Discovering and developing new antibiotics from marine microbes combining genomics and synthetic biology
Lead Research Organisation:
University of St Andrews
Department Name: Chemistry
Abstract
There is an urgent need for new antibiotics preferably with clinically unexploited modes of action. Natural products, compounds generated by plants and microbes, provide an unprecedented starting point for antibiotic discovery with over 70% of antibiotics entering clinical trials, in the last three decades, being based on such compounds. The marine environment represents a particularly rich and largely untapped source of biosynthetically promising bacteria and in very recent years a large number of natural products from the marine environment have progressed into clinical trials.
Through this study a state of the art approach to the discovery and development of new structural classes of antibiotics will be explored. Cosmid libraries of a series of promising marine microbes will be made. The organisms will be sequenced, sequence motifs that potentially encode biosynthetic pathways to structurally interesting natural products will be selected and the cosmids encoding these genes used in their heterologous expression in genetically tractable and ready to ferment host organisms.
In complement to these studies, the genes encoding a known and potent antibiotic from a marine microbe will be cloned from a cosmid library into a heterologous host.
This synthetic biology approach to antibiotic discovery and manipulation will provide a platform for:
.the fermentation, isolation and full structural characterisation of the new antibiotic molecules
.enabling pathway manipulation - particularly using the Genochemetics approach in which gene insertion enables selective chemical modification
New antibiotics will be assayed against pathogenic bacteria including ESKAPE pathogens
Analogues, of key compounds, will be generated and structure activity relationships (SAR) investigated in order to create new and improved medicinally useful compounds.
Through this study a state of the art approach to the discovery and development of new structural classes of antibiotics will be explored. Cosmid libraries of a series of promising marine microbes will be made. The organisms will be sequenced, sequence motifs that potentially encode biosynthetic pathways to structurally interesting natural products will be selected and the cosmids encoding these genes used in their heterologous expression in genetically tractable and ready to ferment host organisms.
In complement to these studies, the genes encoding a known and potent antibiotic from a marine microbe will be cloned from a cosmid library into a heterologous host.
This synthetic biology approach to antibiotic discovery and manipulation will provide a platform for:
.the fermentation, isolation and full structural characterisation of the new antibiotic molecules
.enabling pathway manipulation - particularly using the Genochemetics approach in which gene insertion enables selective chemical modification
New antibiotics will be assayed against pathogenic bacteria including ESKAPE pathogens
Analogues, of key compounds, will be generated and structure activity relationships (SAR) investigated in order to create new and improved medicinally useful compounds.
People |
ORCID iD |
| Rebecca Goss (Primary Supervisor) | |
| Emily Abraham (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/N509759/1 | 01/10/2016 | 30/09/2021 | |||
| 1654346 | Studentship | EP/N509759/1 | 01/10/2015 | 31/03/2019 | Emily Abraham |
| Description | This project so far has used a combination of molecular biology and genome mining approaches to try and identify novel compounds and to improve the bioactivities of known antibiotics by refactoring the antibiotic gene cluster and by creating analogues of the antibiotic. Firstly, this project has used a state-of-the-art data driven genomics approach, whereby in silico analysis has been used to identify biosynthetically rich and sequenced microbial strains which have had little previous work conducted on their secondary metabolism. These strains provide a test-bed to demonstrate the utility of different cloning methods including Gibson assembly, cosmid library construction and BAC library construction, as well as providing the opportunity to discover new antimicrobials. This is particularly important as a significant challenge noted in the field of natural product discovery, and in this work has been those associated with manipulating biosynthetic gene clusters. It is often difficult to clone large PKS and NRPS gene clusters as they can exceed 100 kb, and recombination events can result in the rearrangement of biosynthetic gene clusters (BGCs); therefore, this work has compared cloning techniques to tackle these issues. We have optimised our methods of making cosmid libraries for gene clusters less than 40kb in size and using BAC libraries for gene clusters greater than 50 kb. We are also exploring the use of Gibson Assembly but have found it not to work well with highly repetitive gene clusters. The novel gene clusters have been heterologously expressed and a key challenge has been data analysis. As we do not know the exact structure of the novel compound, we have been using clues from the genome to understand which mass we are expecting. We have also collaborated with Marnix Medema's group at Wageningen University to utilise molecular networking which has helped with the data analysis. Secondly this work has explored using a bioactivity guided approach blended with the genomics approach has been used to explore antibiotics that are known to have promising activity. We have explored the Bacillus 2011SOCCUF3 strain and have shown this strain antibiotic activity against both Pseudomonas aeruginosa and Staphylococcus aureus. We have identified a new Bacillin compound from this strain and subsequent genome sequencing of this strain has revealed that there are about fifteen unknown gene clusters which are being investigated. The final strand of this work has used a structure-led approach to develop new antibiotics. This includes work which has been done on the assembly of a gene cluster that encodes for marinomycin, a known potent antibiotic and anticancer compound. Marinomycin is produced by marinospora CNQ-140 and its highly repetitive 72 kb gene cluster has been cloned using a direct cloning approach utilising Gibson assembly. Initial results suggest that this gene cluster has been cloned; this will pave the way for the marinomycin gene cluster to be heterologously expressed, which will enable for the refactoring of the gene cluster. Moreover, work is being conducted to generate analogues of marinomycin by epoxidating the compound in vivo to enable selectable functionalisation of marinomycin. To do this, a series of known epoxidases have been mined, which have been demonstrated to epoxidate polyene natural products. |
| Exploitation Route | Findings are disseminated regularly across the EMBRIC network, encouraging new ways of working and employment of best practise. |
| Sectors | Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Application for Early Career Researcher Exchanges (PECRE) 2017 |
| Amount | £2,012 (GBP) |
| Organisation | Scottish Universities Life Sciences Alliance |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 02/2018 |
| End | 07/2018 |
| Description | EMBRIC |
| Organisation | Leibniz Association |
| Department | Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures |
| Country | Germany |
| Sector | Public |
| PI Contribution | We have shared data and expertise on cloning methodology. |
| Collaborator Contribution | The partners in this collaberation have provided us with very difficult to culture, rare microbial strains. |
| Impact | We have genomic DNA from rare and hard to culture bacterial strains. These strains would not have been accessible to us without their help. These strains will be investigated, and they may contain entirely new bioactive compounds that we can exploit. |
| Start Year | 2015 |
| Description | Marnix Medema |
| Organisation | Wageningen University & Research |
| Country | Netherlands |
| Sector | Academic/University |
| PI Contribution | We have provided interesting data of novel gene clusters that have been heterologously expressed. |
| Collaborator Contribution | The Medema group have assisted in analysing the data we have generated using molecular networking software. |
| Impact | We have learnt to use molecular networking software to analyse our results. This will be highly useful when more data is generated in the near future as well. |
| Start Year | 2018 |