Our multidisciplinary team, has successfully collaborated on establishing a foundational understanding of WUE determinants in bioenergy grasses, and we have developed the methodologies needed to implement advanced engineering strategies. Leveraging parallel research tracks on Sorghum and the model C4 grass Setaria viridis, our collective work has expanded the understanding of how traits related to CO2 uptake, water loss, and water acquisition interdependently influence WUE in response to changing environmental conditions. We have developed and used methods to phenotype WUE traits and reveal genotype-to-phenotype associations in model C4 species, engineer complex traits into genomes, and integrate and model data through a genomics-enabled systems approach. Our iterative approach to engineering WUE in Sorghum is adopted from the design, build, test, learn (DBTL) cycle traditionally used to by engineers to tackle complex processes. We will leverage this work cycle to advance our novel gene expression control technologies (synthetic genetic circuits) capable of precisely modifying the expression patterns of key regulators. We will create a synthetic biocontainment system with engineered transcription factors to prevent transgene escape. We are also advancing C4 grass transformation and novel virus-dependent methods of tissue-culture-free genome editing to accelerate the development of engineered Sorghum with enhanced WUE traits. We are elucidating the genetic basis of key WUE traits by continuing to improve our understanding of Sorghum and Setaria physiology through genetics, genomics, and implementation of multiscale models to predict the impact of altering specific processes. Through our development of novel synthetic biology tools for C4 grasses, our team is poised to substantially accelerate Sorghum engineering and significantly enhance WUE in this key bioenergy feedstock.

This project is supported by the US Dept. of Energy:

• DE-SC0023160

• DE-SC0018277

• DE-SC0008769