De novo synthesis of a chloroplast genome in Nicotiana tabacum

Project Information

ArtPlast

Grant agreement ID: 101149854

DOI

10.3030/101149854

Project terminated on 31 December 2024

EC signature date 11 March 2024

Start date 1 May 2024

End date 30 April 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 173 847,36

Coordinated by

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 1 - ArtPlast (De novo synthesis of a chloroplast genome in Nicotiana tabacum)

Reporting period: 2024-05-01 to 2026-04-30

Summary of the context and overall objectives of the project

Climate change and food insecurity are among the most urgent global challenges. Plants offer a direct biological solution to both, through their ability to fix carbon and serve as the basis of agricultural systems. However, the tools available for engineering plants remain limited. While progress has been made in molecular breeding and genetic modification, the capacity to systematically design and build plant genomes lags far behind what has been achieved in microbial systems.
This gap is particularly evident at the level of genome-scale engineering. The complexity and slow growth of plants hinder the development of rapid design, build and test cycles. Nonetheless, plants contain chloroplasts, organelles with small genomes and bacterial-like expression systems, which provide a more accessible entry point for synthetic biology. Chloroplasts can be transformed with high efficiency in some plant species and are naturally contained through maternal inheritance. These properties make them a uniquely suitable platform for the development of programmable, biocontained genetic systems in plants.
The ArtPlast project aims to create the first synthetic chloroplast genome in a land plant, Nicotiana tabacum. This genome will feature a compressed and reprogrammed genetic code, allowing the introduction of new functions and creating a platform for future applications in plant biotechnology. The project pursues three main objectives: (1) the in silico design and in vivo synthesis of artificial chloroplast genomes with novel genetic codes, (2) the development of strategies to test and improve genome designs in plants, and (3) the hierarchical assembly and biological characterisation of full synthetic genomes.
By enabling full chloroplast genome synthesis, ArtPlast will allow for the design of new traits that go beyond what is possible with conventional genetic engineering. These include enhanced photosynthesis, the production of high-value biomolecules, and the establishment of genetic firewalls that limit horizontal gene transfer. The project will also generate detailed insights into the design rules and functional constraints of the chloroplast genome.
The expected impact of ArtPlast is significant. Technologically, it will establish a platform for genome-level engineering in plants. Scientifically, it will provide new understanding of chloroplast biology. Strategically, it will contribute to the European Union’s efforts to support green innovation, sustainable agriculture and food security. All results will be shared openly, and public engagement activities will ensure broad societal dialogue around the opportunities and challenges of plant synthetic biology.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project ran for only 8 of the planned 24 months before being terminated. As a result, no full plant transformation has yet been completed. Nonetheless, critical progress was made towards the goal of generating a synthetic chloroplast genome in Nicotiana tabacum. Two major advances should be highlighted.

(1) Genome design (Objective 1, WP1 and WP2)
We have developed a set of synthetic chloroplast genome designs that we believe to be viable, based on current understanding of chloroplast biology and prior transformation studies. These designs follow three guiding principles: (i) compatibility with chloroplast function and regulation in Nicotiana tabacum, (ii) amenability to synthesis and assembly, and (iii) incorporation of codon compression strategies to enable future genetic code expansion and reduce recombination with the wild-type genome.

Our designs include a minimal genome in which non-essential genes and one inverted repeat have been removed, as well as recoded versions where synonymous codons have been consolidated to free specific codons for reassignment. Up to five recoding schemes were developed in silico and assessed for compatibility with known regulatory elements, transcript processing, and translational features of the N. tabacum chloroplast. These genome designs were partitioned into testable fragments, and one is currently being synthesised for experimental validation. The remaining 19 designs will be synthesised and tested through the newly launched SyncSol consortium, a £9.1 million collaborative project funded by the UK’s Advanced Research and Invention Agency, which I initiated and now lead. ArtPlast has been instrumental in enabling this success by providing both the scientific foundation and the institutional credibility necessary to establish and lead this international effort.

(2) Establishing sequencing pipeline (Objective 2, WP3)
We have established a sequencing pipeline based on nanopore sequencing. We began by optimising chloroplast DNA isolation using state-of-the-art chloroplast isolation kits and DNA extraction methods. This now enables the selective isolation of largely intact chloroplast DNA from up to 48 plants in a single day—an effort that previously required more than a week. Only small amounts of leaf material are needed (0.2 g instead of the 5 g previously required).

Using nanopore sequencing, we demonstrated the ability to sequence up to 24 chloroplast genomes in a single run, with potential scalability to 100 genomes per run (three days). We are currently working to establish a full sequencing pipeline capable of de novo chloroplast genome assembly from nanopore reads, supplemented by PacBio or Illumina sequencing.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Although the project was terminated after 8 of the planned 24 months, key technical milestones were achieved that lay important groundwork for future development. Notably, three major advances were made towards the generation of a synthetic chloroplast genome in Nicotiana tabacum: (1) the successful design and partial assembly of synthetic genome sections, (2) the development of a modular workflow for chloroplast genome engineering, and (3) the initial establishment of a state of the art sequencing pipeline for chloroplast transformations. While no complete plant transformation was achieved within the shortened timeframe, these outcomes provide a solid technical foundation.

The potential impact of this work lies in enabling the de novo synthesis and customisation of plastid genomes, with the capacity to transform chloroplast based biotechnology and plant synthetic biology. Ensuring long term success will require sustained investment in foundational research, now secured through ARIA for the coming three to five years, made possible by the conception and support of ArtPlast. This funding will ultimately enable the demonstration of complete synthetic genome function in planta. The development of a platform to accelerate and scale chloroplast transformation and analysis, along with a suite of predictive models for chloroplast biotechnology, could advance the field beyond current technological standards. In the longer term, supportive regulatory frameworks will also be essential to facilitate commercialisation and international collaboration, particularly within the EU.