| Fungi produce terpenoids for a wide variety of functions, primarily as signaling and defense compounds. Several highly bioactive sesquiterpenoids are produced only in fungi, particularly those derived from the protoilludene and hirsutene scaffolds. Many sesquiterpenoids have pharmaceutical applications; however, chemical synthesis of sesquiterpenoids is expensive, and biosynthesis in their native fungal hosts is hindered by low concentration and cultivation that is difficult or impossible. Incredible improvements in engineering terpenoid biosynthetic pathways in heterologous hosts have been made in recent years, rendering terpenoid biosynthesis economically viable. However, developing recombinant engineered systems requires the identification of the enzymes and the respective biosynthetic pathways involved in the production of terpenoids from the native fungal host. The primary goal of this work was to identify and characterize the enzymes producing two classes of sesquiterpenoids, derived from protoilludene and hirsutene scaffolds, from Stereum hirsutum . The first step in understanding terpenoid biosynthesis is the identification and characterization of the key scaffold producing enzymes, sesquiterpene synthases (STS). Previously, we developed a predictive framework based on STS protein sequence to accurately guide identification of fungal sesquiterpene synthases based on their initial cyclization mechanism. I successfully implemented this framework to identify three novel protoilludene synthases in the genome of Stereum hirsutum. Furthermore, data shows that both gene structure as well as protein sequence is highly conserved between fungal STS, reinforcing the hypothesis that fungal STS have evolved based upon initial cyclization mechanisms.;In addition, application of this same predictive framework led to the identification and cloning of the first hirsutene synthase from Stereum hirsutum. Unexpectedly, I discovered that the STS was not a typical, single domain enzyme, but instead an unprecedented two-domain STS, 3-hydroxy-3-methylglutaryl-(HMG)-CoA synthase, HS-HMGS. HMG-CoA synthase is a key enzyme that catalyzes the second step in the isoprenoid-precursor mevalonate (MVA) pathway; duplications of this gene in isoprenoid pathway gene clusters may represent a mechanism for increasing MVA pathway flux in fungi, potentially increasing isoprenoid/sesquiterpenoid yield. Following my discovery of this novel bifunctional HS-HMGS, I conducted a large-scale search of all sequenced fungal genomes for duplications of MVA and isoprenoid pathway genes. I found that duplication of early MVA pathway genes were both common and widespread throughout the fungal kingdom, with many HMG-CoA synthases being found in isoprenoid biosynthetic gene clusters as well. This suggests duplication of early MVA pathway genes may be a common mechanism utilized by fungi to increase production of specific isoprenoids.;This work has identified novel fungal sesquiterpene synthases, and demonstrated success in refactoring sesquiterpenoid biosynthetic pathways. My work has provided insights into evolutionary conservation and adaption of gene and protein structures of STS, which will facilitate future discovery and characterization of novel types of STS. In addition, my work with F. graminearum has highlighted that sesquiterpene(oids) may play a potential role in gene regulation of biosynthetic pathways, an important consideration when rebuilding pathways in a heterologous host. Finally, the conservation of early MVA pathway genes in isoprenoid biosynthetic gene clusters suggests a previously neglected mechanism of isoprenoid pathway regulation in fungi, which has wide implications for fungal isoprenoid biosynthesis, genome architecture, and mechanisms pathway regulation. (Abstract shortened by ProQuest.). |
