Rewriting the genetic code through aminoacyl tRNA synthetase engineering

Lead Research Organisation: University College London
Department Name: Structural Molecular Biology

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description In developing the technology of selection, we also developed a screening platform to monitor tRNA charging. The screening platform is compatible with high-throughput screening and this is an essential step towards the directed evolution of the aminoacyl tRNA synthetase enzymes. The technology of selection has been validated as a proof-of-principle and it is now in place for a challenging selection.
Exploitation Route We are preparing a manuscript to report on the positive results of the project. The selection methodology will be further developed and it is suitable for the selection of ligands and DNA modifying enzymes.
Sectors Chemicals,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Title Arc-based in vivo selection 
Description Arc is a bacteriophage transcriptional regulator that can display very high affinity (< 10 nM) for its recognition sequence. Arc is a small protein and its structure highly sensitive to substitutions, which invariably disrupt DNA binding. Of particular relevance are two residues early in its sequence, L11 and N12. Disruption of either residue results in Arc mutants unable to bind DNA, making this approach a suitable approach for the directed evolution of aaRS and genetic recoding (particularly L->N and N->L). The platform relies on the expression of a fusion protein (Arc-Arc-reporter) including two copies of Arc and a reporter (e.g. GFP, kanR or His-tag). At least one of the Arc proteins must have wild-type sequence for DNA binding. This platform enables: 1. Screening unnatural aminoacid incorporation - placing TAG codons (which can be targeted by orthogonal tRNA/aaRS pairs) between Arc and reporter. 2. Screening reassignment - using a recoded Arc that can subsequently be purified by the reporter (i.e. plasmid capture by reporter capture) 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The platform has been validated and we are currently carrying out additional necessary controls prior to manuscript preparation. 
 
Title In vitro platform for high-throughput screening of aminoacyl-tRNA synthetase activity 
Description A first step towards the directed evolution of tRNA synthetases (aaRS) is the development of a high-throughput platform for screening aaRS actvity. A number of assays are available in the literature but use radioactive compounds, are specific to certain aaRS/tRNA pairs, and are incompatible with in vitro directed evolution of aaRS. By combining multiple published approaches as well as developing some novel custom solutions, we have developed an assay that allows for the high-throughput screening of aaRSs - be it from lysate or purified aaRS. The assay has a wide dynamic range, which allows it to be used to quantify aaRS kinetics and specificity (to both natural and unnatural substrates) as well as to compare activity of different mutants. Key steps include: 1. In vitro synthesis of tRNA (ligation or transcription) 2. Charging reaction 3. tRNA immobilization to solid surfaces (optional) 4. Selective degradation of uncharged tRNAs (periodate) 5. Detection of charged tRNAs by fluorescence (ligation of labelled adaptor) 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact A high-throughput assay underpins the development of selection and directed evolution approaches. We are currently preparing a manuscript for publication of the assay and its development. 
 
Description Marliere 
Organisation University Paris Sud
Department University of √Čvry Val-d'Essonne
Country France 
Sector Academic/University 
PI Contribution None
Collaborator Contribution Partner lab has long experience in gene knock-outs in E. coli, including essential genes. They have generated to date two E. coli knock-outs: LeuRS and OmpT
Impact None yet.
Start Year 2016
 
Description BVL Symposium 2014 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact My talk was the starting point for a debate on genetic engineering and synthetic biology, containment of the technology (GMOs) as well as public acceptance/support - with a view on looking into current German and EU regulation/legislation.

Because of the soft distinction between Genetic Engineering and Synthetic Biology, sections of academics/industry are rebranding themselves Synthetic Biologists to escape anti-GMO movements. The result is that anti-GMO movements are now targetting Synthetic Biologists and important initiatives are being threatened. I was approached by a group of plant Synthetic Biologists who have invited me to join a worldwide debate on Biological diversity that can lead to a potential moratorium on synthetic biology.
Year(s) Of Engagement Activity 2014
URL http://www.bvl.bund.de/EN/07_TheFederalOffice/04_Events/01_Symposium_2014/Symposium_2014_node.html
 
Description Naked Scientists 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Radio interview with the BBC Naked Scientists (29.11.16)
It covered synthetic genetic materials and biocontainment. This is broadcast live to BBC Cambridgeshire, played in BBC Australia and also released as a podcast.
Year(s) Of Engagement Activity 2016
URL https://goo.gl/UUQpKR
 
Description SBUK2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I chaired the Second Synthetic Biology UK conference, which took palce in Edinburgh, on 14-16 November.
The conference with more than 180 participants, included researchers (PIs and PDRAs) and funding organization represenatives (BBSRC, KTN).
Year(s) Of Engagement Activity 2016
URL https://www.biochemistry.org/Events/tabid/379/MeetingNo/SA186/view/Conference/Default.aspx
 
Description Science Week (Birkbeck) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Although the talk was open to general public, students from UCL and Birkbeck were over-represented. The talk raised interesting questions and allowed me to engage with the 2014 UCL iGEM team. Birkbeck also reported the content of the talk in a dedicated blog.

The biggest impact of the talk was the interaction with the 2014 UCL iGEM team (see their entry for specifics on impact) and with a Birkbeck team interested in entering the 2015 iGEM competition.
Year(s) Of Engagement Activity 2014
URL http://www.bbk.ac.uk/science/about-us/events/science-week/science-week-2014
 
Description UK Hub of Responsible Research and Innovation Tools 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Discussion group to identify and formulate strategies (or good practices) to introduce RRI into research. The workshop incorporated academics, media, funders, journals, unions and industry representatives.

None yet (Nov/14).
Year(s) Of Engagement Activity 2014
URL http://www.rri-tools.eu/
 
Description Xenobiology: biosafety, biosecurity and biocontainment 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact We organised a small workshop (approximately 40 participants) including researchers, students, social scientists, funders and government representatives to discuss some of the challenges of releasing genetically engineered microorganisms into the environment. The workshop also explored biological orthogonality and how that can be adapted for biological containment. Some of the issues relating to the development of Xenobiology were also covered. The workshop consisted of a mixture of talks and plenary discussions with participation from the audience.

In addition to it being a consultative exercise, the workshop was also used to establish a network of researchers and social scientists involved in ethics, risk assessment and governance. Our goal was to further develop the engagement between lab-based and social scientists and to improve the dialogue on Xenobiology, Synthetic Biology and biotechnology. Based on feedback from participants, the workshop was a useful introduction ot the area and created a depth of discussion beyond what is currently present in the field.

We are now in the process of generating a podcast based on the workshop for public distribution and creating a webpage to host the talks presented on the day.
Year(s) Of Engagement Activity 2017