Self-Resistance Enzyme Directed Genome-Mining for Fungal Natural Products and Enzyme Catalyses And Cell-free In Vitro Biosynthesis of Plant Terpene Natural Products

With the advances in modern science and technology, humankind leads longer and more comfortable lives than ever before. However, such progress has also given rise to worldwide challenges, one of which is the emergence of increasingly fatal drug-resistances. Since drug and herbicide resistance is inevitable due to overexposure, our need to understand and resolve this issue is an ongoing battle. In parallel, our knowledge of natural products - small molecules derived from secondary metabolism of living organisms which frequently possess crucial bioactivities - has deepened immensely. We continue to appreciate the significance of natural products and the role they play in our lives through natural product derived pharmaceuticals. New methods in discovering bioactive natural products and enzyme-based catalysts have greatly developed in recent years owing to the increasing availability of genomic data, including self-resistance enzyme directed genome-mining (SRE-DGM).This thesis primarily focuses on understanding and utilizing the second-copy self-resistance phenomenon for the discovery of new natural products and enzyme catalysts. First, comparing the co-crystal structure of one housekeeping enzyme to the homology model of its second-copy SRE allowed us to understand part of the mechanism by which the SRE acquired its resistance toward aspterric acid, an herbicidal terpene natural product. Mutational studies on the housekeeping enzyme revealed key amino acids which increase resistance to aspterric acid. This allowed us to anticipate the inevitable naturally occurring herbicide resistance and be prepared to deploy counter measures. We also utilized SRE-DGM to find and elucidate the biosynthetic pathways of the mitochondrial complex II inhibitors, harzianopyridone and atpenin A5, both of which have been the target of investigation for more than four decades. We identified the biosynthetic gene clusters of these compounds from their fungal producers and uncovered the biosynthetic steps which include multiple iterative enzymes. In particular, a methyltransferase and a flavin-dependent monooxygenase are used iteratively to introduce the unique methoxy groups on the 2-pyridone core structure. The pathway unexpectedly requires the installation and removal of a N-methoxy group, which is proposed to be a directing group that tunes the reactivity of the pyridone ring. We also discovered a new type of halogenase that installs the chlorine substitutes in atpenin A5. This halogenase is able to install halogens on an aliphatic carbon and does not belong to any of the known halogenase families. These results signify the importance of SRE and its utilization in DGM for novel enzyme catalysts. Finally, while the discovery of natural products is important, developing a robust and viable production methods of complex natural products is equally crucial. Thus, we developed an in vitro biosynthetic platform for producing plant terpene natural products. We established a one-pot, cell-free biosynthesis platform for nepetalactol and nepetalactone starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD+-dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The overall reaction contains 10 enzymes, four of which are biosynthetic enzymes, including a soluble P450, and five accessory and cofactor regeneration enzymes. Our in vitro platform yielded ~130-fold greater amount of nepetalactol and nepetalactone than the highest producing microbial platform