Investigating the Biosynthesis of Bioactive Compounds in Fungi Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktor der Naturwissenschaften (Dr. rer. nat.) genehmigte Dissertation von Vjaceslavs Hrupins, M. Sc. 2021 0 Referent: Prof. Dr. Russell J. Cox Korreferent: PD Dr. Carsten Zeilinger Tag der Promotion: 02.07.2021 I Abstract Fungal Type I polyketide synthases (PKS) are large multifunctional enzymes with a complex structure consisting of multiple functional domains. They produce complex bioactive products such as squalestatin S1 27 and byssochlamic acid 106 . The main focus of the projects lies on highly reducing iterative polyketide synthases, especially on their structure and function. Previous work has shown that generating structural information from cross-linked polyketide domains can be the first step towards the elucidation of function and programming. In this thesis we have cloned and heterologously expressed the squalestatin tetraketide synthase (SQTKS) KS/AT didomain, standalone DH domain and the ACP. The SQTKS KS/AT didomain and the ACP were heterologously expressed in E. coli but were obtained as insoluble (KS/AT) or inactive (ACP) proteins. The ACP and the standalone DH domain from the Byssochlamys fulva nonadride PKS (bfPKS) were expressed for the first time. Both proteins were successfully expressed in E. coli and showed catalytic activity as measured by mass spectrometry. The bfPKS ACP was used for crosslinking attempts. Biosynthetic enzymes were expressed and used for the enzymatic conversion of chemically synthesized pantetheine linker analogues into CoA linker analogues. A phosphopantetheine transferase (PPTase, also expressed) was used to load the linkers onto the bfPKS ACP. Cross-linking between bfPKS ACP and DH domains of SQTKS, bfPKS and the Strobilurin tenacellus strobilurin PKS (stPKS) were tried but no linking was observed. In vitro investigations of the B. fulva citrate synthase (bfCS) revealed its substrate preference. The bfCS showed conversion of CoA bound substrate but no conversion for the bfPKS ACP bound one. The SQTKS standalone methyltransferase ( C-MeT) domain did not convert the natural substrate which was bound to the bfPKS ACP and was inactive as in previous investigations. Keywords: Polyketides, Squalestatin S1, Acyl Carrier Protein, Byssochlamic Acid II Zusammenfassung Typ I Polyketid Synthasen (PKS) aus Pilzzellen sind große multifunktionale Enzyme, komplex in der Struktur und bestehen aus mehreren funktionalen Domänen. Sie produzieren komplexe, bioaktive Verbindungen wie Squalestatin S1 27 und Byssochlaminsäure 106 . Das Hauptaugenmerk der Projekte liegt auf der Struktur- und Funktionsaufklärung der reduzierenden, iterativ agierenden Polyketid Synthasen. Vorherige Arbeiten haben gezeigt, dass die Strukturaufklärung von miteinander verbundenen Polyketid Domänen der erste Schritt zum Verständnis der Funktion und Programmierung dieser Proteine sein kann. In dieser Arbeit haben wir die Squalestatin Tetraketid Synthase (SQTKS) KS/AT Didomäne, die alleinstehende DH Domäne und das ACP kloniert und heterolog exprimiert. Die SQTKS KS/AT Didomäne und das ACP wurden in E. coli heterolog exprimiert, resultierend in unlöslichem Protein (KS/AT) oder inaktivem (ACP) Protein. Das ACP und die alleinstehende DH Domäne von der Byssochlamys fulva Nonadrid PKS (bfPKS) wurden zum ersten Mal exprimiert. Beide Proteine konnten in E. coli erfolgreich exprimiert und waren katalytisch aktiv, was durch massenspektrometrische Messungen gezeigt werden konnte. Das bfPKS ACP wurde zur Verbindung mit anderen Domänen verwendet. Zusätzliche Enzyme wurden exprimiert, um die chemisch synthetisierten Pantethein Linker in CoA Analoga zu überführen. Eine Phosphopantethein Transferase (PPTase, ebenfalls exprimiert) wurde verwendet, um die Linker auf das bfPKS ACP zu beladen. Die Verbindung mittels Linker zwischen bfPKS ACP und den DH Domänen von SQTKS, bfPKS und der Strobilurin tenacellus strobilurin PKS (stPKS) wurden versucht, scheiterten jedoch. In vitro Untersuchungen der B. fulva Zitrat Synthase (bfCS) gaben Aufschluss über deren Substratpräferenz. Die bfCS akzeptierte CoA gebundene Substrate, jedoch keine bfPKS ACP gebundenen. Die alleinstehende SQTKS Methyltransferase ( C-MeT) konnte das natürliche Substrat, welches an dem bfPKS ACP gebunden war, nicht in das Produkt überführen und war, wie bereits in vorherigen Untersuchungen, inaktiv. Schlagwörter: Polyketide, Squalestatin S1, Acyl Carrier Protein, Byssochlamsäure III Acknowledgement First, I would like to thank Prof. Dr. Russell J. Cox for offering me this interesting and challenging project. I appreciate the good supervision, patience and the professional environment he was offering me. Thank you to PD Dr. Carsten Zeilinger for being the second examiner, sharing his experience in protein expression and purification and for having interesting conversations. A special thanks goes to Prof. Dr. Sascha Beutel for taking part as the third examiner. I would like to thank the former and current Cox group members (Liz, Haili, Dongsong, Chongqing, Steffen, Lei, Yunlong, Mary, Katharina, Hao, Carsten, Karen, Lukas, Eman, Raissa, Verena, Juliane and Henrike) making the time really enjoyable. Moreover, I want to thank especially Oliver Piech for many funny moments inside and outside of the lab. Sen Yin and Jin Feng gave me during my PhD time a small excursus into the chinese culture and language, thanks for this. The time in Frankfurt with Dr. Eric Kuhnert will be kept in good memory, thank you Eric. A special thanks goes to the media kitchen and the store, especially Katja Körner. A fast progress would not be possible without this team. Finally, I want to thank my fiancee, Nina Stehle, and my family for giving me the support during challenging times. IV Abbreviations 2TY 2x tryptone yeast medium ACP acyl carrier protein AT acyl transferase ATP adenosine triphosphate C-MeT C-methyltransferase CoA Coenzyme A Da Dalton DEBS 6-deoxyerythronolide B synthase DH dehydratase DNA desoxyribonucleic acid EDTA ethylenediaminetetraacetic acid EIC extracted ion chromatogram ER enoyl reductase ESI electron spray ionisation ELSD evaporative light scattering detector FAS fatty acid synthase FPLC fast protein liquid chromatography GST glutathione-S-transferase HPLC high performance liquid chromatography hr highly reducing IPTG isopropyl-β-D-1-thiogalactopyranoside KS ketosynthase kb kilo base pairs kDa kilo Daltons KR ketoreductase KS ketosynthase LC liquid chromatography MALDI matrix-assisted laser desorption ionization m/z mass to charge ratio MS mass spectrometry NADPH nicotinamide adenine dinucleotide phosphate Ni-NTA nickel-charged affinity resin V NMR nuclear magnetic resonance nr non-reducing NSAS norsolorinic acid synthase OD 600 optical density at 600 nm PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PDB protein data bank PKS polyketide synthase pr partially-reducing PT product template Q-TOF quadrupole time-of-flight SAM S-adenosylmethionine SAT starter unit:ACP transacylase SAXS small angle light scattering SDS sodium dodecyl sulphate SEC size exclusion chromatography Sfp 4’-phosphopantetheinyl trnasferase SNAC S-N-acetylcysteamine SOC super optimal broth with catabolite repression SQTKS squalestatin tetraketide synthase SUMO small ubiquitin-like modifier TAE tris-acetate-EDTA buffer TE thioesterase TEMED N,N,N,N -tetramethylethylenediamine TIC total ion current Tris tris(hydroxymethyl)aminomethane UV ultraviolet VI Table of Contents Abstract ………………………………………………………………………………... II Zusammenfassung ……………………………………………………………............. III Acknowledgement ……………………………………………………………………. IV Abbreviations …………………………………………………………………………. .V Table of Contents ……………………………………………………………………. VII 1. Introduction ……………………………………………………………….......... 1 1.1 Natural Products……………………………………………………...….. 1 1.2 Natural Products from Fungi……………………………………………..2 1.2.1 Main Classes of Fungal Secondary Metabolites……………….. 2 1.3 Fatty Acid Synthases…………………………………………………….. 4 1.3.1 Architectures of Fatty Acid Synthases…………………………. 4 1.3.2 Function of the Fatty Acid Synthase…………………………… 5 1.4 Polyketides and their Biosynthesis……………………………………… 7 1.4.1 Unusual Starter Units in Fungal Polyketides…………………. 11 1.4.2 Classification of Polyketide Synthases……………………….. 12 1.4.3 Non-Reducing Iterative Polyketide Synthases……………….. 14 1.4.4 Partially Reducing Iterative Polyketide Synthases………….... 14 1.4.5 Highly Reducing Iterative Polyketide Synthases…………..…. 15 1.5 Protein Structure and Mechanisms of Selected Domains…………….... 19 1.5.1 The Ketosynthase Domain………………………………...….. 19 1.5.2 The Acyltransferase Domain…………………………………. 21 1.5.3 The Acyl Carrier Protein…………………………………..….. 23 1.6 Structural Analysis of Crosslinked FAS/PKS Domains…………….…. 24 1.6.1 Crosslinking between an Acyl Carrier Protein and a Ketosynthase Domain…………………………………...……. 25 1.6.2 Crosslinking between an Acyl Carrier Protein and an Acyltransferase Domain……………………………………… 27 1.6.3 Crosslinking between an Acyl Carrier Protein and a Dehydratase Domain………………………………………….. 29 1.7 Main Aims for the Projects………………………………………..…… 30 VII 2. SQTKS KS/AT Di-Domain ……………………………………………..……. 32 2.1 Introduction………………………………………………………..…… 32 2.2 Aims……………………………………………………………….…… 33 2.3 Results………………………………………………………………….. 33 2.3.1 Bioinformatic Solubility Prediction…………………..………. 33 2.3.2 Expression of KS/AT Di-Domain in Bl21 DE3 Cells………... 36 2.3.3 Variation of Expression Strains………………………………. 37