Elucidating and Engineering Legonmycin Biosynthesis: a framework for heterobicyclic biotransformation

Lead Research Organisation: University of Aberdeen
Department Name: Chemistry

Abstract

Many currently used drugs that we rely on for the treatment of cancer, bacterial infection, immune disorders and viral infections are either natural products or are derived from natural products. As such natural products still remain the major source of lead compounds for the development of new drugs and diagnostic molecules. However, natural products are often complex in terms of structure and composition making them difficult for synthetic chemists to access and thus develop further. In contrast bacteria can possess an amazing inventory of complex chemistry that organic chemists only dream of. In making new molecules, a key challenge is often to identify plausible new scaffolds or motifs (also known as chemical diversity) which is at the heart of drug discovery. We are going to study two bacterial enzymes that have the capability to convert linear peptide into heterobicyclic molecules under mild reactive conditions. These heterobicyclic ring systems are a common motif in biologically important compounds but there does not have any good way of making them by synthetic chemistry. The use of the bacterial enzymes to accomplish chemical tasks is well established in food industry and the benefits in terms of sustainable manufacturing are well document. However, the application of enzymatic process/ industrial biotechnology processes are still under developed the fine chemical, pharmaceutical and agricultural industries. By working out the catalytic capabilities of the two enzymes we will gain the ability to develop their industrial biotechnological potential. In doing this, we will be able to make novel building blocks (also known as scaffolds) and, access new biologically active compounds.

Technical Summary

We propose to investigate the capabilities of two novel enzymes, the thioesterase LgnD-Te and FAD-dependent monooxygenase LgnC from the soil bacterium Streptomyces sp. MA37, responsible for the formation of heterobicycles. These heterobicyclic rings are critical components of many biologically important and industrially relevant molecules. We have made significant progress in understanding the chemical mechanism of LgnC, which is the first step towards harnessing the industrial potential this enzyme. We intend to continue this work and elucidate its mechanism in detail through site direct mutagenesis studies to locate the active site. We will also probe the capacity of this enzyme using modified substrates with structural diversity. From our preliminary result we have begun to understand the mechanism of the thioesterase LgnD-Te and our data indicates the formation of a heterobicyclic ring using a surrogate synthetic substrate. We will advance our initial mechanistic work by using substrates with different stereogenic centres and through site direct mutagenesis to locate its active site. Substrate tolerance is a key feature for a successful industrial biocatalyst and we will synthesize peptide thioesters equipped with different chemical handles (i.e. bromine/chlorine/hydroxyl/amine) and probe the substrate flexibility of LgnD-Te. Furthermore, we will identify new homologues of LgnC and LgnD-Te that possess enhanced kinetic profiles and broader substrate tolerance through genome mining and synthetic gene expression. This will not only give us exquisite control of the biochemical experiments but lead to more diverse chemical scaffolds being accessible thus broadening the molecule library generated for bioactivity screening.

Planned Impact

Impact will be delivered by fulfilling the research program's key objective which is to provide stakeholders in various sectors with new bioactive compounds and knowledge in industrial biocatalysis.

The project will deliver impact across four main areas.

People: The program will deliver multidisciplinary training in the chemical and biological sciences to two PDRAs. The UK has identified synthetic biology and industrial biotechnology as key deficits in scientists' training for the future workforce and this program will address that need. The project is at the cutting edge of industrial biotechnology and it will provide excellent training for the PDRAs if they intend to continue to work in this research field (in industry or academia). The PDRAs will also have access to a variety of staff training courses run by both the University of Aberdeen and the University of Durham, which are designed to enhance a wide variety of transferable and career based skills. All of the aforementioned points will ensure that the recruited PDRAs have enhanced job prospects upon completion of the program and this will help to enhance the UK's scientific skill base. The PI's (Deng) lab regularly hosts undergraduates from the UK or Europe as summer placement students. Both Deng and Jaspars are very active in outreach programmes that engage with school children. If funded we will offer summer placements (1-2 per year) to local children to experience research at the chemical-biology interface. We also intend to create a range of practical biochemical experiments suitable for University undergraduates in the first instance at Aberdeen but that will be made open-access via the PI's webpage. The Co-I (Cobb) regularly gives educational lectures to teachers as part of a program of University engagement with Secondary School Education. Interaction with local high schools will also help to enthuse the next generation of scientists. In this area the Co-I (Cobb) has prior experience having led various sixth form projects as part of the annual North East Schools Industry Partnership scheme (NESIP) held in the Durham University Chemistry Department. Students from secondary schools in the North-East of England, accompanied by their teachers, spend a full week undertaking research projects.

Society: The work will generate new biologically active molecules and moreover allow their generation in useful quantities using sustainable manufacturing methods (i.e. biocatalysts). This means they can be used in drug development programmes and in screening campaigns for the identification of new agents such as anti-biotics. The generation of new antimicrobial agents has the potential to reduce the burden of infection both in the National Health Service (NHS), and, more directly in everyday life. Delivering in these areas will in turn have a direct and considerable impact on the health of the UK population.

Economic: The generation of novel IP will help to develop new markets for commercial exploitation which help UK PLC to remain internationally competitive in the areas of chemical-biology and industrial biocatalysis. As part of the work program we will utilise existing links and aim to establish new collaborations with industry to maximise the commercial exploitation of the research. We would expect to transfer technology through service agreements (supply novel compounds) or by technology licensing. We will consider founding our own spin out company as the project develops.

Knowledge: On a fundamental level the work program will deliver exciting new insights into the, biosynthesis of HAs and specifically the mode of action of the novel enzymes, LgnD-Te and LgnC, This new knowledge will be of benefit to researchers both in academia and in industry who are engaged in enhancing biotransformation to access novel molecular scaffolds for biological screening.

Publications

10 25 50
 
Description 1. One of the key enzymes, the monooxygenase LgnC, has been characterized.
2. A series of site-directed mutagenesis experiments to probe the active site of LgnC, has been performed, the putative residues of its active site has been identified.
2. three enzyme homologues have been identified and overexpressed subjected to full characterization.
3. the thioesterase enzyme has been tested with various substrates;
4. Three enzyme homologues of thioesterases have been identified and overexpressed.
Exploitation Route The successful outcome of the project is to develop unique natural product like molecules that have the capacity to be developed into a new therapeutics and agrochemicals. By the end of the project the aim is to have sound, proof of principal data that can be used to further develop the biotechnology developed and the compounds prepared. Ingenza, a biotechnology company based in Edinburgh, showed considerable interest in this biotechnology.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description NPRONET
Amount £10,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2017 
End 01/2018
 
Title monooxygenase 
Description We discovered a series of monooxygenases from different sources that catalyse a similar biochemical transformation which can be used as biocatalysts. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact Not yet 
 
Title thioesterases 
Description Identification and overexpression of a series of thioesterase homologues for characterization. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact We will test these homologues with the aim of finding the enzymes with the best kinetics and wider substrate specificity for potential application as new biocatalysts for the synthesis of new bicyclic alkaloids. 
 
Description Chemo-enzymatic synthesis 
Organisation Durham University
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a collaborative project between Dr Hai Deng , University of Aberdeen and Dr Steven Cobb, Durham University. Aberdeen contribution is mainly focused on the characterization of the targeted enzymes and application of the enzymes of interest for new compound synthesis.
Collaborator Contribution Durham contribution is mainly focused on the chemical synthesis of small molecule substrate analogues for the enzymes of interest.
Impact Not yet but we are accumulating preliminary data right now for future publications.
Start Year 2017
 
Description School visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact St Joseph Primary School organised "Science Day" June, 2017 and invited HD to participate in this event. HD briefly introduced the principles of biochemistry and presented a colorimetric experiment including a series of enzymatic reactions to aspire school pupiles and general audience. The teachers told HD that it appeared that there have been increased interest in the school in biochemistry/chemistry.
Year(s) Of Engagement Activity 2017
 
Description Thai visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The team visited the research teams of Professor Manee Chanama from Mahidol University and Professor Suchart Chanama from Chulaongkorn University, in July, 2018 under the funding of Global Challenge research fund-IPPF scheme. The team hosted a workshop for the local teams and showcased the preliminary results and technologies from both awards. After the workshop, Thai collaborators and the team decided to further strengthen the collaboration by applying Royal Society Challenge-led GCRF scheme on September, 2018 (pending result).
Year(s) Of Engagement Activity 2018
 
Description engagement training 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact 3 day course aimed at enhancing public engagement skills and at designing an event to be held in Oban during West Highland Yachting Week in July where various aspects of current ocean based research will be showcased.
Year(s) Of Engagement Activity 2018
 
Description outreach event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact During 2 days of the West Highland Yachting Week in Oban, the events designed during the training programme were rolled out for the general public to try.
Year(s) Of Engagement Activity 2018