Browsing by Author "Flynn, Christopher"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Identification and characterization of fungal sesquiterpenoid biosynthetic pathways(2016-05) Flynn, ChristopherFungi 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. Following identification of the the protoilludene and hirsutene synthases, I then cloned the P450 and oxidase enzymes found in their gene clusters. These types of enzymes are predicted to be required for the modification of the sesquiterpene scaffold, yielding the final bioactive sesquiterpenoids. Extensive testing of these potential sesquiterpene modifying enzymes identified two, Omp7a and Omp7b, which modify the protoilludene scaffold. Work to purify sufficient quantities of these compounds to determine the exact chemical modifications by NMR is ongoing, and would be the first demonstration of protoilludene scaffold modification to date. Finally, while isolating STS and refactoring their biosynthetic pathways is the major goal of this work, understanding the biological function of sesquiterpenoids in the native host is also key, because it provides insights into the bioactivities of sesquiterpenoids and helps us understand the factors that govern expression of the biosynthetic pathways. Therefore, I initiated a collaboration to identify the sesquiterpenes produced by Fusarium graminearum, the cause of the crop disease Fusarium Head Blight (FHB). F. graminearum is known to produce deoxynivalenol (DON), a toxic sesquiterpenoid derived via trichodiene synthase (Tri5). However, genomic analysis identified eight sesquiterpene synthases, while only two STS had been characterized to date, suggesting genomic potential to produce non-trichothecene sesquiterpenoids (NTS) that may affect pathogenesis, a topic that has not been comprehensively studied. I therefore set out to determine what NTS are produced by F. graminearum cultures, and if their production is increased under pathogenic (inducing) conditions. GC/MS analysis identified several NTS produced only in induced cultures. Surprisingly, induced Δtri5 deletion strains not only ceased production of the anticipated Tri5-derived trichothecenes, but also stopped producing sesquiterpenes not produced by directly by Tri5. Thus, while Tri5 expression is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 is not sufficient to explain the observed diversity of sesquiterpenoids. These findings suggest that tri5 expression, through an as-of-yet unidentified mechanism, is required for production of NTS. To determine the mechanism through which Tri5 influences NTS biosynthesis, either via protein:protein interactions, or via signaling by trichodiene, a catalytically inactive Tri5 was created that retains its secondary structure, and is currently being prepared for testing. While the role of trichothecenes in phytotoxicity is known, the biological function of non-trichothecene sesquiterpenes and their recently discovered co-regulation has not yet been determined. Understanding the role of NTS in pathogenesis may aid in breeding resistant crops, and prove valuable in controlling FHB. In summary, 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.Item Unconquered Louis Rejoiced In Iron": Military History In East Francia Under King Louis The German (C. 825-876)"(2020-05) Flynn, ChristopherThis project is a comprehensive military history of East Francia during the reign of King Louis the German (c. 825-876). It interrogates annals and chronicle sources, royal and ecclesiastical charters, Carolingian capitularies, and papal letters to study all aspects of Louis’s military machine. These include grand strategy, military organization, tactics, training, leadership, morale, and logistics. The work fills a notable lacuna in scholarship between the heavily studied militaries of Charlemagne and the Ottonian period. Louis was not a military innovator. Rather, he looked to his Carolingian predecessors for examples in nearly every aspect of military policy and strategy. His main intent was defensio patriae, which he achieved successfully through a system of what modern scholars would classify as defense in depth and the doctrine of overwhelming force. Both of these essential strategies can be traced readily to the reign of Charlemagne, when offensive warfare aimed at territorial conquest largely gave way to more defensive strategies. Contrary to much of the prevailing ninth century scholarship, the central East Frankish administration did not suffer as a result of the perceived consolidation of power in the hands of local magnates. Rather, Louis proved capable of putting very large armies—often several at once—into the field simultaneously. He fielded well-supplied troops from all across his substantial realm and was able to project effective military force into various theaters. He led his armies on well-organized campaigns to the west of his kingdom, against his Carolingian father, brothers, and nephews, and he defended the long eastern frontier against various Slavic peoples, most prominently the Moravians, whom he subsumed to the Carolingian state through a system of tributary relationships. Louis’s military systems, deliberately modeled upon those established by the early Carolingians and Charlemagne, ensured that East Francia remained intact throughout the course of his reign. The institutions by which he fielded effective armies survived after Louis’s death, during the notable void in royal power in the late ninth and early tenth centuries, to be utilized in recognizable form by the early Ottonian kings.