Genome mining and biosynthetic investigation of secondary metabolites produced by fungal highly reducing polyketide synthases

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dc.contributor.advisorChooi, Heng
dc.contributor.advisorFlematti, Gavin
dc.contributor.advisorShang, Zhuo
dc.contributor.authorArishi, Amr Abker Y
dc.date.accessioned2026-05-12T10:06:11Z
dc.date.issued2026
dc.descriptionFilamentous fungi are remarkable producers of bioactive secondary metabolites with significant pharmaceutical and agricultural value. In particular, fungal polyketides produced by highly reducing polyketide synthases (HR-PKSs) represent an important source of lead compounds for future drug discovery. Polyketide biosynthesis is a complex and highly programmed process that ensures precise control over carbon chain length. Following polyketide chain assembly, the mature product is released from HR-PKSs through well-coordinated mechanisms, often involving collaboration with trans-acting releasing enzymes. Although several HR-PKS pathways have been characterised, the diversity and mechanistic complexity of the reactions they catalyse extend far beyond current understanding. Herein, the aim of thisthesis is to advance the understanding of HR-PKS function in the context of their biosynthetic gene clusters (BGCs) by investigating how interactions with various tailoring enzymes shape polyketide structural diversity. Through genome mining and heterologous expression, this work uncovers novel HR-PKS biosynthetic pathways, elucidates various product-releasing mechanisms, and identifies new metabolites. The results presented in this thesis provide deeper insight into the biosynthetic logic of HR-PKS pathways and the chemical diversity they generate. In Chapter 1, I introduce fungal secondary metabolites in general and then focus on polyketides produced by HR-PKSs, their biosynthesis, and product-releasing mechanisms. I also outline the two main genome-mining strategies used to discover novel polyketides and provide an overview of targeted genome-mining approaches for uncovering bioactive polyketides. In addition, I discuss selected examples of HR-PKSs that employ distinct product-releasing enzymes, which together motivated the work presented in the subsequent chapters. In Chapter 2, I present my work on the metabologenomic characterisation of novel Australian fungus Aspergillus luteorubrus, which led to the discovery of luteodienoside A. In this Chapter, I performed a comprehensive chemical and genomic analyses of A. luteorubrus. Although genome analysis revealed that A. luteorubrus harbours more than 40 BGCs for secondary metabolite biosynthesis, only ten metabolites were isolated from culture extracts, indicating a largely untapped biosynthetic potential. Among these metabolites, a novel glycosylated polyketide, named luteodienoside A was identified. Guided by retrobiosynthetic analysis, I subsequently identified a putative BGC responsible for luteodienoside A biosynthesis (ltb BGC). The ltb BGC encodes an HR-PKS LtbA fused at its C-terminus to a carnitine O-acyl transferase (cAT) domain. Heterologous expressions of the ltb BGC in Aspergillus nidulans led to the production of luteodienoside A alongside pathway-related intermediates and revealed that the HR-PKS LtbA cAT domain utilises glucinol as the releasing substrate for product offloading. This study reports the first example of an HR-PKS cAT domain employing an unusual carbocyclic sugar, glucinol, as a product-releasing substrate. In Chapter 3, I describe my contribution to the discovery of the biosynthetic genes responsible for cerulenin biosynthesis, the first reported fatty acid synthase inhibitor, in two Sarocladium species. Beyond its irreversible inhibition of fatty acid biosynthesis, cerulenin exhibits a broad spectrum of bioactivities, including antifungal, anti-obesity, and anticancer effects. Despite decades of study, the molecular basis of cerulenin biosynthesis had remained unresolved for over sixty years. In this work, I employed a self-resistance gene-guided genome-mining strategy to identify the cerulenin BGC (the cer BGC), comprising 10 genes conserved in both Sarocladium attenuatum and Sarocladium oryzae. Heterologous expression of the cer BGC in Aspergillus nidulans, followed by incubation with XAD-16 resin, enabled elucidation of the complete cerulenin biosynthetic pathway. The pathway begins with formation of a C12 polyketide-derived polyene featuring a remarkable 2Z,4E,6Z,8Z,10Z double-bond configuration and proceeds through a series of complex tailoring steps, including amidation, epoxidation, double-bond migration, E/Z isomerisation, and epoxide reduction. In addition to cerulenin, I isolated and characterised 18 new analogues and pathway shunt products. These results reveal new insight into the complexity of HR-PKS tailoring chemistry and open new opportunities for the future biosynthetic engineering of cerulenin and its analogues. Following characterisation of the cerulenin biosynthetic pathway in Chapter 3, I noticed that many BGCs encoded in the two Sarocladium species were associated with unknown metabolites. This observation motivated me to perform targeted genome-mining strategy aimed at identifying unusual HR-PKS BGCs featuring atypical enzymatic collaborations within these fungal genomes. In Chapter 4, I describe the genome mining and functional characterisation of a conserved, previously uncharacterised BGC (the sarc BGC) from the two cerulenin producing Sarocladium species. The sarc BGC encodes co-localised HR-and type III PKSs (T3PKS) enzymes. Heterologous expression of the sarc BGC in Aspergillus nidulans led to the discovery of novel alkylresorcinols, sarocladones A-I, produced through collaboration between the HR-PKS SarcA and the T3PKS SarcB. To further expand structural diversity, I identified a homologue BGC in Colletotrichum fructicola (the col BGC) and co-expressed the HR-PKS colA with the T3PKS sarcB. This resulted in the production of additional sarocladone analogues, collecladone A and B, which lack the C2- C3 double bond present in sarocladones. Biological evaluation revealed that selected sarocladones exhibited mild cytotoxicity against murine NS-1 myeloma cells and anti-phagocytotic activity against THP-1 macrophages, highlighting the biosynthetic and biological potential of metabolites produced through collaboration between HR-PKS and T3PKS. Fungal polyketide-derived β-lactones represent an important class of secondary metabolites with antibacterial, antifungal, and anticancer properties. Despite their pharmaceutical properties, the enzymatic mechanisms underlying β-lactone formation remain poorly understood. In Chapter 5, I investigated the biosynthesis of the fungal β-lactone hymeglusin, a highly specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS). Analysis of culture extracts from Neocosmospora suttoniana cultivated on rice revealed hymeglusin as a major metabolite. Accordingly, the genome of N. suttoniana was sequenced, annotated and mined for a BGC responsible for hymeglusin production. This analysis led to the identification of a candidate BGC, designated the hym BGC. Reconstitution of the hym BGC in Aspergillus nidulans revealed that two genes are essential for hymeglusin biosynthesis, including HR-PKS hymA and cytochrome P450 hymB. Expression of the HR-PKS hymA alone resulted in the accumulation of new polyunsaturated polyketides, suttonic acids A–C. A self-resistance assay demonstrated that the cluster-associated HMGCS HymC, confers resistance to hymeglusin, distinguishing it from housekeeping HMGCS homologues. Overall, characterisation of the hym pathway provides new insights into the fungal β-lactone biosynthesis by fungal HR-PKSs and advances our understanding of hymeglusin biosynthesis, paving the way for future engineering and large-scale production of novel analogues. In Chapter 6, I summarise the key findings of this thesis and discuss their significance within the broader context of fungal HR-PKSs. This Chapter also provides a general conclusion and a personal perspective on fungal HR-PKSs, as well as on the utility of Aspergillus nidulans as a heterologous host for expressing and characterising novel HR-PKSs pathways. Together, this work expands current understanding of secondary metabolites biosynthesis by HR-PKSs, addresses key questions related to polyketide biosynthesis and highlights complex chemical modifications catalysed by fungal HR-PKSs.
dc.description.abstractFungi produce diverse bioactive metabolites with pharmaceutical and agricultural relevance. Among these, polyketides generated by highly reducing polyketide synthases (HR-PKSs) are promising drug leads, although their biosynthetic mechanisms remain only partially understood. This thesis employs genome mining and heterologous expression to investigate selected fungal HR-PKS pathways. It reports the discovery of luteodienoside A, released via a glucinol-utilising cAT domain; elucidates the cerulenin biosynthetic pathway; characterises collaborative HR-PKS and type III PKS systems producing alkylresorcinols; and identifies genes essential for hymeglusin biosynthesis. Collectively, this work advances our understanding of HR-PKS-mediated biosynthesis and expands the known chemical diversity of fungal polyketides.
dc.format.extent615
dc.identifier.urihttps://hdl.handle.net/20.500.14154/78946
dc.language.isoen
dc.publisherSaudi Digital Library
dc.subjectfilamentous fungi
dc.subjectgenome mining
dc.subjecthighly reducing polyketide synthases
dc.subjectsecondary metabolites
dc.subjectbiosynthetic gene clusters
dc.subjectpolyketide biosynthesis
dc.subjectAustralian fung
dc.titleGenome mining and biosynthetic investigation of secondary metabolites produced by fungal highly reducing polyketide synthases
dc.typeThesis
sdl.degree.departmentSchool of Molecular Sciences
sdl.degree.disciplineMycology-Fungal Genetics and Biochemistry
sdl.degree.grantorThe University of Western Australia
sdl.degree.nameDoctor of Philosophy
sdl.thesis.sourceSACM - Australia

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