathway. Expression trials are on-going with the current iteration of the refactored pathway. Specialised skills learned include LC-MS for characterisation of antibiotic, CRISPR-Cas9 for cluster modification, a variety of DNA assembly methods, as well as building upon known molecular biology techniques. The research thus far has been underpinned by a close working relationship with the CASE partner, Demuris, who have provided comprehensive feedback on project planning and experiments. |
| Exploitation Route | It is too early to say where the production of the specific antibiotics might be built upon. In terms of the DNA assembly techniques used, a modification of ligase cycling reaction (LCR) so that it is able to work at higher efficiency with GC-rich DNA such as those of Actinomycetes would be very valuable for the community as the search for new antibiotics continues. This could be done by investigating whether different temperature conditions are optimal, as well as whether linker regions designed for the protocol could be modified so that they do not preferentially bind to each other instead of the dsDNA template parts (oligo hairpins/self- and cross-dimerization). |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Demuris collaboration |
| Organisation | Demuris Limited |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | The award for the PhD project was granted in collaboration with Demuris Ltd, a SME based in Newcastle focused on drug discovery. The entire project has been done with this collaboration in mind, with all lab work towards generating novel antimicrobials being presented to and approved by their principal scientist as well as the supervisory team at the University of Manchester. |
| Collaborator Contribution | Demuris Ltd provided bacterial strains and training in techniques such as antibiotic bioassays and analytical chemistry over three days in March 2018. Since then, work has been presented at a monthly meeting where the future directions are discussed and feedback contributions from their principal scientist (Nick Allenby) is incorporated. |
| Impact | No current outcomes |
| Start Year | 2017 |
| Description | iGEM Jamboree Judging |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Undergraduate students |
| Results and Impact | The iGEM Jamboree is a competition for undergraduates, postgraduates and high schools to use synthetic biology to solve real world problems. The competition culminates in an annual 'Giant Jamboree' which requires other members of the community to judge students on their projects. Through the PhD study I was given the opportunity to contribute to the competition as a judge in 2018 (in person) and 2020 (online). This involved ~3 days of attending talks, speaking with teams about their work at a poster presentation and scoring based on a rubric. Through this, I was able to learn more about various subfields of synthetic biology and improve my critical analysis, while also contributing ideas to these students' work. |
| Year(s) Of Engagement Activity | 2018,2020 |
| URL | https://igem.org/Main_Page |