Biosynthetic studies on the fungal metabolite tenelln

This thesis is divided into seven chapters plus appendices. The first chapter introduces tenellin (1), its origins, structure and biogenetic relationships to other metabolites. The second chapter concentrates on polyketides and through three representative examples explains their relationship to the fatty acids and current theories and evidence for a processive mechanism during their biosynthesis. An investigation carried out into the biosynthesis of the polyketide sidechain of tenellin (1) from sodium [l-(^13)C, 2-(^2)H(_3)] and [2-(^13)C, 2-(^2)H(_3)] acetates and L-[S-methyl-(^13)C(^2)2H(_3)]-methionine is described. The possible intermediacy of singly methylated diketides and doubly methylated triketides carrying stable isotopic labels during the biosynthesis of tenellin (1) is also tested experimentally and discussed. In the third chapter possible intermediates to both the 2-pyridone moiety of tenellin (1) and the tropic acid (42) moiety of the tropane alkaloids are discussed and tested experimentally. 3-Amino-2-phenylpropionic acid (104) was synthesised and rejected as an intermediate to tenellin (1) and tropic acid (42). In the case of tenellin (1) a tetramic add (139) was implicated as a possible intermediate by a biosynthetic feeding experiment. An alternative mechanism to account for 2-pyridone formation from a tetramic acid is put forward. In the fourth chapter the determination of the absolute configuration of tenellin (1) is described. The configuration of the chiral methyl bearing centre of tenellin (1) is compared to that of similar fungal polyketide derived molecules and the mode of reduction by various fungal PKS enoyl reductase systems is assessed. In the fifth chapter the syntheses and characterisations of the compounds labelled with (^14)c, (^13)c and (^2)H that were used in the biosynthetic studies on tenellin (1) and tropic acid (42) are described. Chapter six describes in detail the experimental procedures used in the syntheses of these compounds. Chapter seven consists of a description of the biological materials and methods used during this study. The appendices consist of abbreviations used in the text, and a list of lectures, seminars and coUoquia arranged within the Department of Chemistry and attended by the author

Generalized Model of Resonant Polymer-Coated Microcantilevers in Viscous Liquid Media

Expressions describing the resonant frequency and quality factor of a dynamically driven, polymer-coated microcantilever in a viscous liquid medium have been obtained. These generalized formulas are used to describe the effects the operational medium and the viscoelastic coating have on the device sensitivity when used in liquid-phase chemical sensing applications. Shifts in the resonant frequency are normally assumed proportional to the mass of sorbed analyte in the sensing layer. However, the expression for the frequency shift derived in this work indicates that the frequency shift is also dependent on changes in the sensing layer’s loss and storage moduli, changes in the moment of inertia, and changes in the medium of operation’s viscosity and density. Not accounting for these factors will lead to incorrect analyte concentration predictions. The derived expressions are shown to reduce to well-known formulas found in the literature for the case of an uncoated cantilever in a viscous liquid medium and the case of a coated cantilever in air or in a vacuum. The theoretical results presented are then compared to available chemical sensor data in aqueous and viscous solutions

Evidence for enzyme catalysed intramolecular [4+2] Diels-Alder cyclization during the biosynthesis of pyrichalasin H

Cytochalasans are highly complex fungal metabolites which exhibit diverse biological activities. Little is known of the chemical steps involved in the construction of the tricyclic core, which consists of an octahydro-isoindole skeleton fused to a macrocyclic ring. Here, using a directed gene knockout and complementation strategy, we show that PyiF is implicated as the proposed intramolecular [4+2] Diels-Alderase required for construction of the tricyclic core of pyrichalasin H 1. © 2020 The Royal Society of Chemistry

Reductive Release from a Hybrid PKS-NRPS during the Biosynthesis of Pyrichalasin H

Three central steps during the biosynthesis of cytochalasan precursors, including reductive release, Knoevenagel cyclisation and Diels Alder cyclisation are not yet understood at a detailed molecular level. In this work we investigated the reductive release step catalysed by a hybrid polyketide synthase non-ribosomal peptide synthetase (PKS-NRPS) from the pyrichalasin H pathway. Synthetic thiolesters were used as substrate mimics for in vitro studies with the isolated reduction (R) and holo-thiolation (T) domains of the PKS-NRPS hybrid PyiS. These assays demonstrate that the PyiS R-domain mainly catalyses an NADPH-dependent reductive release of an aldehyde intermediate that quickly undergoes spontaneous Knoevenagel cyclisation. The R-domain can only process substrates that are covalently bound to the phosphopantetheine thiol of the upstream T-domain, but it shows little selectivity for the polyketide

Trichoderma reesei Contains a Biosynthetic Gene Cluster That Encodes the Antifungal Agent Ilicicolin H

The trili biosynthetic gene cluster (BGC) from the well-studied organism Trichoderma reesei was studied by heterologous expression in the fungal host Aspergillus oryzae. Coexpression of triliA and triliB produces two new acyl tetramic acids. Addition of the ring-expanding cytochrome P450 encoded by triliC then yields a known pyridone intermediate to ilicicolin H and a new chain-truncated shunt metabolite. Finally, addition of the intramolecular Diels-Alderase encoded by triliD affords a mixture of 8-epi ilicicolin H and ilicicolin H itself, showing that the T. reesei trili BGC encodes biosynthesis of this potent antifungal agent. Unexpected A. oryzae shunt pathways are responsible for the production of the new compounds, emphasising the role of fungal hosts in catalysing diversification reactions

Biosynthesis of 6-Hydroxymellein Requires a Collaborating Polyketide Synthase-like Enzyme

The polyketide synthase (PKS)-like protein TerB, consisting of inactive dehydratase, inactive C-methyltransferase, and functional ketoreductase domains collaborates with the iterative non reducing PKS TerA to produce 6-hydroxymellein, a key pathway intermediate during the biosynthesis of various fungal natural products. The catalytically inactive dehydratase domain of TerB appears to mediate productive interactions with TerA, demonstrating a new mode of trans-interaction between iterative PKS components

Engineering Aspergillus oryzae for the Heterologous Expression of a Bacterial Modular Polyketide Synthase

Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may lead to molecules with enhanced biological activities. In this study, we modified the metabolism of the fungus Aspergillus oryzae to enable the synthesis of triketide lactone (TKL), the product of the modular polyketide synthase DEBS1-TE engineered from bacteria. We established (2S)-methylmalonyl-CoA biosynthesis via introducing a propionyl-CoA carboxylase complex (PCC); reassembled the 11.2 kb DEBS1-TE coding region from synthetic codon-optimized gene fragments using yeast recombination; introduced bacterial phosphopantetheinyltransferase SePptII; investigated propionyl-CoA synthesis and degradation pathways; and developed improved delivery of exogenous propionate. Depending on the conditions used titers of TKL ranged from <0.01–7.4 mg/L. In conclusion, we have demonstrated that A. oryzae can be used as an alternative host for the synthesis of polyketides from bacteria, even those that require toxic or non-native substrates. Our metabolically engineered A. oryzae may offer advantages over current heterologous platforms for producing valuable and complex natural products

Azaphilone Pigments from Hypoxylon rubiginosum and H. texense: Absolute Configuration, Bioactivity, and Biosynthesis

We report new stromatal azaphilone pigments rubiginosins Z-X from the ascomycete Hypoxylon rubiginosum, as well as rubiginosins Z and W from H. texense, which were isolated along with known monomeric and dimeric congeners. Structures were elucidated using comprehensive HRMS, NMR, and ECD analysis, revealing azaphilones from both fungi to be exclusively C-8(S)-configured. The orsellinic acid (OA)-carrying rubiginosins A, Z and dimeric rutilins A-B exhibited cytotoxicity. Rubiginosins X-W bearing linear polyketide side chains as well as rutilins A-B were antimicrobial. Structures of the differently-substituted azaphilones were linked to two putative biosynthetic gene clusters (BGCs; hraza1/2) in H. rubiginosum, which are proposed to collaboratively synthesize the OA-substituted azaphilones. These share high homology with the azaphilone-forming BGCs hfaza1/2 from H. fragiforme. Comparison of hraza and hfaza suggests that lack of an FAD-dependent monooxygenase and acyltransferase gene in hraza1 prevent formation of C-8(R)-configured fatty acid-substituted azaphilones in H. rubiginosum. The polyketide synthase-derived side chain of rubiginosins C and X-W is not encoded in the respective BGCs, showing that a third BGC is hypothetically involved in their formation. Cross-interaction of three BGCs which are forming a single molecule is unprecedented in fungal natural product biosynthesis

Diels–Alder Reactions During the Biosynthesis of Sorbicillinoids

The sorbicillinoids are a class of biologically active and structurally diverse fungal polyketides arising from sorbicillin. Through co-expression of sorA, sorB, sorC, and sorD from Trichoderma reesei QM6a, the biosynthetic pathway to epoxysorbicillinol and dimeric sorbicillinoids, which resemble Diels–Alder-like and Michael-addition-like products, was reconstituted in Aspergillus oryzae NSAR1. Expression and feeding experiments demonstrated the crucial requirement of the flavin-dependent monooxygenase SorD for the formation of dimeric sorbicillinoids, hybrid sorbicillinoids, and epoxysorbicillinol in vivo. In contrast to prior reports, SorD catalyses neither the oxidation of 2',3'-dihydrosorbicillin to sorbicillin nor the oxidation of sorbicillinol to oxosorbicillinol. This is the first report that both the intermolecular Diels–Alder and Michael dimerization reactions, as well as the epoxidation of sorbicillinol are catalysed in vivo by SorD. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA

Heterologous Expression of Secondary Metabolite Genes in Trichoderma reesei for Waste Valorization

Trichoderma reesei (Hypocrea jecorina) was developed as a microbial cell factory for the heterologous expression of fungal secondary metabolites. This was achieved by inactivation of sorbicillinoid biosynthesis and construction of vectors for the rapid cloning and expression of heterologous fungal biosynthetic genes. Two types of megasynth(et)ases were used to test the strain and vectors, namely a non-reducing polyketide synthase (nr-PKS, aspks1) from Acremonium strictum and a hybrid highly-reducing PKS non-ribosomal peptide synthetase (hr-PKS-NRPS, tenS + tenC) from Beauveria bassiana. The resulting engineered T. reesei strains were able to produce the expected natural products 3-methylorcinaldehyde and pretenellin A on waste materials including potato, orange, banana and kiwi peels and barley straw. Developing T. reesei as a heterologous host for secondary metabolite production represents a new method for waste valorization by the direct conversion of waste biomass into secondary metabolites