Directed Evolution of Biosynthetic Pathways to Carotenoids with Unnatural Carbon Backbones

Over the course of evolution, nature continually discovers new small molecules through the alteration of biosynthetic enzymes and pathways by mutation and gene transfer. Hundreds of these natural products have proven indispensable to medicine, culture, and technology, greatly contributing to increases in the length and quality of human lives. Chemists have found that the “chemical space” surrounding natural products is especially rich in functional molecules, and synthesis of natural product analogs has uncovered many with new or improved properties. Inspired by nature’s search algorithm, we and others have conducted our own evolution of carotenoid biosynthetic pathways in the laboratory. Chapter 1 comprehensively reviews the motivations, accomplishments, and challenges of this research area as of early 2005, and describes in detail how biosynthetic routes to dozens of new carotenoids have been established. To expand the number of carotenoid backbones beyond the C30 and C40 carbon scaffolds that give rise to the ~700 known natural carotenoids, we subjected a carotenoid synthase, the enzyme responsible for carotenoid backbone synthesis, to directed evolution. Chapter 2 describes the evolution of the C30 carotenoid synthase CrtM from Staphylococcus aureus for the ability to synthesize C40 carotenoids. This work also resulted in novel carotenoids with C35 backbones. We later found that some of the CrtM mutants generated in this laboratory evolution experiment, as well as several secondgeneration variants, are also capable of synthesizing unnatural C45 and C50 carotenoid backbones when supplied with appropriate prenyl diphosphate precursors. ix Chapter 3 describes the creation of full-fledged pathways to carotenoid pigments based on the C45 and C50 scaffolds. Coexpression of the carotenoid desaturase CrtI from Erwinia uredovora resulted in the biosynthesis of at least 10 new C45 and C50 carotenoids with different systems of conjugated double bonds. We also present evidence of an unnatural asymmetric C40 carotenoid pathway beginning with the condensation of farnesyl diphosphate (FPP, C15PP) and farnesylgeranyl diphosphate (FGPP, C25PP). In addition to clarifying how CrtM and CrtI achieve their product specificities, this work also sheds light on the molecular mechanisms used by evolution to access new chemical diversity and the selective pressures that have shaped natural product biosynthesis.