PlantSynBio: Regulatory Systems to Tune Gene Expression in Synthetic Chloroplast Operons

Investments in basic research over the past five decades have been rewarded with remarkable advances in understanding fundamental biological processes, including mechanisms of heredity, metabolism, and organismal development. The discipline of synthetic biology applies this understanding to practical problems within a framework of engineering design principles.

This project will use this framework to apply recent advances in understanding mechanisms of gene expression in plants to improve agronomic and nutritional traits, and to foster the use of plants as “green” factories for pharmaceuticals and other beneficial biochemicals. Foundational principles inferred from basic research will be used to design genetic “parts” to optimize the expression of genes that confer beneficial properties.

This project will also provide research training for undergraduate and postdoctoral students, and it will engage at-risk high school students in hands-on workshops in nutritional biochemistry as part of a broader program at the University of Oregon that aims to encourage such students to enroll and succeed in college.

The overarching goal of this project is to develop tools for metabolic engineering, biotechnology, and enhancement of agronomic traits in plants. Many such applications require expression of multiple genes, with optimal outcomes depending upon their suitably balanced expression. The chloroplast genetic system offers particular promise as a chassis for the expression of multiprotein assemblies and multi-enzyme metabolic pathways because chloroplast genes are naturally expressed in operon-like polycistronic transcription units, they do not experience epigenetic effects, and they can achieve very high expression levels.

A current limitation, however, is a paucity of characterized genetic elements to program predictable expression over a wide dynamic range, especially for the purpose of multigene applications. This project aims to fill this gap by leveraging recent advances in (i) understanding mechanisms that determine expression levels in natural chloroplast operons, (ii) elucidating the basis for sequence-specific RNA recognition by pentatricopeptide repeat proteins, which are key to this regulation, and (iii) cataloging translational efficiencies of native chloroplast genes.

This deep knowledge base will be used for the rational design of a palette of cis-elements and cognate trans-factors to achieve predictable, regulatable, and suitably tuned gene outputs in synthetic multigene transcription units.

This award was co-funded by the Plant Genome Research Program in the Division of Integrative Organismal Systems and the Systems and Synthetic Biology Cluster in the Division of Molecular and Cellular Biosciences.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.