Thoroughly Optimised Production Chassis for Advanced Pharmaceutical Ingredients

Project Information

TOPCAPI

Grant agreement ID: 720793

DOI

10.3030/720793

Project closed

EC signature date 9 November 2016

Start date 1 January 2017

End date 30 September 2021

Funded under

• INDUSTRIAL LEADERSHIP - Leadership in enabling and industrial technologies – Biotechnology

Total cost

€ 5 063 361,25

EU contribution

€ 5 063 361,25

Coordinated by

THE UNIVERSITY OF MANCHESTER
United Kingdom

Periodic Reporting for period 3 - TOPCAPI (Thoroughly Optimised Production Chassis for Advanced Pharmaceutical Ingredients)

Reporting period: 2020-01-01 to 2021-09-30

Summary of the context and overall objectives of the project

Chemical products from natural sources, i.e. natural products, are an extremely diverse set of compounds with a seemingly infinite variety of chemical structures and bioactivities. Many of these compounds are very successful pharmaceuticals, and the discovery of novel natural products is obviously of great importance. Infectious diseases are believed to be the second leading cause of death worldwide, with antibiotic-resistant infections affecting 4.1 million patients each year, leading to 147,000 deaths globally.

In TOPCAPI, we addressed the problems of:
1) the lack of an industry-strength actinomycete production host for bioactive compounds;
2) the lack of a generic host system for the identification and heterologous production of new antimicrobials/natural products;
3) the need for greener production of bioactive compounds and compounds that are effective to antimicrobial resistant infections.

TOPCAPI exploited the natural fabrication power of actinomycetes as microbial cell factories and aimed to produce two high-value compounds: GE2270, a starter compound for the semi-synthesis of NAI-Acne, a new topical anti-acne drug in Phase II clinical trials, and tetracycline derivatives, intermediates for semi-synthetic conversion to medically important type II polyketide tetracyclines (TC), some of which have been used for decades and new derivatives at present are in advanced clinical trials, to be used against methicillin-resistant Staphylococcus aureus infections.

The objectives of the TOPCAPI project were to apply an integrative systems and synthetic biology approach to a range of actinomycete species to create high-production chassis for a number of important antibiotics.

We characterised the chassis, applying integrated data analysis to transcriptomics and metabolomics experiments, combined with predictive mathematical modelling to drive the rapid improvement of these microbial cell factories for industrial drug production using advanced metabolic and biosynthetic engineering. At the same time, we sought an expanded toolbox for the engineering of actinomycete bacteria as cell factories for other high added-value compounds.

Conclusion:
The TOPCAPI project successfully achieved its objectives and was able to exploit the strength of the integration of systems and synthetic biology approaches for the improvement of antibiotic production chassis.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Major achievements:
• Sequenced and analysed genome of four Streptomyces rimosus strains, including release of WT genome and preparation of a joint publication
• Performed quantitative metabolic flux estimation for several wild type and engineered strains and used these to further constrain metabolic models
• Determined structures of new compounds by HRMS, 1D and 2D NMR.
• Created and improved several generations of genome-scale metabolic models in a range of actinomycete species
• Performed metabolomics data/flux analysis and integration for several strains and improved correlation between predicted flux and experimental data
• Identified knockout and/or overexpression gene targets by transcriptomic-driven analysis to inform the creation of improved production strains
• Optimised CRISPR-Cas9 method and integrated cosmid excision in S. coelicolor
• Developed innovative tools for chassis genetic manipulation and expression
• Validated and quantitatively analysed several improved production strains in various actinomycete species
• Performed pilot-scale fermentation and production analysis of improved strains
• Developed and exploited innovative DNA design software, the MoCLO and Gibson wizards
• Established data analysis and dissemination using the new on-line bioinformatics tool MORF
• Published a major multi-omics study of Planobispora rosea
• Co-hosted an international webinar series “Microbial Cell Factories 2021 (MCF2021)” in March and April 2021, together with another H2020 project (CHASSY http://chassy.eu/).

Overview:
We successfully validated and analysed strains for the production of our two target compounds: GE2270 and a tetracycline derivative. GE2270 production was improved more than 4-fold, compared to the initial heterologous production strain, using an iterative process of multi-omics, metabolite modelling and engineering of the host, and this result was successfully reproduced in pilot-scale fermentation. We successfully used the principles of synthetic biology to generalise and apply the insights obtained during the engineering for establishing a novel production chassis.
Of the two tetracycline derivatives, one was promising in the earlier stages and, therefore, became the focus for strain development. After iterative cycles of multi-omics, metabolite modelling and engineering of the host, production levels for substantially improved. This result was successfully reproduced at larger-scale production.

We disseminated our activities and results to non-academic groups though our website and Twitter, by participation in community events (e.g. Festival of Ideas in York, European Researchers Nights, collaboration with The Mikro Collective), and media briefings contributing to local and regional newspapers.

We engaged with the business sector though commercially oriented events/fairs and individual B2B discussions. We took advantage of virtual platforms and meetings in order to keep communicating with potential business partners during Covid pandemic. The three SME partners included the developments of TOPCAPI in their regular B2B communication and held focused discussions about opportunities arising from TOPCAPI chassis strains and technologies.
Communication with the business sector will continue in the post-project period, based on the desire of TOPCAPI partners to continue the development work towards commercial partnerships and exploitation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

One important target of TOPCAPI were the tetracycline (TC) derivatives. TCs are the fifth most prescribed class of antibiotics. The growing medical problem of drug-resistant bacteria represents an expanding market opportunity. On the other hand, in recent years the cost of antibiotics is rapidly growing. For example, the cost of a 250mg tablet of TC soared by more than 70-fold in just two years. Therefore, competitiveness of the EU-based pharma industry will in coming years greatly depend on the efficacy of fermentation and semi-synthetic processes for the production of these important drugs as addressed in TOPCAPI.

The global market for acne products is around $2B/year. The global market is expected to steadily increase due to lifestyle changes in developing nations. Importantly, none of the currently used antibiotics is particularly effective against acne. In contrast, NAI-Acne, the second focus drug of TOPCAPI, is highly selective against Cutibacterium acnes, the etiological agent of acne, and might provide improved efficacy over comparator drugs (e.g. clindamycin, erythromycin).

TOPCAPI has been able to progress beyond the state of art in antibiotic production by utilising systems and synthetic biology approaches and integrating the multi-omics data with bioinformatics analysis and modelling and chassis engineering. The tools developed for the engineering of the target production chassis will be of wider utility for the enhanced production of valuable anti-infectives and other high-value compounds, and the computational tools of the TOPCAPI pipeline are already used by the wider academic and biotechnological community.

TOPCAPI 4th Annual Meeting Sept2021

TOPCAPI Partners 2nd Annual Meeting