Enhanced productivity and functionality of Modified Ribosomally Produced Peptides (M-RIPPs)
Lead Research Organisation:
University of Aberdeen
Department Name: Chemistry
Abstract
Ribosomally Produced Peptides (RIPPs) are widely recognised as one of the most promising classes of compounds with the potential to treat many diseases including infection, cancer & inflammation. They are of great interest to the pharma industry, but are extremely costly to produce/modify - even in milligram amounts. Through the utilisation of cutting-edge techniques in combinatorial synthetic biology, this project sets out to achieve a world first; namely, to produce bespoke libraries of Modified RIPPs (M-RIPPs) in vivo and in sufficient quantities to permit drug discovery screening. The project combines the fundamental knowledge of the natural processes involved in RIPP biosynthesis of the two premier UK academic groups active in the field with the applied expertise in industrial biosynthesis of a leading UK IB company. It will deliver a versatile yet robust technology platform for the production of M-RIPPs in vivo that will be transferred to a spinoff company to be formed around 18 months after project start.
Technical Summary
Ribosomally Produced Peptides (RIPPs) as a biosynthetic class contains many different families such as the cyanobactins, lanthipeptides, proteusins and lasso peptides amongst others. They have a range of biological activities and have a common biosynthesis in which a the core peptide, a small sequences within a larger precursor peptide is modified by tailoring enzymes. The modified core peptide is freed from the leader and additional signal sequences and often undergoes further modification (macrocycle formation, heterocycle oxidation, O/N prenylation) to produce the final modified peptide. The common RIPP biosynthetic pathway indicates that it should be possible to apply multiple types of chemical tailoring from different RIPP families to core peptides, thus generating hybrid molecules with features from multiple RIPPs. This project aims to generate such modified RIPPs (M-RIPPS) in vivo and overcome several barriers to their scaleable production. We will incorporate modifications common in cyanobactins (heterocycles, macrocycles, O/N prenylation), lanthipeptides (lanthionine and labionin bridges) and lass peptides. We will use this methodology to generate libraries of unique compounds with novel bioactivities. To assist with M-RIPPS that may not be producible using standard expression systems, we will also use one alternative expression system. We will scale up production to 1-3 L scale and improve downstream processing using a variety of methods, including the incorporation of a cyclic peptide exporter in the producing cells and cultivating them in a biphasic system to allow easy compound extraction and subsequent purification. The final step is technical marketing in consultation with big Pharma with a view to establishing a spinoff company based on this technology.
Planned Impact
As described in proposal submitted to IUK
Publications
Alexandru-Crivac CN
(2017)
Cyclic peptide production using a macrocyclase with enhanced substrate promiscuity and relaxed recognition determinants.
in Chemical communications (Cambridge, England)
Dalponte L
(2018)
N-Prenylation of Tryptophan by an Aromatic Prenyltransferase from the Cyanobactin Biosynthetic Pathway
in Biochemistry
| Description | The aim of this project was to show that genes that direct the synthesis of cyclic peptides of high biomedical relevance could be transferred from their poorly culturable and non-manipulable native hosts to prepare cyclic peptides in a recombinant microbe suitable for future scalable manufacture. To date, this had never been successful, due to the difficulties of expressing sufficiently active enzymes to allow the two peptide cyclisation events (hetero- and macro-cyclization) to occur. This was fully achieved following multiple iterations to overcome natural limitations of the system and employ novel enzymes development during the collaboration. Natural peptides of both the patellamide and lantibiotic classes were individually expressed, modified post-translationally (heterocylised and macrocylised or lanthionine bridged respectively) within the recombinant host. Practice to date has required addition of the purified macrocyclase enzyme to the heterocyclised patellamide peptide intermediate that had been isolated from bacteria. This step adds cost and complexity. By testing a range of macrocyclase enzymes and diverse operational conditions, we have succeeded in allowing the entire cyclisation process to occur in vivo, removing the requirement for the additional costly step to enable macrocyclization. Building upon this success, we combined the individual enzymatic steps of the two biosynthetic routes to demonstrate the ability to macrocyclise peptides that would not naturally undergo this step in nature. The further engineered recombinant host carried out in vivo synthesis of a completely novel cyclic peptide structure that comprises macrocyclisation of a lanthipeptide core sequence. Such novel cyclic peptides may embody desirable pharmacological characteristics, such as increased stability, often observed with cyclised peptides and crucially, they offer the possibility to explore and further diversify completely novel structures that show potential efficacy as pharmaceutical leads. In addition to these key biosynthetic successes, many enabling technologies were advanced during this project including the isolation and structural/mechanistic characterisation of relevant biosynthetic enzymes, generations of genetic fragment libraries that can be readily reconfigured to concertedly express relevant genes of interest and the use of heterologous leader sequences to direct enzyme activities to peptide targets of interest. |
| Exploitation Route | The main barrier to exploitation is the support of pharmaceutical company end-users looking to commercialise cyclic peptides as therapeutic molecules. To date, difficulties in scalable production have been a significant limitation of commercial interests in cyclic peptides as pharmaceutical targets. Our demonstration of the adaptable and scalable in vivo production method for cyclic peptides will certainly help to change this attitude for the better. However, further funding will be required to advance the project outputs to a level of de-risking necessary to encourage aggressive investment from pharmaceutical companies. This barrier can be addressed by further work to demonstrate i) increased productivity of the recombinant hosts constructed in this work, ii) synthesis of many further novel targets by straightforward changes to the primary amino acid sequence of the target classes as well as substitution of the modifying enzymes, iii) synergy between cyclic peptide biosynthesis and direct screening of activity and efficacy, a key driver of pharmaceutical company interest as noted above. Ingenza has sought further funding support to this end, reaching the panel interview stage with an SBRI proposal to develop cyclic peptides to address antimicrobial resistance in pathogenic fungi. Unfortunately the proposal was not ultimately funded. We will continue to pursue this objective and to build the necessary relationships with the pharma majors. In addition to the exploitation partner there will be a need for appropriate academic partners with ongoing knowledge of the discovery and development of the relevant biosynthetic enzymes as well as expertise in the appropriate assays of efficacy and safety in the areas of application. |
| Sectors | Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | We are able to offer a new service to use scalable fermentation based manufacturing methods to produce chemical compounds of high pharmaceutical interest for oncology and antimicrobial applications. Such processes could also be out-licensed to end-users in the pharmaceutical industry. The project has strengthened the relationship between all collaborators. Ingenza and the researchers at the Universities of Aberdeen and St. Andrews (the latter now at Oxford) have worked together on several projects related to cyclic peptides (one was previously funded by IUK). Together, we are continuing to investigate appropriate avenues of funding to continue developing means to produce and fully exploit the recognised therapeutic potential of cyclic peptides. Employees at Ingenza have benefitted from the training and expertise available through working on this project. One member of the project team has been promoted to a higher position within Ingenza, due largely to her contribution to the project. Relationships were developed with academic staff highly skilled in areas of biochemistry and structural biology not available at Ingenza and the project provided opportunities to engage with big pharma that could well lead to strong future relationships and exploitation of this or related synthetic biology approaches. Big pharma is not going to be able to develop therapies for a whole range of intractable conditions without novel structures that are active in relevant biochemical space. If we are able to achieve this with RIPPs the payback will be enormous. |
| First Year Of Impact | 2018 |
| Sector | Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |
| Description | Collaborative research with Ingenza Ltd |
| Organisation | Ingenza Ltd |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | It was joint research as summarised in the results and impact sections. |
| Collaborator Contribution | See results and impact sections |
| Impact | TSB and IBCatalyst funding (this project) |
| Start Year | 2012 |