dc.identifier.uri |
http://dx.doi.org/10.15488/11361 |
|
dc.identifier.uri |
https://www.repo.uni-hannover.de/handle/123456789/11448 |
|
dc.contributor.author |
Schotte, Carsten
|
eng |
dc.date.accessioned |
2021-10-14T07:36:44Z |
|
dc.date.available |
2021-10-14T07:36:44Z |
|
dc.date.issued |
2021 |
|
dc.identifier.citation |
Schotte, Carsten: Understanding and engineering the biosynthesis of tropolone sesquiterpenoids in fungi. Hannover : Gottfried Wilhelm Leibniz Universität, Diss., 2021, VII, 219 S. DOI: https://doi.org/10.15488/11361 |
eng |
dc.description.abstract |
Tropolone sesquiterpenoids (TS) are meroterpenoid natural products that share the conserved structural feature of a polyketide-derived tropolone nucleus connected to a humulene-derived macrocycle via a bridging dihydropyran ring. Here, the biosynthesis of the TS xenovulene A [1] was investigated using a combination of heterologous gene expression in the fungal host Aspergillus oryzae NSAR1 and in E. coli BL21. Heterologous expression experiments validated a minimal xenovulene A [1] biosynthetic gene set encoding eight dedicated enzymes involved in tropolone formation, humulene formation and DIELS-ALDER chemistry. Reconstitution of key enzymatic steps in vitro identified a new type of class I terpene cyclase (AsR6), that catalyzes the stereoselective formation of ɑ-humulene [2] from farnesyl pyrophosphate [3] or either enantiomer of nerolidyl pyrophosphate [4]. The biosynthesis of the structurally related bistropolones eupenifeldin [5] and noreupenifeldin B [6] in Phaeosphaeriaceae sp. CF-150626 was also investigated. Isotopic labelling studies identified an unusual oxidative ring contraction that putatively converts [5] into [6]. Through a combined genetic and chemical approach, a candidate biosynthetic gene cluster for [5]-biosynthesis was identified (the eup2 BGC). With EupR3 a homologue of AsR6 was characterized that stereoselectively produces 2Z-humulene [7], a geometric isomer of [2]. In cooperation with the Helmholtz Institute for Infection Research (Braunschweig, Germany) the crystal structure of AsR6 in the unliganded state and in complex with thiolo-S-diphosphate [8] and an in crystallo cyclized reaction product was obtained. A new pyrophosphate binding site was identified that consists of a binuclear magnesium cluster and a conserved lysine residue. Site- directed mutagenesis validated the motif and identified a key amino acid residue, L/M285, that drives the stereoselective formation of either [2] or [7]. New-to-nature TS natural products were produced through heterologous expression of different combinations of biosynthetic enzymes from the xenovulene A [1], eupenifeldin [5] and pycnidione [9] pathways. The rational design of expression experiments resulted in the formation and characterization of seven new derivatives. The obtained non-natural products differ in the nature of the polyketide moiety, the substitution pattern of the humulene macrocycle and the degree of hydroxylation. |
eng |
dc.language.iso |
eng |
eng |
dc.publisher |
Hannover : Institutionelles Repositorium der Leibniz Universität Hannover |
|
dc.rights |
CC BY 3.0 DE |
eng |
dc.rights.uri |
http://creativecommons.org/licenses/by/3.0/de/ |
eng |
dc.subject |
tropolone sesquiterpenoid |
eng |
dc.subject |
biosynthesis |
eng |
dc.subject |
pathway engineering |
eng |
dc.subject |
humulene |
eng |
dc.subject |
fungi |
eng |
dc.subject |
Tropolonsesquiterpenoid |
ger |
dc.subject |
Biosynthese |
ger |
dc.subject |
Pathway Engineering |
ger |
dc.subject |
Humulene |
ger |
dc.subject |
Pilz |
ger |
dc.subject.ddc |
500 | Naturwissenschaften
|
eng |
dc.title |
Understanding and engineering the biosynthesis of tropolone sesquiterpenoids in fungi |
eng |
dc.type |
DoctoralThesis |
eng |
dc.type |
Text |
eng |
dcterms.extent |
VII, 219 S. |
|
dc.description.version |
publishedVersion |
eng |
tib.accessRights |
frei zug�nglich |
eng |