Synthetic biology and organelle genomics: A rubisco library as a case study in evolutionary landscapes and organellar engineering

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Food security is a critical issue facing human health in the 21st century as the global population approaches 10 billion and climate change threatens the food supply. Photosynthetic engineering has been successful in improving crop yields but the central enzyme of carbon fixation, rubisco, has remained intractable as a target of molecular evolution. The few plants tested with heterologous rubiscos grow uniformly slower than their wild-type counterparts. The goal of this proposal is to generate a chloroplast genome library in plants to explore the limits of rubisco function and organellar genome organization. In Aim 1 of this proposal I will explore the sequence-function landscape of a model, homodimeric rubisco enzyme and a novel, linked variant I have generated. I have generated a deep-mutational scan (OMS: all possible point-mutants) library and will assay their function in a uniquely suited rubisco-dependent E. coli strain. I have shown that these linked rubiscos function in vivo and in vitro and have already constructed the mutant library; in the K99 phase of this project I will perform selections on this library and analyze the data with a machine learning model. In the R00 phase I will generate further libraries to explore regions of the sequence landscape predicted to be rich in improved rubiscos with the goal of testing them in plants. In Aim 2 I will improve chloroplast transformation technology in order to enable the generation of chloroplast genome libraries of unprecedented size in the chloroplast-editing model plant N. tabacum. To do this I will generate sequence-specific TALEN nucleases that will be expressed from the nuclear genome and trafficked to the chloroplast where they will cut and disrupt the genome, preferencing homologous recombination of transgenic donor ONA. After demonstrating TALEN-cutting in the K99 phase I will develop this technology into an intracellular gene drive which will accelerate chloroplast genome editing. In Aim 3, with the technology developed in Aim 2 and the rubisco variants discovered in Aim 1, I will generate libraries of plants with altered rubisco genes and promoters in order to demonstrate the potential of rubisco engineering to improve plant growth. In addition, in the R00 phase, I will produce a comprehensive chloroplast gene knockout library in order to answer fundamental questions about the evolutionary flow of genes from organellar genomes to nuclear genomes. My goal as an independent investigator is to study organellar biology in model plants in order to explore the limits of engineering in individual proteins and metabolic pathways. My training in organellar transformation, protein library generation and laboratory management during the K99 phase will prepare me well for the R00 phase. The Innovative Genomics Institute at UC Berkeley and the laboratories of Ors. Oavid Savage and Brian Staskawicz in particular are the ideal environment for my career transition, combining precisely the necessary mixture of expertise - protein evolution and plant transformation, respectively.