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ProteinFactory Report Summary

Project ID: 642836
Funded under: H2020-EU.1.3.1.

Periodic Reporting for period 1 - ProteinFactory (Engineering of new-generation protein secretion systems)

Reporting period: 2015-01-01 to 2016-12-31

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 three
broad forms of protein recognized: therapeutic proteins, industrial enzymes, and membrane proteins.
The global market for these proteins was worth €78 billion in 2010 and is forecast to exceed €130
billion by 2017.
A high proportion of the target proteins are produced in bacteria, where secretion out of the cytoplasm
is a favored strategy. However, current production platforms have severe limitations and cannot handle
many secreted enzymes and membrane proteins. There is an overwhelming need for new production
systems that can 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
product group. A single anti-cancer antibody, Herceptin, achieved sales of €4 billion in 2010 alone.
• Industrial enzymes such as amylases, lipases and proteases, are usually produced in bacilli. These
enzymes are, among others, used in detergent and food industries and, as such, they play an
important role in the daily life of many people. Efficient enzymes are also the key to sustainable
biotech-based alternatives to traditional chemical processes.
• Membrane proteins are of huge biomedical importance (potential drug targets) and they are heavily
exploited in the secretion of therapeutic proteins and industrial enzymes by bacteria.

1.3 Overall objective
Europe's current lead in the production of target proteins via bacterial protein secretion systems can
only be maintained using new protein secretion platforms that either bypass limitations with traditional
systems, or allow bulk protein production at minimal cost.
The overall objective of ProteinFactory is to address this major challenge by:
• Improving the sustainability and economic viability of industrial protein production in bacteria;
• Training a new generation researchers, versed in Systems/Synthetic Biology approaches.

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. The planned research activities are well on schedule and the progress has been
WP1 aims to achieve an understanding of the fundamental principles governing membrane protein
biogenesis and protein secretion. For this purpose, ESRs 2/15, 7 and 8 have teamed up to develop
improved predictive algorithms and good progress in this direction has been made. Initial computation
protocols for description/-annotation of interacting residues in TM regions and of membrane protein
complexes have been developed and a pipeline for fast and accurate prediction of membrane protein
structure has been developed.
ESRs 1, 5, 10 and 11 are working on the characterization of membrane protein complexes. New
proteins interacting with, or affected by, the Sec (ESRs5&10) and Tat (ESRs1&11) secretion
pathways have already been detected.
WP2 aims at understanding protein production stress and, to this end, ESR10 has set up the methods
and technologies for the quantitation of selected membrane proteins. This facility is available for the
entire ProteinFactory consortium. ESRs 1, 3, 4, 11, 12, 13, 14 and 16 have established the
experimental setups needed to define protein production stress in E. coli and B. subtilis. A new
inventory of candidate proteins affected by protein production stress in E. coli was made (ESRs3&11).
A novel assay for the proofreading activity of the Tat export system was set up, and specific effects of
charge and length of cargo proteins on their export were already detected (ESR4). New folding
catalysts for proteins secreted by B. subtilis were identified by ESR12 using a bioinformatics
approach, and they are currently being screened for function and specificity.
In WP3, ESRs 6 and 9 work on the development of chassis strain models for predicting secretion
bottlenecks and the balanced use of cellular resources. A computational model has been developed,
featuring a simplistic model of cellular growth, which is used as an extensible hub into which
pathways of interest can be added (ESR9). In addition, a model is being developed for the reallocation
of cellular resources during (high-level) protein secretion (ESR6). New protein interactions
within the Sec secretion pathway have been detected by ESR5.
In WP4 and WP5, ESRs 2/15, 3, 12, 13, 14 and 16 have established assays and experimental setups
towards the development and industrial exploitation of E. coli and Bacillus strains in which secretion
bottlenecks are being removed. A new inventory of candidate proteins affected by protein production
stress was made (ESR3), and ESR12 has established a new library of protein folding catalysts in B.
subtilis. ESR13 is engineering a set of E. coli strains for improved production of vaccines, proteinbased
drugs and membrane proteins. ESR14 is setting up methods for the secretion of “difficult”
proteins, using the Tat pathway in E. coli. ESR16 has made excellent progress in setting up a nanoliter-
reactor screening assay for quantification of proteins secreted by B. subtilis. Lastly, ESR2/15 has
made good progress in genome-wide analyses to identify optimal secretion signals in Bacillus.

2.2 Training. The training programme for ProteinFactory has been very effective. All ESRs have
participated in the four project meetings of the consortium in years 1 and 2, where they have presented
the plans and progress of their projects. All ESRs have followed the network-wide training events on
Bioinformatics + Ethics; Proteomics Summer School; and IPR, patenting + project management,
which have been given in year 2.
All ESRs, together with their supervisors, have prepared personal Career Development Plans and for
all ESRs a scheme was set up for secondments.

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)

• The results obtained so far in ProteinFactory are promising with respect to progress beyond the
state of the art. Several relevant new technologies and methods have been, or are being developed
and improved production systems for secreted proteins in E. coli and bacilli are likely to emerge.
This will be of considerable significance for European biotech industries using bacterial systems
for the production of secreted proteins.
• The progress in the project forms a good basis for increasing fundamental knowledge of protein
export systems in general and of bacterial secretion systems in particular. Several new components
interacting with, or affected by both the Sec- and the Tat-secretion pathways have already been
detected and their roles in secretion are currently under investigation.
• New and improved protein secretion systems open up the way for better and safer food production
systems. Moreover, such systems will contribute to the enhancement of the economic sustainability
of bacterial cell factories. This will result in a reduction of the use of fossil energy resources.
• ProteinFactory includes an intensive training programme for the appointed PhD students. This is
expected to deliver a cohort of young scientists well trained in scientific and entrepreneurial aspects
of (bacterial) cell factories.
• ProteinFactory has initiated various outreach activities for the general public, including a public

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