Periodic Reporting for period 4 - GENEVOSYN (Reshuffling genes and genomes: from experimental evolution to synthetic biology in plants) Reporting period: 2020-04-01 to 2021-03-31 Summary of the context and overall objectives of the project 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 aimed 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 had three highly ambitious objectives: (i) the development of chloroplasts as platform for synthetic biology applications in plants, (ii) the development of technologies for mitochondrial genome engineering, and (iii) the exploration of the potential of recently discovered horizontal genome transfer processes for the creation of novel crop species. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In a synthetic biology approach, we have 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. To facilitate the engineering of the whole pathway, we developed a new synthetic biology approach termed COSTREL (COmbinatorial Supertransformation of Transplastomic REcipient Lines’). First, we transferred the genes for the core pathway into the tobacco chloroplast genome. The resulting plants were then combinatorially supertransformed with genes for additional enzymes, including all accessory enzymes known to affect the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 mg artemisinic acid per kg biomass. This work provides a novel and highly efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output. It also demonstrates how a 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 (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. Additional pathways engineered into chloroplasts include the pathway for the stress-protective compound dhurrin and the high-value ketocarotenoid astaxanthin. Implementation of the pathway for astaxanthin, a non-natural photosynthetic pigment, has revealed that it can completely replace the pigments naturally present in the photosynthetic apparatus, thus representing an important breakthrough in engineering of artificial photosystems with simpler pigment composition. Additionally, a synthetic biology approach has been developed to facilitate the large-scale engineering of signal transduction pathways in plants, leading to plant varieties with improved resistance to abiotic stresses (drought, salt stress, osmotic stress). Finally, a major breakthrough has been achieved with the development of a chloroplast transformation technology for the model plant Arabidopsis.To develop a technology for mitochondrial genome engineering in plants, large sets of vectors for mitochondrial transformation were constructed. Large-scale mitochondrial transformation and selection experiments with all vectors have been conducted and analysis of candidate lines is underway. In addition, we recently achieved a breakthrough in mitochondrial genome engineering by developing of a new technology for site-directed mutagenesis of the plant mitochondrial genome. Mutant plants have been isolated and comprehensively characterized genetically and biochemically (publication in preparation).Exploiting our recent discovery that entire genomes can be horizontally transferred between plant species across graft junctions, we aim to create novel (synthetic) crop species as well as new ornamental and medicinal plants. To facilitate horizontal genome transfer between species in the nightshade family (Solanaceae), we have developed transgenic lines with different selectable marker genes for nearly 20 different species. Large-scale grafting experiments and selection for horizontal genome transfer have been performed, and several candidate events have been isolated. In parallel, we elucidated the cellular mechanisms underlying horizontal genome transfer. 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) The development of the COSTREL technology offers a new synthetic biology method that allows the 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. The work on the artemisinin pathway stirred enormous attention in the public, due to artemisinin combination therapies currently being the only effective cure of malaria. Transfer of the pathway to tobacco, a high-biomass non-food/non-feed crop, can potentially provide a stable supply of the feedstock that can be scaled up flexibly, and moreover, take full advantage of the existing agricultural infrastructure. We have extended the strategy to other metabolic pathways and, for the first time, also to complex signal transduction pathways that we successfully engineered to create plants with improved stress tolerance. Finally, a major breakthrough had been the successful development of a chloroplast transformation technology for the model plant Arabidopsis, which now makes this species accessible to chloroplast genome engineering for synthetic biology. With the development of a transformation technology for plant mitochondria, we expect to pave the way to studying all aspects of mitochondrial gene expression and physiology in vivo, and open up entirely new possibilities in plant biotechnology by (i) engineering novel biochemical pathways into mitochondria, (ii) using the mitochondrion as expression factory for recombinant proteins, and (iii) engineering traits that are determined by mitochondrial genes (e.g. cytoplasmic male sterility, an extremely important trait in plant breeding). An important milestone has been achieved with the successful development of a technology for site-directed mutagenesis of the plant mitochondrial genome.With the production of new plant species by horizontal genome transfer, we provide a novel approach towards the generation of new crop plants as well as medicinal and ornamental plants. This work opens up exciting opportunities in plant breeding, by making synthetic plants with novel properties (e.g. improved growth, stress tolerance and disease resistance). An added benefit from this work has been the development of transformation technologies for a large number of species in the nightshade family. In addition to these practical applications, work on the underlying cellular mechanisms has uncovered a pathway of organelle movement from cell to cell and provided a mechanistic framework for horizontal genome transfer. Twitter graphic on artemisinin work Facebook graphic on artemisinin work Media graphic on artemisinin work
