Details
Original language | English |
---|---|
Qualification | Doctor rerum naturalium |
Awarding Institution | |
Supervised by |
|
Date of Award | 10 Sept 2021 |
Place of Publication | Hannover |
Publication status | Published - 2021 |
Abstract
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
Hannover, 2021. 241 p.
Research output: Thesis › Doctoral thesis
}
TY - BOOK
T1 - Understanding and Engineering the Biosynthesis of Tropolone Sesquiterpenoids in Fungi
AU - Schotte, Carsten
N1 - Doctoral thesis
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
U2 - 10.15488/11361
DO - 10.15488/11361
M3 - Doctoral thesis
CY - Hannover
ER -