Details
| Originalsprache | Englisch |
|---|---|
| Qualifikation | Doctor rerum naturalium |
| Gradverleihende Hochschule | |
| Betreut von |
|
| Datum der Verleihung des Grades | 1 Feb. 2024 |
| Erscheinungsort | Hannover |
| Publikationsstatus | Veröffentlicht - 25 Apr. 2024 |
Abstract
Kombination von biosynthetischen Genen aus mehreren Wegen in einem einzigen heterologen Wirt, um neue Verbindungen zu schaffen. Um den Titer von Tetraketid 25 in A. oryzae NSAR1 durch SQTKS-Expression zu verbessern, haben
wir zwei Hydrolase-kodierende Gene koexprimiert. Die 7-tägige Fermentation in DPY-Medium beinhaltete tägliche Titermessungen. Obwohl die Hydrolasen einen bescheidenen Einfluss hatten, erreichte der Titer etwa 3,2 mg/L, was eine Vervierfachung im Vergleich zum vorherigen Experiment darstellt.
Wir führten eine Protein-Level-Engineering des cis-ER-Domäne in SQTKS durch, indem wir 10 Mutationsgruppen über Heferekombination in das gesamte SQTKS einschleusten. Diese Mutationen wurden auf der Grundlage isolierter ER-Mutanten von SQTKS entworfen, die durch eine Kombination aus computergestützter Modellierung und experimentellen Assays mit verschiedenen Imitat-Substraten von vorherigen Kollegen analysiert wurden. Nach ihrer Expression in A. oryzae NSAR1 haben wir nach korrekten Transformanten gescreent, Fermentation und chemische Extraktion durchgeführt und LCMS zur Verbindungserkennung genutzt. Jedoch wurden in diesen Expressionsversuchen keine neuen Verbindungen erzeugt.
Wir führten ein Engineering auf der Pathway-Ebene durch, indem wir genomgeförderte Gene aus mehreren biosynthetischen Genclustern (BGCs) in A. oryzae NSAR1 integrierten. Wir co-exprimierten Gene aus vier BGCs von sporogen AO-1 27, Hypoxylan A 73, Eremoxylarin D 123, PR-Toxin 107.
Diese vier BGCs teilen einen gemeinsamen Kernkohlenstoff-Skelett, und besitzen gleichzeitig unterschiedliche Anpassungsenzyme. Es wurden zwanzig oxygenierte Aristolochene-Kongenere synthetisiert und charakterisiert, wobei ihre Strukturen herausgearbeitet wurden. Hervorzuhebende Verbindungen sind dabei das Naturprodukt Hypoxylan A 73 und ein Epimer von Guignaderemophilane C 37. Des Weiteren wurde eine neue pilzliche Aromatase entdeckt, die durch oxidative Demethylierung Phenole produziert. Wir haben die pilzlichen Wege für Tetraketid-Multiforisine und mit Islandic-Säure verwandte
Verbindungen untersucht. Heterologe Expressionsversuche ergaben hohe Titel dieser Verbindungen und Zwischenprodukte des Wegs. Dies führte zur Strukturcharakterisierung von 14 isolierten Verbindungen, einschließlich Multiforisin H 143h und I 143i. Diese Ergebnisse klärten nicht nur den
Weg, sondern legten auch den Grundstein für die Gesamtbiosynthese dieser Metabolitenklasse. Versuche, Gene aus dem Islandic-Säure-BGC in A. oryzae einzufügen, um Islandic-Säure 144 oder Allantopyron A 145 zu synthetisieren, waren erfolglos, da keiner der Transformanten Verbindungen erzeugte, die über diejenigen hinausgingen, die bereits von den A. oryzae-Wirten synthetisiert wurden.
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Hannover, 2024. 214 S.
Publikation: Qualifikations-/Studienabschlussarbeit › Dissertation
}
TY - BOOK
T1 - Engineering Aspergillus oryzae for the production of biosynthetic proteins
AU - Sun, Yunlong
PY - 2024/4/25
Y1 - 2024/4/25
N2 - This investigation aims to strategically engineer the biosynthesis of fungal natural products through heterologous expression. It includes modifications to the heterologous host to boost productivity; leveraging the product diversity of an individual enzyme via mutasynthesis and combining biosynthetic genes from multiple pathways in a single heterologous host to creat new compounds. To improve tetraketide 25 titer in A. oryzae NSAR1 through squalestatin tetraketide synthase (SQTKS) expression, we co-expressed two hydrolase-encoding genes. The 7-day fermentation in DPY medium involved daily titer measurements. Although the hydrolases had a modest impact, the titer reached around 3.2 mg/L, marking a fourfold increase from the previous experiment. We conducted protein-level engineering of the cis-ER domain in SQTKS, introducing 10 mutation groups via yeast recombination into the complete SQTKS. These mutations were designed based on isolated ER mutants of SQTKS, analyzed through a combination of computational modeling and experimental assays with various mimic substrates by previous co-worker. After expressing them in A. oryzae NSAR1, we screened for correct transformants, performed fermentation and chemical extraction, and utilized LCMS for compound detection. However, no new compounds were generated in these expression experiments. We performed pathway-level engineering by integrating genome-mined genes from multiple biosynthetic gene clusters (BGCs) into A. oryzae NSAR1. We co-expressed genes from the four BGCs of sporogen AO1 27, hypoxylan A 73, eremoxylarin D 123 and PR-toxin 107. These four BGCs share a common core carbon skeleton, while possessing distinct tailoring enzymes. Twenty oxygenated aristolochene congeners were synthesized, and their structures were characterized, featuring notable compounds such as the natural product hypoxylan A 73 and an epimer of guignaderemophilane C 37. A novel fungal aromatase enzyme has been identified, which catalyses the production of phenols via oxidative demethylation. We investigated fungal pathways for tetraketide multiforisins and islandic acid-related compounds. Heterologous expression experiments yielded high titers of these compounds and pathway intermediates, leading to the structure characterization of 14 isolated compounds, including multiforisin H 143h and I 143i. These results not only clarified the pathway but also laid the groundwork for the total biosynthesis of this metabolite class. Attempts to add genes from the islandic acid BGC into A. oryzae for synthesizing islandic acid 144 or Allantopyrone A 145 proved unsuccessful, as none of the transformants generated compounds beyond those already synthesized by the A. oryzae hosts.
AB - This investigation aims to strategically engineer the biosynthesis of fungal natural products through heterologous expression. It includes modifications to the heterologous host to boost productivity; leveraging the product diversity of an individual enzyme via mutasynthesis and combining biosynthetic genes from multiple pathways in a single heterologous host to creat new compounds. To improve tetraketide 25 titer in A. oryzae NSAR1 through squalestatin tetraketide synthase (SQTKS) expression, we co-expressed two hydrolase-encoding genes. The 7-day fermentation in DPY medium involved daily titer measurements. Although the hydrolases had a modest impact, the titer reached around 3.2 mg/L, marking a fourfold increase from the previous experiment. We conducted protein-level engineering of the cis-ER domain in SQTKS, introducing 10 mutation groups via yeast recombination into the complete SQTKS. These mutations were designed based on isolated ER mutants of SQTKS, analyzed through a combination of computational modeling and experimental assays with various mimic substrates by previous co-worker. After expressing them in A. oryzae NSAR1, we screened for correct transformants, performed fermentation and chemical extraction, and utilized LCMS for compound detection. However, no new compounds were generated in these expression experiments. We performed pathway-level engineering by integrating genome-mined genes from multiple biosynthetic gene clusters (BGCs) into A. oryzae NSAR1. We co-expressed genes from the four BGCs of sporogen AO1 27, hypoxylan A 73, eremoxylarin D 123 and PR-toxin 107. These four BGCs share a common core carbon skeleton, while possessing distinct tailoring enzymes. Twenty oxygenated aristolochene congeners were synthesized, and their structures were characterized, featuring notable compounds such as the natural product hypoxylan A 73 and an epimer of guignaderemophilane C 37. A novel fungal aromatase enzyme has been identified, which catalyses the production of phenols via oxidative demethylation. We investigated fungal pathways for tetraketide multiforisins and islandic acid-related compounds. Heterologous expression experiments yielded high titers of these compounds and pathway intermediates, leading to the structure characterization of 14 isolated compounds, including multiforisin H 143h and I 143i. These results not only clarified the pathway but also laid the groundwork for the total biosynthesis of this metabolite class. Attempts to add genes from the islandic acid BGC into A. oryzae for synthesizing islandic acid 144 or Allantopyrone A 145 proved unsuccessful, as none of the transformants generated compounds beyond those already synthesized by the A. oryzae hosts.
U2 - 10.15488/17169
DO - 10.15488/17169
M3 - Doctoral thesis
CY - Hannover
ER -