Exploitation of Tat export machinery for protein production by bacteria

Numerous therapeutic proteins (biopharmaceuticals) are currently produced in bacteria by the biotechnology industry, and this form of production has been in constant use for over 20 years. The Gram-negative bacterium Escherichia coli is the usual host organism for these processes. In some cases the proteins are made in the interior cytoplasm of the bacteria and then extracted, but a favoured approach is to ensure that the proteins of interest are exported out of the cytoplasm into the periplasmic space between the two membranes surrounding the E. coli cell. Once here, the proteins can be purified with relative ease by selectively rupturing the outer membrane. This process has almost always involved exploiting the so-called secretory pathway for protein export, in which the substrate protein is 'threaded' through a pore in the inner membrane in an unfolded state. However, a major problem is that many foreign proteins simply cannot be transported in this manner - they fold too rapidly or tightly before they can be transported. We and others have recently shown that a second protein export system operates in the bacterial inner membrane: the twin-arginine translocation (Tat) system. This system exports proteins that bear transient peptide signals and the Principal Applicant's group has shown that foreign proteins can be efficiently exported by this system. Crucially, the system exports pre-folded proteins. The Tat system thus has huge potential as a platform for the bacterial production of the many recombinant proteins that cannot be exported by traditional means because of folding problems. The main aim of this project is to systematically engineer and analyse E. coli strains that export proteins via the Tat pathway at the high rates demanded by industry. In a concerted effort, the Warwick group will carry out strain improvement and the UCL group will rigorously test the fitness of these strains for industrial use; this is viewed as vital because industrial fermentation systems demand the use of strains that are not prone to lysis or other stress damage. Key elements of the proposal are to (i) identify the most efficient Tat-specific targeting signals, (ii) test the ability of the Tat system to export a range of foreign proteins, (iii) increase Tat-dependent export capacity by over-expressing tat genes and manipulating the levels of key chaperones and proteases, and (iv) develop methods to assess the fitness of engineered strains through ultra-scale-down methods that accurately mimic industrial process conditions. The final section of the E. coli work will be the generation of strains that exhibit an optimal balance of increased export flux and high cell integrity, by identifying the ideal combination of engineered characteristics. These strains will be suitable for use in industrial production systems. The second overall aim of project is to test the feasibility of exploiting Tat-dependent export for recombinant protein production in Gram-positive bacteria. These organisms are not extensively used for production of bipharmaceuticals, but are heavily used for the production of industrial enzymes. Once again, the protein products are ideally exported out of the cell to separate them from the cytoplasmic compartment, but the absence of an outer membrane means that the products are secreted into the growth medium and then purified (there is no periplasmic compartment). The Tat system has real longer-term potential for the production of many recombinant proteins in Gram-positive hosts, and we propose to carry out a feasibility to study to directly assess its potential in the Gram-positive species Bacillus subtilis.

The proposed research will generate a new platform for the production of recombinant biopharmaceuticals in bacteria. Many recombinant proteins are currently produced in bacteria, especially Escherichia coli, and a favoured approach is to target ('export') the protein of interest into the periplasmic space by the secretory (Sec) pathway. We propose to develop an entirely novel platform that exploits the unique abilities of the more recently-discovered Tat export pathway. This system exports fully folded proteins to the periplasm, thereby bypassing major technical problems associated with the Sec system's need to transport unfolded proteins. The project has been designed as follows: Phase 1 will involve a detailed assessment of E. coli strains that already export proteins at moderate rates via the Tat system, together with an initial step-wise enhancement of export rates. The aims are to understand the physiological consequences of tat gene overexpression, and to enhance the export capacity of the cells through a combination of efficient Tat signal peptide and overexpression of tat genes and substrates. Phase 2 will further improve export capacities through a systematic overexpression of cytoplasmic chaperones (essential for substrate folding), reduction of periplasmic proteases and manipulation of redox levels to support export of disulfide-bonded proteins. In parallel, each stepwise improvement will be assessed under industrial fermentation conditions to provide continual feedback of the effects on key physiological / process parameters. Phase 3 will generate super-secreting strains using a combination of engineered characteristics identified in Phases 1 and 2, with the ultimate aim of achieving an optimised balance of high export flux vs minimised downstream processing complications. In this Phase we will also carry out a feasibility study in which we explore the potential of Tat-dependent export in the Gram-positive organism Bacillus subtilis.