Orthogonal riboswitches as tools for controlling gene expression in bacteria

Gene expression is the process by which DNA sequence information is first transcribed in to messenger RNA (mRNA), and then translated to produce proteins. The expression of genes is tightly controlled (regulated) by a number of mechanisms, which affect the timing and levels by which the gene products (RNAs and proteins) are synthesised in response to environmental conditions and other signals. For example, it was recently discovered that certain metabolites in cells can turn gene expression on or off by triggering switches present within mRNA. These so called riboswitches are found in all domains of life, and are particularly wide spread in bacteria. Typically these switches regulate the production, degradation or transport of specific metabolites. For example the add A-riboswitch binds adenine to activate translation of the mRNA that encodes the enzyme adenine deaminase, which degrades adenine. Through this feedback mechanism the bacteria can control the cellular levels of adenine a key building block in DNA synthesis. Recently we succeeded in re-engineering (or rewiring) add A-riboswitches, so that they are no longer triggered by the natural metabolites present in the cell, but instead can be controlled by the addition of various synthetic molecules (ligands). In this project we aim to develop new orthogonal riboswitches through further genetic manipulation and by developing new and more effective synthetic ligands which will allow more precise and dynamic control of gene expression in bacteria. In addition to riboswitches that can activate gene expression on binding synthetic ligands, we will also apply our strategy to re-engineer riboswitches which can block gene expression in response to selected synthetic molecules. We will also couple multiple switches together, in a tandem arrangement, to enable a more digital control of gene expression. For example we propose to develop a genetic switch which can be turned on with one ligand, activating gene expression, before being switched off with a second distinct ligand. In addition we will also show how the mutually orthogonal riboswitches can be employed to affect the simultaneous and differential control of multiple genes in bacteria. The new genetic switches we develop could be used to study the function of genes in bacteria. In the case of pathogenic bacteria, which cause disease, expression tools based on orthogonal riboswitches could be used to validate new targets for antibiotics that could lead to the development of new antimicrobial treatments. The riboswitch expression tools could also be used to aid production of proteins in bacteria, which could include therapeutically important proteins (biopharmaceuticals) or industrial important enzymes (biocatalysts). In addition, orthogonal riboswitches could be used to control multiple genes which encode metabolic pathways leading to natural product based drugs, biofuels and other commercially important products which are emerging as targets for synthetic biology. During this project we will be working to develop orthogonal riboswitches with the required properties that will allow us to demonstrate their utility in these important applications.

Riboswitches are regulatory elements found within mRNA, across all domains of life, which modulate gene expression on binding specific metabolites by relatively simple protein-independent mechanisms. We recently developed an approach for re-engineering orthogonal riboswitches that no longer respond to the natural cellular metabolites, but instead can be controlled by synthetic ligands, with desirable physicochemical properties, that readily enter the cell. In this proposal, we aim to engineer a range of new orthogonal riboswitches which can induce as well as repress gene expression, in a precise dose-dependent response to low concentrations of synthetic small molecules, allowing access to a wide dynamic range of expression levels. Tandem arrangements of orthogonal riboswitches will also be employed enabling more digital control of gene expression in response to two distinct ligands. We will also demonstrate how orthogonal riboswitches can be used to control the differential and simultaneous expression of multiple genes, including synthetic operons. Given that riboswitches are wide-spread in nature and operate by simple protein-free mechanisms, they are potentially more transferable across a wider range of bacteria than existing expression systems (e.g. lac, ara & tet). As such they could offer many advantages as tools for gene functional analysis, establishing conditional mutants and other important applications. For example we will demonstrate how orthogonal riboswitches can be used for protein production, metabolic engineering and in synthetic biology. In addition to E. coli, we will also show how the orthogonal riboswitches can be utilised in other bacteria including industrially important Streptomyces sp. and clinically relevant enterococci pathogens where there are currently few reliable inducible expression systems available.

WHO WILL BENEFIT: In addition to the academic beneficiaries, scientists in pharmaceutical and biotech companies who are studying pathogenic bacteria could use orthogonal riboswitches to generate conditional mutants to screen for new antibiotics targeted at specific essential gene products. Pharmaceutical and biotech companies could also use riboswitch based expression systems to produce biopharmaceuticals including antibodies in E. coli or other bacterial hosts. Many chemical companies that employ biocatalysts such as DSM, Lonza, BASF, Dr. Reddy's could use new expression tools for the production of enzymes. Several biotech and pharmaceutical companies (e.g. Biotica, Bristol-Myers Squibb, Novartis, Cubist) are engineering bacteria to produce antibiotics and other therapeutically important molecules. Similarly, oil companies such as Shell and BP have invested heavily in synthetic biology programmes to engineer bacteria to produce new biofuels, where new expression tools would be equally important. Finally there are many companies such as Invitrogen, Qiagen, Promega, Stratagene, EMD Bioscience, Thermo Fisher, Bio-Rad, GE healthcare and Sigma-Adrich who sell commercial expression systems for use in bacteria. Any number of these companies could benefit through licensing agreements to use new systems based on orthogonal riboswitches. HOW WILL THEY BENEFIT: We will actively seek to communicate our findings to the wider community through scientific meetings and scholarly publications (We consistently publish in top journals JACS, PNAS, Angew. Chem. & Nature Chem. Biol.). However, in order for the technology we develop to become widely adopted, particularly in industry, it will be important to secure intellectual property rights for all new inventions we discover. To this end, we will work closely with University KT staff and when possible we will seek follow-on funding to allow development work to be undertaken with industrial partners. We have already established that the potential market for new gene expression tools is significant. In 2008 the market for protein expression tools in the US alone was reported to be $200 Million, of which 70% was for bacteria. We have identified and opened up discussions with several potential partners in the biotechnology industry whose business could be bolstered by new riboswitch technologies. Having secured IP we would aim to negotiate licensing deals for new orthogonal riboswitch technologies with one or more of these companies. Indeed we envisage that the maximum commercial value of orthogonal riboswitches will best be realised in combination with existing technologies including proprietary engineered bacterial host strains and plasmids. Moreover the integration of orthogonal riboswitch tools with existing technologies should make them more available to the wider industrial and academic community enabling greater impact to be realised in the many scientific applications that have been described.