A new generation of microbial expression hosts and tools for the production of biotherapeutics and high-value enzymes

Project Information

Secreters

Grant agreement ID: 813979

DOI

10.3030/813979

Project closed

EC signature date 30 August 2018

Start date 1 January 2019

End date 30 June 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 4 087 420,92

EU contribution

€ 4 087 420,92

Coordinated by

ACADEMISCH ZIEKENHUIS GRONINGEN
Netherlands

Periodic Reporting for period 2 - Secreters (A new generation of microbial expression hosts and tools for the production of biotherapeutics and high-value enzymes)

Reporting period: 2021-01-01 to 2023-06-30

Summary of the context and overall objectives of the project

1.1 Problem being addressed

The production of proteins is of paramount importance within the biotechnology industry, with two
broad forms of protein recognized: therapeutic proteins and industrial enzymes. A high proportion of the target proteins are produced in microbial systems, where secretion is a favored strategy. However, current production platforms have severe limitations and cannot handle many secreted enzymes, in particular those carrying disulphide bonds. Hence there is an overwhelming need for new production systems to deliver these products in greater yields, with higher quality and at lower costs.

1.2 Importance for society

Microbial protein secretion systems are of great importance for society:
• Therapeutic proteins (biopharmaceuticals): Escherichia coli is used to produce over 30% of the therapeutic proteins approved to date, among which recombinant antibodies are the fastest growing group. Many of these proteins are difficult to produce and carry disulphide bonds.
• Industrial enzymes such as amylases, lipases and proteases, are usually produced in bacilli. These enzymes are used in detergent and food industries and play important roles in the daily life of many people. Efficient enzymes are also key to sustainable biotech-based alternatives to traditional chemical processes.
• The yeast Pichia pastoris is an upcoming emerging platform because of its high secretion capacity.

1.3 Overall objectives

The overall objective of Secreters research was to improve the sustainability and economic viability of industrial protein production in bacteria and P. pastoris and to enhance both product quality and quantity with particular focus on disulphide bonded proteins.

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

2.1 Scientific progress

All research in this project was carried out with three well-known microbial cell factories: the Gram-negative bacterium Escherichia coli, the Gram-positive bacterium Bacillus subtilis and the yeast Pichia pastoris. Importantly, the progress has been excellent. All scheduled Milestones and Deliverables have been reached.

Four interlinked Work Packages (WPs) addressed the key problems in this field:
WP1 aimed at the development of new strategies to express target proteins in the three selected host chassis, using a series of innovations to (i) produce such proteins in high amounts in the cytoplasm, (ii) export them to the periplasm or growth medium and (iii), secrete high amounts of biotherapeutics and enzymes in P. pastoris. WP2 served as the ‘strain clinic’ to improve the host strains by counteracting the stress and adaptive responses induced in WP1. WP3 was aimed as a “bridge” between WP1 and WP2, by integrating the acquired data with previously published data on stress responses and construct computational models that identify mitigation strategies. WP4 was aimed at benchmarking the new strains for industrial applications.

2.2 Training

The ESR training included:
• Training by research.
• Network-wide training courses.
• In-house transferable skills training.
• Secondments.
Although the training programme for Secreters has been affected by Covid 19-measures, effective mitigation strategies have been set up. All ESRs have followed the consortiums’ network-wide training events (physically, or on-line), where they have presented the plans and progress of their research.

2.3. Exploitation

Several E. coli, B. subtilis and P. pastoris strains have been constructed with improved protein secretion properties, in particular concerning proteins containing disulphide bonds. Several systems and technologies were developed which have a high potential for exploitation. In total, between eight and 10 project results have the potential for exploitation.

2.4. Dissemination

The ESRs in Secreters have written a highly valued review paper. Further, the project already resulted in 12 publications in peer-reviewed open access journals and 21 additional manuscripts are in preparation. About half of the publications from Secreters are joint publications between partner groups.
All ESRs have contributed to (inter)national scientific meetings where about 50 oral or poster presentations were given.

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)

3.1. Progress beyond the state of the art

• Major progress was achieved in understanding the principles of two major protein secretion pathways in E. coli and B. subtilis, namely the Sec- and Tat pathways and the general secretion pathway in P. pastoris. This has enabled the development of new production strategies and new strains, in particular for the production of proteins carrying disulphide bonds.
• An enduring cooperation between the scientific and the industrial groups in Secreters has been established.
• The project has put the industrial partners at the forefront of global competition, reinforcing European innovation potential in terms of growth and job creation.
• The project has trained a new generation of researchers, versed in Systems and Synthetic Biology approaches.
• All ESRs have been working successfully on their PhD theses.

3.2. Socio-economic impact

Secreters was designed to have maximum impact in terms of enhancing ESRs' career prospects, provision of innovative new protein production platforms, and stimulation of the European recombinant protein production sector.
The results from Secreters have resulted in several scientifically and commercially relevant findings with respect to protein production and the delivery of new production platforms. The industrial partners in Secreters have benefitted greatly from the interactions between them and with the academic partners.

3.3. Wider societal implications

A primary aim of Secreters was to train a next generation of scientists and leaders with an innovative and entrepreneurial mind-set. Considerable emphasis was, therefore, placed on educating the ESRs to be (i) scientifically excellent and (ii) well-prepared for business development. This has been achieved through original fundamental and applied research in an open multidisciplinary, intersectoral and international setting. As anticipated, the programme has indeed delivered a cohort of young researchers who understand secretion systems at all levels.

Altogether, the Secreters project has generated new concepts, technologies, and novel production strategies and strains that are of direct relevance to participant companies (ACIB, Lonza, AB Enzymes, Celltech, Boehringer). The chassis platforms established, together with the massive knowledge base generated, are likely to have far-reaching impacts on the European bio-based economy, opening up new horizons and opportunities for European innovation, growth and job creation.

ESRs at recruitment symposium Groningen, May 2019

ESRs team building activity, Vienna, October 2019

ESRs at final consortium meeting, Dubrovnik , July 2022

ESRs at consortium meeting, Catania, March 2022

Secreters consortium at kick-off meeting Vienna, October 2019

ESRs at consortium meeting, Berlin, October 2021