Exploitation of Tat export machinery for protein production by bacteria

Lead Research Organisation: University College London
Department Name: Biochemical Engineering

Abstract

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.

Technical Summary

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.
 
Description Ultra scale-down analysis of strains over-expressing the Tat system and heterologous protein substrate.
The key strains developed capable of high-level protein export of 'standard proteins (defined here as those that do not contain disulphide bonds) have been studied in detail using a range of USD tools at UCL. These studies involved extensive analysis of the cells' propensity for lysis using a range of USD tools developed at UCL, and the data were carefully analysed in terms of implications for the early stages of downstream processing including the centrifugation and periplasmic extraction phases. The net result is that these strains are highly robust in overall terms, even when over-expressing both the tatABC operon and the target protein of interest. These data have been published in early 2012

the CyDisCo system developed by Ruddock can be modified to export prefolded, disulphide-containing proteins by the Tat system, and the strains can be used in fermentation systems. The cells reach OD values of up to 150 and are as intact as wild type cells; they are thus suitable for industrial applicationthe CyDisCo system developed by Ruddock can be modified to export prefolded, disulphide-containing proteins by the Tat system, and the strains can be used in fermentation systems. The cells reach OD values of up to 150 and are as intact as wild type cells; they are thus suitable for industrial application


we have developed a new strain comprising an E. coli tat null mutant expressing a B. subtilis Tat system. The strain is able to export a heterologous protein to levels of 0.5 g/l culture and 90% of the protein is released into the culture medium. This strain offers unique possibilities form simple recovery of proteins without the need for periplasmic extraction.
Exploitation Route We have demosstrated an easy and simpler way to manufacture high value proteins using a rang eof changes made tot eh cell in teh study . teh outcome was validated under pilot scale envirnment. they can be used by biopharmceutical and industrial biotechnology companies .
Academia can use teh techniques for further development of comaptible strains.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Dr Cristina Matos (Warwick). Destination: University of Oulu and then University of Kent Dr Steven Branston (left slightly early in November 2011 for position with health market research firm IMS Health .now employded by AmpliPhi Biosciences ). Participation at the Biotechnology YES competition: Dr. Branston and Dr. Matos formed a BRIC team to compete in this year's Biotechnology YES competition, together with other 2 other BRIC PDRAs (Flavius C. Pascut and Malkey Verma). Dr Arjun Dhanoya (appointed January 2012 to complete final tasks. currently working for )
Sector Education,Manufacturing, including Industrial Biotechology
Impact Types Societal,Economic

 
Description Strategy for Emerging Biotechnologies
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact Important regarding development of new strategies for emerging technologies
URL http://www.nuffieldbioethics.org
 
Description industry
Amount £100,000 (GBP)
Organisation PT Bio Farma 
Start 03/2018 
End 03/2021
 
Description COLLABORATION WITH INDSUTTRY 
Organisation Fujifilm
Department Fujifilm Diosynth Biotechnologies
Country United States 
Sector Private 
PI Contribution research testing and demonstration of processing for the production of given molecules
Collaborator Contribution provision of industrially relevant molecules
Impact Successful completion of a PhD leading to the graduate obtaining a position in a related industry.
Start Year 2014
 
Description Provision of industrially relevant molecules 
Organisation Cobra Biologics
Country United Kingdom 
Sector Private 
PI Contribution Research testing and demonstration of process options
Collaborator Contribution Provision of industrially relevant molecules
Impact application in manufacturing
Start Year 2014