Developing New Strategies for Engineering Novel Natural Product Metabolic Pathways

Natural products represent a large and diverse array of molecules. Natural products and their derivatives play important roles in the human sphere, serving as pharmaceuticals, biofuels, and more. However, the structural complexity of many promising natural products prohibits industrial production sufficient to make full use of their capabilities. The challenge posed by natural products has spurred many advances in multiple fields. Despite these achievements, ignorance of the native metabolic pathways and inefficiencies in manipulating the genes involved has slowed the ability of science to capitalize on the enormous potential of natural products. In Chapter 1, we begin by surveying the fields concerned with the production or variation of natural products. This begins with organic synthesis, continues with in vivo and in vitro biocatalytic methods, and concludes with the “combination” techniques that seek to unite the strengths of biocatalysis and organic chemistry: precursor-driven biosynthesis, mutasynthesis and semi-synthesis. After examining the advantages and disadvantages of the extant technologies, in Chapter 2 we describe a novel strategy to develop semi-synthetic routes to underexplored classes of natural products. While it employs features of existing techniques, our strategy originates from a fundamentally different conception of natural product production, which looks away from the native precursors of a single target, and towards versatile precursors amenable to multiple forms of chemical modification. We then carry out a demonstration of this strategy by first biosynthetically producing 2Z,7E-farnesol from heterologously expressed Mycobacterium tuberculosis synthetases, and subsequently derivatizing this unnatural precursor into a set of novel Ambrox© analogs. Complex biocatalytic applications rely on DNA manipulation technologies to rapidly construct and diversify metabolic pathways. When components of the targeted pathway are unknown or poorly understood, the creation of large libraries of variant pathways can be employed to circumvent these limitations and rapidly develop the desired phenotype. In Chapter 3, we harness our existing library building technology, Reiterative Recombination, to the yeast sexual reproduction cycle for the purpose of combining separately constructed library strains via simple mating and chromosome segregation into an exponentially larger combinatorial library. This chapter describes the design, construction, and initial validation of this system, termed Reiterative Segregation. Finally, in Chapter 4, we explore possible elaborations of the Reiterative Segregation design, and work towards combining libraries of alternative sugar metabolic pathways as an application relevant to biofuel production.