Abstract

Bacteria export numerous proteins across the plasma membrane and this process has been heavily exploited for protein production on an industrial scale. However, the standard general secretory pathway' transports proteins in an unfolded state and is inapplicable for the export of the majority of proteins that have a tendency to fold prematurely. Recent studies, primarily by this partnership, have revealed the operation of a novel bacterial protein export system that has the remarkable ability to export large, fully-folded proteins, synthesised with signal peptides bearing twin-arginine motifs ('RR-signal peptides'). This application is aimed at the exploitation of this twin-arginine translocation (Tat) system to export active, high value-added proteins from Escherichia coli and Bacillus subtilis. We propose to engineer RR-signal peptides capable of directing highly specific and efficient export in these organisms, and to produce super-exporting strains capable of producing heterologous proteins on a commercial scale.

Objectives

The objective of this project is the provision of bacterial cell factories capable of the highly efficient production of an unprecedented range of high value-added proteins via export into the E. coli periplasmic space or the B. subtilis culture medium. Protein export/secretion from these organisms is a major industrial production tool with a market of over $1 billion p.a. However, standard export technologies are totally inapplicable for the production of a large number of proteins because folding problems preclude transport in an obligatorily unfolded state by the well-characterised general secretory pathway. The recently discovered twin-arginine trans-location (Tat) pathway offers enormous potential because of its ability to transport fully folded proteins. The proposed research is aimed at a full exploitation of this potential through the identification of all relevant components of the pathway, the engineering of strains capable of high export rates and the provision of highly specific RR-signal peptides capable of the efficient export of an array of heterologous proteins for the first time.

Description of the work

The project aims to provide a powerful and cost-effective new tool for the EU biotechnology industry through the development of technologies and expertise required for the exploitation of the Tat pathway for protein production.

Workpackage 1 involves the development of analytical techniques whereby the properties and potential of the Tat pathway can be rapidly and efficiently assessed in cell-free assays.
Workpackage 2 will identify and engineer RR-signal peptides that direct efficient export through this pathway in E. coli and B. subtilis while exhibiting zero or minimal interference with other cellular processes.
Workpackage 3 will identify all of the major components of the pathway and any potential export bottlenecks, while
Workpackage 4 will systematically explore the applicability of the pathway by testing its ability to export a range of proteins that cannot be exported by traditional strategies.
Workpackage 5 will use the accumulated knowledge to construct vectors containing optimised RR-signal peptides and engineered strains expressing all relevant Tat components and ancillary folding factors at levels required to support the export of heterologous proteins at very high flux rates.
Workpackage 6 will test the full potential of the system in fermentation analysis and develop production strategies accordingly.

Ultimate aim: the provision of a bioprocess that meets a major market need and which addresses the criteria specified in the work programme of QLK3, The Cell Factory.

Deliverables

The major project deliverables are:

• The development of analytical tools to assay Tat-dependent protein transport.
• The identification and optimisation of RR-signal peptides.
• The determination of the subunit structure of the Tat export system.
• Understanding of the roles of folding catalysts.
• The construction of secretion vectors and engineered super-exporting strains of E. coli and B. subtilis;
• The development of fermentation strategies for full commercial production of heterologous proteins.

Contacts

Coordinator

• Jan Maarten VAN DIJL / University of Groningen Dept. Pharmaceutical Biology / NETHERLANDS

Participant

• Long-Fei WU / CNRS, IBSM Laboratory of Bacterial Chemistry UPR9043 / FRANCE
• Joop van der LAAN / Genencor International B.V. Microbial and Molecular Screening / NETHERLANDS
• Tracy PALMER / John Innes Centre Department of Molecular Microbiology / UNITED KINGDOM
• Ben C. BERKS / University of East Anglia Centre for Metalloprotein Spectroscopy and Biology / UNITED KINGDOM
• Matthias MÜLLER / University of Freiburg Institute of Biochemistry and Molecular Biology / GERMANY
• Sierd BRON / University of Groningen Department of Genetics / NETHERLANDS
• University of Warwick Department of Biological Sciences / UNITED KINGDOM