Industrial scale production of cyanobactin enzymes for fast and efficient cyclic peptide synthesis

Lead Research Organisation: University of Aberdeen
Department Name: Chemistry


The pharmaceutical industry needs cleaner and greener ways to produce complex compounds that will form the basis of future pharmaceuticals. In this project we will use enzymatic methods to generate cyclic compounds which may be applied as potential treatments for a range of diseases (e.g. fungal infection, epilepsy). The enzymes are derived from a marine blue-green alga and have been modified to make them more efficient. Combining these enzymes enables us to generate the compounds of interest in a few days compared to chemical synthesis which may take months. One aim of this project is to optimise the production of these enzymes with industry input, as well as streamline the whole process, including practical purification procedures. A key aim is to generate some compounds in reasonable quantity for testing against a number of disease models. Using this process we anticipate starting a company based around the ability to generate these unique cyclic compounds in a sustainable way.


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Description The main objective was to make our chemo-enzymatic approach (Angew. Chem. Int. Ed. 53, 14171-14174.; Patent WO/2014/001822) to synthesize heterocycle-containing cyclic peptides more economic and scalable. Cyclic and modified cyclic peptides are very appealing scaffolds for pharmaceutical industry as they can tackle challenging therapeutic targets such as protein-protein interaction while being amenable for oral administration and are much cheaper than biologics. However chemical synthesis of these compounds is very challenging. Our method relies on the use of marine-derived biosynthetic enzymes to process a recombinantly produced substrate that contains the linear sequence of the cyclic product and a leader sequence containing the recognition determinants for the processing enzymes. The method was proved to be:
1) Efficient: several sequences have been successfully processed including those containing unnatural amino acids. Incorporation of selanozoline heterocycles was also successful.
2) Fast: a single compound can be produced in three weeks. This is compared to 6 months using conventional chemical synthesis.
3) Eco-friendly: the method does not involve the use of any toxic reagents nor produce toxic waste as it relies mainly on the use of enzymes to catalyse chemical transformations.
However, the main impediment for the translation of this approach was the relatively high cost of enzyme/engineered substrate production and purification. Purification protocol involved the use of size exclusion after affinity chromatography on Histrap columns. Size exclusion is slow and inapplicable for large scale production of enzymes. We used the BBSRC Follow on Fund to achieve the following milestones.
1) We designed a set of heterocyclases that do not need the leader sequence in the substrate (Nat. Chem. Biol. 11, 558-563; Patent No. WO/2016/071422). The new heterocyclases are fused to the leader sequence and were engineered based on the crystal structure that identified the site of leader binding in the enzyme. The new enzymes allow processing of leaderless (short) substrates that can be made using solid phase synthesis at a scale that is very challenging to be matched by recombinant expression.
2) We developed a simple and fast method to purify the enzymes and exclude the requirement of the slow size exclusion chromatography. Purified enzymes were tested and proved functional and stable.
3) We developed an in-house fermentation protocol for every enzyme involved in the process based on the experience we gained from our industrial partner Chirotech. We obtained between 90-150 g/L cell mass compared to a max of 30g/L using cultures in shaking flasks.
4) We used RT-PCR and a thermoshift assay to determine the stability of each enzyme involved under different conditions (different salt concentrations, pH values, buffer composition and co-factors) and thus managed to identify the best recipe for a one pot reaction. This saves the time previously used to purify the individual reaction products.
5) We have developed a couple of methods for accurate quantitation of the final cyclic product using ICPMS and NMR. The former method requires the presence of sulfur in the product while the later can be used for quantification of cyclic peptides that don't contain sulfur containing residues.
6) We have developed two protocols for purification of the final products, one relies on the use of SPE and the other relies on the liquid-liquid partition with Butanol.
7) We have showed that we can use the modified technology to produce compounds at 1 mg scale and we have NMR data for these products.
8) We have expanded our enzymatic tool box to include two thiazoline oxidases and a tryptophan C-3 prenylase. This allows us to incorporate additional functionalities and explore a wider chemical space.
Exploitation Route Our findings will allow us and others to generate libraries of cyclic peptides at scale that allows further biological testing. These compounds are therapeutically promising but the difficulty in their synthesis impeded their development.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

Description We have modified our chemo-enzymatic technology to make azole-based cyclic peptides and make it more economic, faster, and scalable. In addition to meeting all the objectives of the FoF award, the modified technology has been successfully used to create two compound libraries for inhibition of the efflux membrane transporter, P-glycoprotein involved in multidrug resistance in cancer and the HIV integrase. The construction of focussed libraries to tackle these two targets was the main objective of the awarded Scottish Enterprise high-growth spinout programme that led to the formation of a spin out Ripptide Pharma.
First Year Of Impact 2015
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

Description IB Catalyst
Amount £1,600,000 (GBP)
Funding ID 48487-341231 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 09/2015 
End 08/2018
Description IBIOIC Exemplar
Amount £142,000 (GBP)
Funding ID NA 
Organisation Industrial Biotechnology Innovation Centre 
Sector Academic/University
Country United Kingdom
Start 08/2015 
End 07/2016
Description Sarcoma UK Project Grants
Amount £119,000 (GBP)
Organisation Sarcoma UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 12/2017
Description Scottis Enterprise High Growth Spinout Programme
Amount £455,000 (GBP)
Funding ID NA 
Organisation Scottish Enterprise 
Sector Public
Country United Kingdom
Start 04/2015 
End 06/2016
Description GyreOx's proprietary discovery platform creates unique and highly modified macrocyclic peptides called 'gyrocycles', which combine the target-engagement power of biologics with the cell-entry ability of small molecules.Treating complex diseases with solutions inspired by nature, GyreOx offers rapid production of designed libraries of gyrocycles to hit 'undruggable' targets. GyreOx has developed a pipeline to design and rapidly generate focused libraries of molecules that can hit previously undruggable targets. Our combination of computational design, automation and unique engineered enzymes allows us to deliver novel drugs in the chemical space beyond the 'rule of 5'. Focussed library design enables better compound-target interaction and the tuning of important drug properties. No other technology offers GyreOx's degree of flexibility in design and production. GyreOx has engineered nature to create designer molecules to treat complex human diseases with unmet medical need. We aim to bring new drugs to the clinic. We use a computational strategy to design 'gyrocycles', complex modified cyclic peptides against extended drug targets such as protein-protein interactions. An automated platform (MACRO), employing our engineered enzymes, creates libraries of gyrocycles with tuneable physicochemical properties. These enzymes carry out complex chemical transformations in minutes or hours compared to the days, or not at all, for equivalent synthetic chemistry processes. Delivery of designed focussed libraries is possible in weeks from the start of the process. Our base at the Research Complex at Harwell provides access to state-of-the-art facilities and equipment to deliver the GyreOx mission. Our technology is based on ground-breaking science carried out by Professor James Naismith (University of Oxford) and Professor Marcel Jaspars (University of Aberdeen). 
Year Established 2019 
Impact Secured ca £1.1 M investment
Company Name Ripptide Pharma 
Description Ripptide Pharma has developed a chemoenzymatic process for the efficient production of macrocycles and cyclic peptides, addressing entirely novel areas of chemical space for drug discovery and development. Macrocycles are new therapeutics in areas only currently accessible through biologics, such as protein-protein interactions. Unlike biologics they can be administered orally and access therapeutic targets within the cell. Our technology is available to partners to discover or optimise macrocycles in their areas of therapeutic interest Ripptide is advancing its in-house drug discovery/development activities in unmet areas of inflammation, autoimmune disease and cancer. 
Year Established 2015 
Impact None yet, we will in-license IP formally in July 2016