Rewriting the genetic code through aminoacyl tRNA synthetase engineering

Two classes of molecules are essential to all life on Earth: nucleic acids and proteins. Nucleic acids, such as DNA, are our genetic material and serve as a repository for all information required by each cell to function. That information includes the instructions for the synthesis of all proteins, which are the cellular workhorses and catalyse essential chemical reactions as well as orchestrating how the cells sense and respond to the environment. The genetic code describes how specific DNA sequences are translated into specific protein sequences - such that given a DNA sequence, it is possible to exactly deduce the sequence of the protein generated. The genetic code emerged so early in evolution that, bar minor exceptions, it is universal - a given DNA sequence will generate the same protein in most organisms.

The universal nature of the code is of particular concern when it comes to genetically modified organisms (GMOs). Although great efforts are placed in containment and in ensuring that GMOs cannot outcompete natural organisms in the wild, little has so far been done to minimize the risk of "genetic pollution" - the possibility of DNA from GMOs, that codes for particular traits, being taken up by other organisms with unpredictable consequences. Although such risk is thought to be small, it will rise as GMOs increasingly become part of our environment and as the genetically engineered traits become more complex. One way to minimise the risk of genetic pollution is by creating GMOs with a modified genetic code - sufficiently different from the natural one so that the information encoded in a GMO's DNA cannot be used by natural organisms to synthesise active enzymes and vice-versa.

Although conversion of genetic information into a specific protein sequence is a complex multi-step process, a single family of enzymes, aminoacyl-tRNA synthetases (aaRSs), lie at the heart of it - they enforce the genetic code by linking specific nucleic acid sequences to specific protein building blocks (amino acids). We propose to engineer these synthetases to modify the nucleic acid sequence to amino acid assignment, thus changing the genetic code. The project will focus on developing novel directed evolution methodologies and applying them to a well-characterised synthetase, to reassign its amino acid specificity in vitro - providing the first step towards rewriting the natural genetic code.

Multiple synthetase reassignments as well as wholesale synthesis of the modified genomes will be required to establish a GMO carrying a modified genetic code. However, once such an organism is obtained, the resulting GMO, being substantially safer, can readily replace current GMOs used in the large scale synthesis of fine chemicals, novel materials and other synthetic biology applications. In addition to increasing our understanding of these enzymes, the directed evolution of synthetases may provide insights on how the genetic code was first established and how it evolved.

Having emerged early in evolution, the genetic code, bar minor exceptions, is universal - an RNA message will give rise to the same protein in most living organisms, if translated. Aminoacyl-tRNA synthetases (aaRSs) enforce the code by creating the link between RNA and protein sequences through charging precise sets of tRNAs with specific amino acids. That exact link between RNA and protein sequence can be rewritten through altering the substrate specificity of aaRSs. Genetically modified organisms (GMOs) are increasingly a part of our environment but containment remains our most effective measure to limit any potential danger to the environment. We propose a viable approach which will lead to the development of substantially safer GMOs: rewriting the genetic code by systematically altering the substrate spectrum of individual aaRSs to create alternative genetic codes (capable of encoding the 20 natural amino acids). Rewriting of the genetic code would allow auxotrophic GMOs that cannot exchange information with nature to be developed - a substantially safer GMO. To do so, we will develop novel in vitro selection methods based on linking function of variant synthetases to their genotypes in water-in-oil emulsions. We will focus on isolating an engineered LeuRS capable of efficient and specific tRNA(Leu) charging with asparagine; a modification we expect will lead to non-functional proteins if it were introduced in vivo and thus suitable for the engineering of safer GMOs.

The proposed research programme will develop an in vitro selection platform for the directed evolution of aminoacyl-tRNA synthetases (aaRSs) but that is general and could be applied to other bond forming enzymes. The project will also focus on using the selection platform developed to isolate an asparaginyl-tRNA synthetase from a leucyl-tRNA backbone. This reassignment is a first step towards the systematic engineering of multiple synthetases and the generation of a novel genetic code not expected to cross-talk with the natural one. The long-term aim of the project is to implement a novel genetic code in an organism with a view to creating an auxotrophic genetically modified organism (GMO) that is unable to exchange genetic information with natural organisms - a safer GMO. These aims are of significant impact not only to academia but could also benefit the general public, policy makers and industry.
Very few groups internationally have successfully engineered aaRSs - all using in vivo selection methodologies to target incorporation of amino acid analogues at rare codons. As such, an in vitro selection method for the directed evolution of aaRSs that is not limited to rare codons and has the potential to bypass substrate delivery (which may be an issue for chemically divergent and for potentially toxic analogues) while allowing the step-wise engineering of function through the isolation of evolutionary intermediates, can have considerable academic impact. Thus, the project is likely to have international scientific impact with a number of possible avenues for collaboration towards the development of our long-term goals. An organism with a substantially altered genetic code would be a landmark result in synthetic biology, allowing exploration of reduced and expanded genetic codes as well as genetic codes with different chemical functionalities.
By being dependent on essential chemical compounds (which limits an organism's ecological risk) and by being unable to exchange genetic material with natural organisms (which limits the escape of any genetic information), an auxotrophic GMO operating under a different genetic code would significantly increase GMO biosafety. Such an organism could be adopted as a standard safety measure in both academia and industry to develop GMOs for large-scale use, such as in fine chemicals or biofuel syntheses, or GMOs that will be used in the environment, such as for bioremediation or as biosensors - thus minimising potential risks of environmental damage and addressing some of the public concerns about GMOs and "genetic pollution". Such GMOs could also be used by policy-makers as a tool to restrict (or license) research with potential biosecurity implications (e.g. antibiotic resistance).
Having established the in vitro selection technology, it can be modified to select for synthetases that incorporate unnatural amino acid analogues with a view towards the in vitro synthesis of sequence-defined polymers. It may also be possible to develop alternative chemistries leading to the synthesis of polymers without an amide backbone. Similarly, the technology can be adapted for the selection of novel bond forming enzymes. As such, the technology has potential economic value and any enzymes (or compounds) isolated would have academic and commercial applications. Commercial potential could be exploited through patents and licensing agreements or through spin out companies, contributing to the British economy.
The proposed research is multi-disciplinary and will provide researchers with ample training and learning opportunities in biochemistry, chemistry, mathematics and physics. Knowledge in these areas can be easily accessed in the ISMB as well as other UCL and Birkbeck departments and would foster scientific dialogue. Multi-disciplinary training is essential in synthetic biology and would equip researchers with a wide range of skills applicable to both academic and industrial careers.