A mini foxtail millet with an Arabidopsis-like life cycle as a C4 model system

Over the past few decades, several plant species, including Arabidopsis thaliana, Brachypodium distachyon and rice (Oryza sativa), have been adopted as model plants for various aspects of research. These species, especially Arabidopsis, have had vital roles in making fundamental discoveries and technological advances 1. However, all these model plants use C 3 photosynthe-sis, and discoveries made in these species are not always transferable to, or representative of, C 4 plants such as maize (Zea mays), sor-ghum (Sorghum bicolor) and millets, which are efficient fixers of atmospheric CO 2 into biomass. Thus, it is critical to develop a new model system for studies in these and many other C 4 plants 2. Foxtail millet (S. italica) is a cereal crop that was domesticated from its wild ancestor, green foxtail (Setaria viridis). These two species are evolutionarily close to several bioenergy crops, including switchgrass (Panicum virgatum), napiergrass (Pennisetum purpu-reum) and pearl millet (Pennisetum glaucum), and major cereals such as sorghum, maize and rice 3. In addition, extensive genetic diversity exists in Setaria, with approximately 30,000 accessions preserved in China, India, Japan and the United States 3 as valuable resources for gene-function dissection and elite-allele mining 4. In recent years, the whole-genome sequences of foxtail millet and green foxtail have been made available 5-9 , and both species have been proposed as C 4 model plant systems 3,6. Between these two species, foxtail millet is more suitable as a model plant due to the seed shattering and dor-mancy in green foxtail. Nevertheless, the relatively long life cycle (usually 4-5 months per generation) and large plant size (1-2 m in height) limit the use of foxtail millet as a model plant 3,10-12. To overcome such limitations, we have recently developed a large fox-tail millet ethyl methane sulfonate (EMS)-mutagenized population using Jingu21, a high-yield, high-grain-quality elite variety widely grown in north China in the past few decades. From the mutant population, we identified a miniature mutant (dubbed xiaomi) with a life cycle similar to that of Arabidopsis. Subsequently, we developed genomics and transcriptomics resources and a protocol for efficient transformation of xiaomi, as essential parts of the toolbox for the research community

Validation of RetroPath, a computer-aided design tool for metabolic pathway engineering.

International audienceMetabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer-aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top-ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top-ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17-fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds