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Engineering of new-generation protein secretion systems

Periodic Reporting for period 2 - ProteinFactory (Engineering of new-generation protein secretion systems)

Reporting period: 2017-01-01 to 2018-12-31

1.1 Problem addressed
The production of proteins is of paramount importance within the biotechnology industry, with three broad forms of protein recognized: therapeutic proteins, industrial enzymes, and membrane proteins. Many target proteins are produced in bacteria, where secretion is a favored strategy. However, current production platforms have severe limitations and cannot handle many secreted enzymes and membrane proteins. 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
Bacterial 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.
• 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.
• Membrane proteins are of huge biomedical importance as potential drug targets.

1.3. Overall objectives
The overall objective of ProteinFactory research was to improve the sustainability and economic viability of industrial protein production in bacteria and to enhance both product quality and quantity.
2. ESR training
The ESR training included:
• Training by research.
• Network-wide training courses.
• In-house transferable skills training.
• Secondments.
The training programme for ProteinFactory has been completed. All ESRs have followed the consortium’s network-wide training events, where they have presented the plans and progress of their research.

3. Scientific progress
All research in this project was carried out with two well-known bacterial cell factories: the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. Importantly, all scheduled Milestones and Deliverables have been reached.
3.1 Progress per WP
Five interlinked Work Packages (WPs) addressed the key problems in this field:
• WP1 aimed at a fundamental understanding of basic principles of membrane protein insertion. New molecular interactions relevant for membrane protein biogenesis were identified and algorithms were developed to predict interacting amino acid residues and membrane protein structures.
• WP2 aimed to deliver a time-resolved proteomic analysis of chassis strains' responses to protein production stress. Strategies to counteract or avoid detrimental effects of secretion stress were developed along with a high-throughput screening technology for strain improvement.
• WP3 aimed to integrate information from WP1 and WP2 in models that accurately predict cellular responses to overproduction stress. Several predictive models for the general protein secretion pathway and the utilization of resources by protein-producing bacteria were developed.
• WP4 used the acquired knowledge to generate industrially-useable E. coli strains for secretion of therapeutic proteins and membrane proteins. Indeed, the work has delivered several E. coli strains with improved properties in the production of membrane and secretory proteins.
• WP5 was aimed to generate industrially-useable B. subtilis strains for enzyme secretion and, indeed, the work has delivered strains and strategies for improved secreted protein production.

4. Exploitation
Several E. coli and B. subtilis strains have been constructed with improved protein secretion properties and several systems and technologies were developed which have a high potential for exploitation. In total, eight project results have the potential for exploitation.

5. Dissemination
The project already resulted in 6 publications in peer-reviewed open access journals and five additional manuscripts have been submitted. Approximately 30 further publications will appear in the next few years. About half of the publications from ProteinFactory are joint publications between partner groups.
All ESRs have contributed to (inter)national scientific meetings where about 50 oral or poster presentations were given. A particularly high exposure of ProteinFactory was realized through the organization of the “International Conference on Recombinant Protein Production’ (RPP9), organized in Dubrovnik, Croatia, from 22 to 25 April, 2017 ( At the end of the meeting, the ESRs of ProteinFactory were called to the stage to receive credits for their hard work and dedication to make RPP9 a great success.
A joint networking event was organized in conjunction with the RPP9 Conference with two other doctoral training programmes: the EU-funded eCHO ITN and the Vienna-based BioToP at BOKU.
Likewise, a joint networking event was organized on 24 April 2019 with the BioToP programe in conjuction with the RPP10 Conference on Crete, Greece.
• 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. This has enabled the development of new production strategies and new strains.
• An enduring cooperation between the scientific and the industrial groups in ProteinFactory has been established.
• The project has put the industrial and SME 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. The first two PhD thesis were successfully defended in September 2018 and April 2019, and 12 additional PhD theses will be completed in 2019.

ProteinFactory 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 ProteinFactory 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 ProteinFactory have benefitted greatly from the interactions between them and with the academic partners.
A primary aim of ProteinFactory 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 ProteinFactory project has generated new concepts, technologies, and novel production strategies and strains that are of direct relevance to participant companies (Novozymes, DSM, Abera, UCB and FGen). 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.
ESR10 at Proteomics facility Greifswald
ESRs called to stage RPP9, Dubrovnik
Registration desk at RPP9, Dubrovnik
ProteinFactory Consortium
ESR5 presenting her poster
Cover page of website
ProteinFactory consortium at final meeting, Amsterdam
ProteinFacotory consortium at Mid-term review meeting
ESRs 10 & 12 at work
ESR Presentations