Marna Yandeau-Nelson, Iowa State University
Erin Sparks, University of Delaware
Rajib Saha, University of Nebraska, Lincoln
Basil Nikolau, Iowa State University

As stationary organisms that are faced with surviving constantly changing environments, plants have evolved specialized features to protect against environmental stresses. One of these features is an exterior protective barrier on aerial plant surfaces called the cuticle. The cuticle acts as a physical barrier between the plant and its environment, functioning to limit the loss of water and gasses. Although many key genes that function in making the cuticle have been identified, a holistic view of how the cuticle is built is missing. This project will engineer two novel, parallel synthetic biology systems that are normally devoid of a cuticle (yeast cells and plant roots) to build a cuticle from scratch and decipher the complexities of the biochemical pathways underlying this unique plant feature. Systematically determining how a cuticle is built will lead to important applications such as the breeding of crop plants with customized cuticles that may have enhanced tolerance to environmental stresses, as well as cuticle-inspired chemicals for the biorenewables industry. Moreover, this project will train the next generation of multi-disciplinary scientists, and build teaching and research initiatives with the ultimate goal of increasing the proportion of the scientific workforce who are from STEM-underrepresented backgrounds.

This multi-disciplinary project will build and test two synergistic synthetic biology chassis in systems that do not naturally produce a cuticle (i.e., plant roots and the yeast Saccharomyces cerevisiae) to systematically refactor the transcriptional regulatory network, and the metabolic pathways that assemble the protective, hydrophobic cuticle barrier. These two synthetic chassis will be used to comprehensively model and quantitatively understand the integrated mechanisms that assemble a functional plant cuticle. The root chassis will be used to study the coordinated activation of cuticle assembly by plant transcription factors. This chassis will provide temporal transcriptional and metabolic data to enable the development of dynamic predictive models that provide a holistic view of cuticle metabolism and its associated regulation. A second chassis relies on multiple engineered yeast plug-and-play systems expressing different genetic complements capturing the gene redundancies within the pathway that will be assessed for the production of synthetic cuticle constituents. The metabolic data generated from these strains will be the inputs for kinetic modeling, which will provide the first kinetic understanding of this complex pathway. The coordinated development of the plant root and yeast chassis in combination with the proposed computational framework will provide a novel platform for discovery, and systematic analysis of cuticle assembly.