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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - GENEVOSYN (Reshuffling genes and genomes: from experimental evolution to synthetic biology in plants)

Teaser

Summary

To provide global food security, the development of new generations of genetically improved crops with optimized properties and improved resilience to more extreme growth conditions is of utmost importance. The GENEVOSYN project aims at developing a new set of enabling technologies for synthetic biology and plant biotechnology that will greatly expand our capabilities of engineering and ultimately redesigning plant genomes.
GENEVOSYN has three highly ambitious objectives that will enable the engineering of new generations of crop plants. The project will (i) develop the chloroplast as a platform for synthetic biology applications in plants, by pursuing both bottom-up and top-down approaches, (ii) develop technologies for mitochondrial genome engineering to harness the unique potential of mitochondrial biotechnology, and (iii) explore the potential of recently discovered horizontal genome transfer processes for the creation of novel crop species and the improvement of existing ones.

Work performed

WP1 of GENEVOSYN aims at developing the plastid (chloroplast) a highly efficient platform for synthetic biology applications in plants. In a top-down synthetic biology approach, we have already successfully introduced into chloroplasts the entire biochemical pathway for artemisinin, the most effective antimalarial compound which currently is not accessible to many patients in the poorest counties in Africa and Asia, due to limited supply and relatively high costs. To facilitate the engineering of the whole pathway, we developed a new synthetic biology approach termed COSTREL (for â€˜combinatorial supertransformation of transplastomic recipient linesâ€™). The first step in the process was to transfer the genes for the core pathway of artemisinic acid biosynthesis into the tobacco chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for additional enzymes, including all accessory enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. This work, published in the high-impact open access journal eLife (Fuentes et al., 2016), provides a novel and highly efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output. It also demonstrates how an complex pathway in secondary metabolism can be transplanted from a medicinal herb into a high-biomass crop and, moreover, showed that the pathway can be relocated from the cytosol of specialized cells (glandular trichomes of Artimisia annua, the natural source of artemisinin) into leaf chloroplasts (of tobacco). Publication of this work received enormous media attention and was also highlighted in several multidisciplinary journals, including Nature and Science. As suggested in the proposal, we are currently continuing this work by introducing additional enzymes of the isoprenoid pathway to address suspected bottlenecks in precursor provision.
In a bottom-up synthetic biology approach, we have computationally designed a synthetic plastid genome that represents the minimum non-photosynthetic genome version (i.e., the smallest possible genome that supports cell survival under heterotrophic conditions). This in silico designed synthetic genome is based on rational design principles and on minimum-size cis-acting elements for plastid gene expression. As the next step, it will now be synthesize and assembled. The ultimate goal is to determine whether the synthetic genome can be booted up in the plant.
WP2 aims at developing a technology for mitochondrial genome engineering in plants. A major achievement (published very recently) was the identification of a new selectable marker gene for plastid transformation that provides an additional candidate selectable marker that is potentially suitable for mitochondrial transformation (Tabatabaei et al., 2017). In addition, three sets of vectors for mitochondrial transformation were constructed during the reporting period: (i) a set based on the previously identified candidate marker chloramphenicol acetyltransferase, (ii) a set based on the newly identified tobramycin resistance marker (Tabatabaei et al., 2017), and (iii) a set of vectors for an innovative novel approach that we refer to as â€œchloroplast-protected mitochondrial transformationâ€ (that is based on creation of a recipient line for mitochondrial transformation in which the chloroplast compartment is protected from the inhibitory action of a drug that affects both organelles). The sets comprise vectors with different mitochondrial expression signals and targeting regions in the mitochondrial genome. Mitochondrial transformation experiments with all three sets of vectors (using the biolistic method) have commenced.
WP3 plans to exploit recently discovered horizontal genome transfer processes for the creation of novel crop species and the improvemen

Final results

The development of the COSTREL technology (see above) offers a new synthetic biology method that allows the efficient transfer of complex biochemical pathways between species and the relocation of pathways to new cellular compartments. It potentially can be applied to many other pathways and complex traits (and is not limited to biochemical pathway). The work on the artemisinin pathway, recently published in eLife, stirred enormous attention in the public, due to artemisinin combination therapies (ACTs) currently being the only effective cure of malaria. As ACTs are the mainstay of malaria treatment and no alternative to artemisinin derivatives is expected to enter the market in the foreseeable future, there is a steadily increasing demand for ACTs which reached nearly 400 million treatment courses in 2013. Transfer of the pathway to tobacco, a high-biomass non-food/non-feed crop, as accomplished in our work, can potentially provide a stable supply of the feedstock that can be scaled up at will and at short notice, and moreover, take full advantage of the existing agricultural infrastructure. Tobacco is grown in large acreages, and alternative uses for tobacco biomass (that are unrelated to smoking) have long been sought. Since tobacco is well suited for cultivation at high cropping densities and multiple harvests (4â€“5) per season are possible, 40 t of biomass can be obtained from a single acre of tobacco field at a cost of only around $100 per ton. Thus, with our currently best-performing COSTREL lines (Fuentes et al., 2016), production levels of ~4.8 kg artemisinic acid per acre can be obtained, suggesting that the current world demand (of ~100 t artemisinin) can be met by cultivating tobacco on an area of ~200 km2, which is less than the area of the city of Boston (assuming ~50% loss during extraction and conversion).