Plants have been the highest volume source of biomass and biomaterials for human use since the agricultural revolution, and even before that in most societies. Despite incredible advances in synthetic chemistry and microbial synthetic biology, plants remain the key source for calories and nutrients, and critical feedstocks for materials, fuel, and drugs. This thesis consists of four body chapters. The first outlines the background of plant biotechnology and highlights a useful principle used in plant breeding but not well-known among plant biotechnologists. The second outlines how natural plant engineers outperform and might inspire the plant synthetic biologists of the future. The third chapter reports the first independent replication of an extremely exciting and surprising finding first reported halfway through my PhD. The fourth presents a large suite of genetic parts, specifically transcriptional repressors. Each chapter is intended to stand largely on its own, but I nonetheless believe the whole is greater than the sum of its parts, combining a conceptual advance, a look at nature’s plant synthetic biologists, a replication project which lies at the heart of the scientific method, and the development of a novel suite of tools which lies at the heart of synthetic biology.

The ability of plants to produce an incredible diversity of small molecules and complex polymers has made them an integral component of lives and societies. We have long harnessed the diversity of chemicals found in plants as food, commodities, and medicine. While plants innately produce millions of useful chemicals, biotechnology has allowed us to further tailor plants to our needs. An integral component of plant anabolism is the attachment of sugars to various chemicals, whether it be to small molecules or complex glycans. This unique specialty of plants creates novel opportunities to expand the ways in which we can improve their properties. In this work, we utilized the innate metabolism of plants to generate a variety of glycosides and glycans. First, we optimized the production of a glycoside, glucoraphanin, to permit expanding the portfolio of plants that produce this beneficial compound. Second, we developed transgenic crops capable of converting glucoraphanin from an inactive form to a chemopreventive form following cooking, thereby improving the nutritional quality of cooked foods. Last, we demonstrated the ability of plants to economically produce milk oligosaccharides, complex glycans that are normally produced only by mammals. Together, these results demonstrate our ability to engineer plants for improved nutritional quality and the production of novel compounds.

The invention of oxygenic photosynthesis has forever changed the face of the Earth by producing the oxygen present in our atmosphere. It is thought that this unique metabolism arose from the bacterial phylum, Cyanobacteria. Moreover, this group has significantly contributed to eukaryotic diversity via endosymbiosis, as a cyanobacterium is considered to be the progenitor of the original plastid organelle, more commonly recognized as chloroplasts in plants. The antiquity of these events, dearth of convincing methodologies, and lack of conclusive evidence all contribute to the considerable challenges we face in understanding the timing of these major evolutionary and geological transitions. In order to address these problems, I have employed various techniques focusing on improving our understanding of the role of cyanobacteria and photosynthesis in shaping the world we have today.

Evolutionary relationships are difficult to reconstruct due to phylogenetic noise (e.g. horizontal gene transfer and homoplasy), resulting in uncertainty in our ability to build accurate phylogenetic trees. In order to address this issue, fifty-four strains of cyanobacteria were chosen for genome sequencing based on improving the phylogenetic coverage of the phylum. Not only does the diversity-driven and phylum-level approach identify many novel genes, but it also clarifies the phylogenetic placement of various cyanobacterial subclades, protein families, and endosymbiosis events.

The timing of ancient events, such as primary endosymbiosis events, has primarily been dependent on the fossil record. This is problematic as microfossils are difficult to interpret and assign to extant lineages. Conversely, molecular clock methods have been just as widely varying. We devised a new approach to increase the amount of dating information incorporated into molecular clock analyses, improving the accuracy of the predicted dates. We date the plastid and mitochondrial endosymbiosis events to approximately 900 and 1200 million years ago, respectively.

Finally, I focus on the protein evolution of a specific protein crucial to photosynthesis: RuBisCO. Here, I use ancestral sequence reconstruction methods to predict, synthesize, and characterize ancestral versions of RuBisCO. We show that ancestral RuBisCOs have lower rates of carboxylation, reflective of the high CO2 and low O2 Precambrian atmosphere.