Expanding Biological Engineering from Single Enzymes to Cellular Pathways

The emerging field of synthetic biology evolved from biological research much the same way synthetic chemistry evolved from chemical research; with accumulated knowledge of the structure of single genes and proteins and the methodologies to manipulate them, researchers turn to forward engineer complex biological systems to effectively manipulate living systems. Much like in the case of enzyme engineering, a rationally designed biological network is currently beyond our reach, and we turn to directed evolution to circumvent this gap in knowledge. Yet the unique nature of live biological networks uncovered new challenges previously unmet by single-gene molecular technologies, and extrapolation of current technologies to the manipulation of multi-component has proven laborious and inefficient. To establish engineering technologies for living cells, novel directed evolution techniques are sought for that are compatible with simultaneous manipulation of multiple biological components in vivo. In this work, we explore techniques for library DNA mutagenesis in the context of single and multiple genes. Chapter 1 provides an overview of the challenges in expanding current in vivo directed evolution methods from single enzymes, to the design pathways and cells. Chapter 2 describes the design and characterization of an assay for combinatorial directed evolution of a single metabolic enzyme. In Chapter 3 we present the utilization of our DNA assembly system, Reiterative Recombination, for attenuation of metabolic pathways. We use a library of promoters to combinatorially vary the expression of genes in the heterologous lycopene biosynthetic pathway in S. cerevisiae. Finally, Chapter 4 explores the calibration of the dynamic range of genetic selection, using metabolic enzyme activity as probe for cell survival