Simplified engineering of Acinetobacter baylyi ADP1 and evolutionary strategies for genome minimization

Our ability to engineer and domesticate microbes to give them useful properties promises grand rewards in the energy, agriculture, chemical and health industries. Yet, synthetic biologists often struggle to engineer bacterial genomes despite ever-improving genome-scale models of how they function. Often, they are stymied by the sheer complexity of the cell’s underlying systems biology and by how these continue to evolve rapidly after they are engineered. Recent advances in genome stabilization and genome simplification promise to overcome these barriers and profoundly extend our understanding of basic molecular biology and cellular life. Both the natural instability of bacterial genomes and their unexplored complexity (e.g., the presence of many genes with unknown functions) underlie major challenges to be reckoned with that often lead synthetic biologists to rely on extensive experimental trial and error. The construction of cells with minimal genomes to make microbiology more predictable is riddled with difficulties. There are sometimes advantages and sometimes disadvantages for removing more and more genes to simplify a bacterial cell. Similarly, evolution is a process that may both frustrate or enable synthetic biology. It can be slowed down by removing selfish DNA elements from a genome or it can be applied to compensate for suboptimal designs. The work in this thesis explores these interactions between genome design and evolution. It asserts that rational engineering and simplification principles can lay stronger foundations for engineering microbial cells so that more complex and ambitious designs can be successfully built, but that evolution is also a necessary tool to achieve extreme simplification of a living cell to make it robust enough for research and industrial demands to achieve the potential of synthetic biology. Our model organism is Acinetobacter baylyi ADP1, a highly naturally transformable and metabolically versatile soil bacterium. Chapter 1 provides an introduction to A. baylyi genetic engineering and the current state-of-the-art in bacterial genome stabilizing and streamlining projects. Chapter 2 describes our rational engineering efforts to reduce A. baylyi ADP1 genome instability– mainly by deleting all transposable elements from its genome–and the beneficial phenotypes in the ADP1-ISx strain that resulted from this work. Chapter 3 describes improved A. baylyi genome engineering methods and how they were used in the first stage of a genome streamlining project. We also describe a “Golden Transformation” protocol that speeds up and simplifies the steps needed to make precise edits to the A. baylyi genome and also show that the native CRISPR-Cas system is functional and can be reprogrammed using this method. Chapter 4 describes how we begin to test how compensatory evolution of reduced genomes can open new pathways to more extreme genome minimization by restoring fitness that is lost after deleting many dispensable genes from a genome. Chapter 5 discusses future directions for making improvements that further stabilize and streamline the A. baylyi genome. Together, the work presented in this dissertation presents concepts, tools, and insights into strategies that were successful and unsuccessful for building a better and simpler Acinetobacter baylyi ADP1 genome. These approaches can also be applied to other bacterial species to propel the goals of synthetic biology forward