Rewriting The Genetic Code: The Algal Plastome As A Testbed For Basic And Applied Studies

All cellular life uses a universal genetic code that provides the informational link between genes and their protein outputs. The code has 64 codons to enable 20 amino acids to be assembled into proteins, with between 1 and 6 codons per amino acid. But the assignment of the codons has remained essentially unchanged despite over three billion years of evolution. This raises several fundamental questions: i) is the genetic code the product of early optimisation, or a 'frozen accident' with other arrangements of the codon table equally viable? Theoretical analyses suggest that the code is optimised to minimise errors, but alternative arrangements have not been tested experimentally. ii) Can the code be expanded to include other amino acids that are not currently found in proteins (so called ncAAs) through the reassignment of one or more codons? To-date, such engineering has been achieved only to a limited degree. iii) Could industrial biotechnology exploit cells with a rearranged and expanded code to make ncAA-containing proteins with novel features (e.g. hormone drugs with longer half-lives, or enzymes with new catalytic functions)? iv) Could such recoding address issues and public concerns around escape and horizontal transfer of synthetic genes given that they would use a code that is unreadable by any other organism?

The creation of 'genome recoded organisms' represents an extremely challenging endeavour, even when considering a 'simple' cell such as E. coli. In this project we will reduce the complexity of this challenge by focussing on the chloroplast (or 'plastid'). Whilst algal and plant cells have most of their genes in the nucleus, their plastids possess a self-contained genetic system with a tiny genome of only a hundred-or-so genes known as the plastome. Recent advances in synthetic biology and genetic engineering using the single-cell alga Chlamydomonas now offer the potential for genome recoding, expansion, and exploitation using this model system.

In this project we bring together a consortium of leading experts in the fields of chloroplast synthetic biology, genome recoding and ncAAs, plant synthetic biology, and algal genetic engineering and gene editing. We will undertake an ambitious programme of work with five main goals:

1. We will generate a synthetic plastome (SynPlast1.0) in which all non-essential genes have been removed and the remaining genes use a minimal set of 51 codons. This will demonstrate the principle of codon compression and also serve as the basis for redesign of the system.
2. Having establish the pipeline for plastome design and delivery, we will reassign codons to different amino acids to make SynPlast2.0. We will then assess the 'fitness' of cells containing this new genetic code in terms of the biology of the cell and its plastid.
3. We will extend the genetic code to include ncAAs by making use of the spare codons released in step 1. We will demonstrate that multiple ncAAs can be added to the code, and that proteins with novel properties can be synthesised.
4. In order to develop the engineered plastid as a sub-cellular factory, we need to precisely control plastome gene expression. We will develop genetic switches in the nucleus that allow the tuneable expression of plastid genes.
5. We will exploit the fact that the alga is photosynthetic to test different re-engineering strategies for improving photosynthesis. We will then use the collective knowledge from the project to demonstrate the light-driven synthesis of two pharmaceutical proteins, incorporating ncAAs that have previously been shown to confer valuable new therapeutic properties. This will allow future technology where such proteins are made simply, cheaply and sustainably using CO2 and sunlight.

The genetic code, which defines the rules of translation from the 4-letter nucleic acid alphabet to the 20-letter protein alphabet, is universal to all life. Investigating why the code is set as it is and how it might be rearranged or expanded to include additional amino acids requires a simple and easily tractable genetic system. Current efforts to create 'genome recoded organisms' using bacterial models represent an extremely challenging endeavour, even when considering a 'simple' cell such as E. coli. In this project we will reduce the complexity of this challenge by focusing on the genetic system found in the plastids of plants and algae. This organelle contains a highly reduced genome (= 'plastome') that possess only a hundred-or-so genes, and recent advances in plastid synthetic biology and genetic engineering using the single-cell alga Chlamydomonas now offer the potential for genome recoding and exploitation using this model system. We will explore this potential by developing a system for reprogramming the plastid with a synthetic plastome, and demonstrate that a cell with minimal plastome of ~96 kb and using as few as 51 codons, can be created. This will release up to 13 codons for rearranging the code, or for assignment to non-canonical amino acids (ncAAs). This plastome will also serve as a naïve chassis onto which we can bolt new transgene modules for novel plastid functions. We will introduce plastomes with different codons arrangements to test whether such alternative codes are viable, and will extend the code to include several ncAAs. To control expression within the synthetic plastid, we will develop genetic switches encoded in the nucleus that allow precise orthogonal control of plastid transgenes. Finally, we will test strategies for improving photosynthesis through plastome engineering to demonstrate the utility of the system as a test bed for such applied research, and as a light-driven cell platform for synthesis of new-to-Nature proteins.