Enhanced productivity and functionality of Modified Ribosomally Produced Peptides (M-RIPPs)

Lead Research Organisation: University of Aberdeen
Department Name: Chemistry


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


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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
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