First-class - biosynthesis of 6-MSA and bostrycoidin type I polyketides in Yarrowia lipolytica.

Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in filamentous fungi presents some challenges: small yield and low-purity titers. To tackle these issues, we switched to the yeast Yarrowia lipolytica, an easily cultivable heterologous host. As an oleaginous yeast, Y. lipolytica displays a high flux of acetyl- and malonyl-CoA precursors used in lipid synthesis. Likewise, acetyl- and malonyl-CoA are the building blocks of many natural polyketides, and we explored the possibility of redirecting this flux toward polyketide production. Despite its promising prospect, Y. lipolytica has so far only been used for heterologous expression of simple type III polyketide synthases (PKSs) from plants. Therefore, we decided to evaluate the potential of Y. lipolytica by targeting the more complex fungal polyketides synthesized by type I PKSs. We employed a CRISPR-Cas9-mediated genome editing method to achieve markerless gene integration of the genes responsible for bostrycoidin biosynthesis in Fusarium solani (fsr1, fsr2, and fsr3) and 6-methylsalicylic acid (6-MSA) biosynthesis in Aspergillus hancockii (6MSAS). Moreover, we attempted titer optimization through metabolic engineering by overexpressing two enzymes, TGL4 and AOX2, involved in lipid ß-oxidation, but we did not observe an effect on polyketide production. With maximum titers of 403 mg/L 6-MSA and 35 mg/L bostrycoidin, the latter being substantially higher than our previous results in Saccharomyces cerevisiae (2.2 mg/L), this work demonstrates the potential of Y. lipolytica as a platform for heterologous production of complex fungal polyketides.

Yeast recombinational cloning for heterologous biosynthesis of polyketides: a molecular microbiology laboratory module for undergraduate students.

Recombinant plasmids are essential tools in molecular biotechnology, and reliable plasmid assembly methods have, therefore, become a prerequisite for the successful cloning and transfer of genes. Among the multitude of available plasmid assembly strategies, in vivo homologous recombinational cloning in yeast has emerged as a cost-effective and relatively simple method. Since we use this method routinely in our group for assembling large plasmids with secondary metabolite gene clusters and for direct heterologous production of polyketides in Saccharomyces cerevisiae, we developed an exercise module for undergraduate students where they would get hands-on experience with these molecular practices. The exercises target several molecular techniques, including PCR, restriction enzyme digestion, and yeast recombinational cloning. The students will learn about plasmid assembly and yeast transformation methods by performing these experiments while inherently acquiring new skills valuable for their subsequent laboratory work or projects.

Filling out the gaps - identification of fugralins as products of the PKS2 cluster in Fusarium graminearum.

As one of the grain crop pathogenic fungi with the greatest impacts on agricultural economical as well as human health, an elaborate understanding of the life cycle and subsequent metabolome of Fusarium graminearum is of great interest. Throughout the lifetime of the fungus, it is known to produce a wide array of secondary metabolites, including polyketides. One of the F. graminearum polyketides which has remained a mystery until now has been elucidated in this work. Previously, it was suggested that the biosynthetic product of the PKS2 gene cluster was involved in active mycelial growth, the exact mechanism, however, remained unclear. In our work, disruption and overexpression of the PKS2 gene in F. graminearum enabled structural elucidation of a linear and a cyclic tetraketide with a double methyl group, named fugralin A and B, respectively. Further functional characterization showed that the compounds are not produced during infection, and that deletion and overexpression did not affect pathogenicity or visual growth. The compounds were shown to be volatile, which could point to possible functions that can be investigated further in future studies.

6-MSA, a secondary metabolite distribution hub with multiple fungal destinations.

6-methylsalicylic acid (6-MSA) is a small, simple polyketide produced by a broad spectrum of fungal species. Since fungi obtained the ability to synthesize 6-MSA from bacteria through a horizontal gene transfer event, it has developed into a multipurpose metabolic hub from where numerous complex compounds are produced. The most relevant metabolite from a human perspective is the small lactone patulin as it is one of the most potent mycotoxins. Other important end products derived from 6-MSA include the small quinone epoxide terreic acid and the prenylated yanuthones. The most advanced modification of 6-MSA is observed in the aculin biosynthetic pathway, which is mediated by a non-ribosomal peptide synthase and a terpene cyclase. In this short review, we summarize for the first time all the possible pathways that takes their onset from 6-MSA and provide a synopsis of the responsible gene clusters and derive the resulting biosynthetic pathways.

Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Fusarium sp. as a Heterologous Host.

Heterologous expression of uncharacterized biosynthetic gene clusters is a popular strategy for exploring the chemical potential of filamentous fungi. Here, we describe the process of PCR-amplifying fungal gene clusters and re-assembling them in a cloning vector via target-associated recombination in Saccharomyces cerevisiae . The gene cluster-carrying construct is validated and used to transform protoplasts of Fusarium graminearum , a well-studied host that is able to express the gene cluster. Chemical analysis of transformants expressing biosynthetic genes can lead to the detection and isolation of novel compounds, such as polyketides.

Targeted Genetic Engineering via Agrobacterium-Mediated Transformation in Fusarium solani.

Members of the Fusarium solani species complex are filamentous fungi that can act as pathogens to many crops and animals. Although relevant, a robust molecular toolbox is missing for the investigation of gene function and metabolism. In this chapter, we describe how Agrobacterium-mediated transformation can be used to facilitate gene targeting. A flexible vector system, based on in vivo recombination in Saccharomyces cerevisiae, is utilized to achieve overexpression and gene deletion of targeted biosynthetic genes in F. solani f. sp. pisi.

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase.

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS's, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.

The H4K20 methyltransferase Kmt5 is involved in secondary metabolism and stress response in phytopathogenic Fusarium species.

Fusarium fujikuroi and Fusarium graminearum are agronomically important plant pathogens, both infecting important staple food plants and thus leading to huge economic losses worldwide. F.fujikuroi belongs to the Fusarium fujikuroi species complex (FFSC) and causes bakanae disease on rice, whereas F.graminearum, a member of the Fusarium graminearum species complex (FGSC), is the causal agent of Fusarium Head Blight (FHB) disease on wheat, barley and maize. In recent years, the importance of chromatin regulation became evident in the plant-pathogen interaction. Several processes, including posttranslational modifications of histones, have been described as regulators of virulence and the biosynthesis of secondary metabolites. In this study, we have functionally characterised methylation of lysine 20 histone 4 (H4K20me) in both Fusarium species. We identified the respective genes solely responsible for H4K20 mono-, di- and trimethylation in F.fujikuroi (FfKMT5) and F.graminearum (FgKMT5). We show that loss of Kmt5 affects colony growth in F.graminearum while this is not the case for F.fujikuroi. Similarly, FgKmt5 is required for full virulence in F.graminearum as Δfgkmt5 is hypovirulent on wheat, whereas the F.fujikuroi Δffkmt5 strain did not deviate from the wild type during rice infection. Lack of Kmt5 had distinct effects on the secondary metabolism in both plant pathogens with the most pronounced effects on fusarin biosynthesis in F.fujikuroi and zearalenone biosynthesis in F.graminearum. Next to this, loss of Kmt5 resulted in an increased tolerance towards oxidative and osmotic stress in both species.

Production and Selectivity of Key Fusarubins from Fusarium solani due to Media Composition.

Natural products display a large structural variation and different uses within a broad spectrum of industries. In this study, we investigate the influence of carbohydrates and nitrogen sources on the production and selectivity of production of four different polyketides produced by Fusarium solani, fusarubin, javanicin, bostrycoidin and anhydrofusarubin. We introduce four different carbohydrates and two types of nitrogen sources. Hereafter, a full factorial design was applied using combinations of three levels of sucrose and three levels of the two types of nitrogen. Each combination displayed different selectivity and production yields for all the compounds of interest. Response surface design was utilized to investigate possible maximum yields for the surrounding combinations of media. It was also shown that the maximum yields were not always the ones illustrating high selectivity, which is an important factor for making purification steps easier. We visualized the production over time for one of the media types, illustrating high yields and selectivity.

The effects of different potato dextrose agar media on secondary metabolite production in Fusarium.

Potatoes contain several nutrients essential for fungal growth, making them an excellent component of media such as the popular Potato Dextrose Agar (PDA) medium. Commercially, PDA is available from multiple retailers offering virtually the same product. These media, however, could contain small differences in composition of nutrients affecting the expression of secondary metabolites. This study aims to investigate the use of four PDA media from different manufacturers (Fluka, Oxoid, Sigma, and VWR) and their effect on the metabolite profile of four species of Fusarium (F. fujikuroi, F. graminearum, F. pseudograminearum and F. avenaceum). Secondary metabolites were analysed using HPLC-HRMS, from which statistically significant differences in intensities were observed for 9 out of 10 metabolites.

Heterologous Expression of the Core Genes in the Complex Fusarubin Gene Cluster of Fusarium Solani.

Through stepwise recreation of the biosynthetic gene cluster containing PKS3 from Fusarium solani, it was possible to produce the core scaffold compound of bostrycoidin, a red aza-anthraquinone pigment in Saccharomyces cerevisiae. This was achieved through sequential transformation associated recombination (TAR) cloning of FvPPT, fsr1, fsr2, and fsr3 into the pESC-vector system, utilizing the inducible bidirectional galactose promoter for heterologous expression in S. cerevisiae. The production of the core metabolite bostrycoidin was investigated through triplicate growth cultures for 1-4 days, where the maximum titer of bostrycoidin was achieved after 2 days of induction, yielding 2.2 mg/L.

Simulation of electrochemical properties of naturally occurring quinones.

Quinones are produced in organisms and are utilized as electron transfer agents, pigments and in defence mechanisms. Furthermore, naturally occurring quinones can also be cytotoxins with antibacterial properties. These properties can be linked to their redox properties. Recent studies have also shown that quinones can be utilized in flow battery technology, though naturally occurring quinones have not yet been investigated. Here, we have analyzed the properties of 990 different quinones of various biological sources through a computation approach to determine their standard reduction potentials and aqueous solubility. The screening was performed using the PBE functional and the 6-31G** basis set, providing a distribution of reduction potentials of the naturally occurring quinones varying from - 1.4 V to 1.5 V vs. the standard hydrogen electrode. The solvation energy for each quinone, which indicates the solubility in aqueous solution, was calculated at the same level. A large distribution of solubilities was obtained, containing both molecules that show tendencies of good solubilities and molecules that do not. The solubilities are dependent on the nature of the side groups and the size of the molecules. Our study shows that the group containing the quinones of fungal origin, which is also the largest of the groups considered, has the largest antimicrobial and electrochemical potential, when considering the distribution of reduction potentials for the compounds.

Characterization of Eight Novel Spiroleptosphols from Fusarium avenaceum.

Chemical analyses of Fusarium avenaceum grown on banana medium resulted in eight novel spiroleptosphols, T1, T2 and U-Z (1-8). The structures were elucidated by a combination of high-resolution mass spectrometric data and 1- and 2-D NMR experiments. The relative stereochemistry was assigned by 1H coupling and NOESY/ROESY experiments. Absolute stereochemistry established for 7 by vibrational circular dichroism was found analogous to that of the putative polyketide spiroleptosphol from Leptosphaeria doliolum.

Evidence of a Demethylase-Independent Role for the H3K4-Specific Histone Demethylases in Aspergillus nidulans and Fusarium graminearum Secondary Metabolism.

Fungi produce a plethora of secondary metabolites (SMs) involved in cellular protection, defense, and signaling. Like other metabolic processes, transcription of SM biosynthesis genes is tightly regulated to prevent an unnecessary use of resources. Genes involved in SM biosynthesis are usually physically linked, arranged in secondary metabolite gene clusters (SMGCs). Research over the last decades has shown that chromatin structure and posttranslational modifications (PTMs) of histones represent important layers of SMGC regulation. For instance, trimethylation of histone H3 lysine 4 (H3K4me3) is a PTM typically associated with promoter regions of actively transcribed genes. Previously, we have shown that the H3K4me3-specific, JmjC domain-containing histone demethylase KdmB functions not only in repression but also in activation of secondary metabolism in Aspergillus nidulans, suggesting that KdmB has additional functions apart from histone demethylation. In this study, we identified demethylase-independent functions of KdmB in transcriptional regulation of SM gene clusters. Furthermore, we show that this activating and demethylase-independent role of the H3K4 demethylase is also conserved in the phytopathogenic fungus Fusarium graminearum. Lack of FgKdm5 resulted in significant downregulation of five of seven analyzed SMs, whereby only one SMGC depends on a functional JmjC-domain. In A. nidulans strains deficient in H3K4 methylation, i.e., cclA∆, largely phenocopied kdmB∆, while this is not the case for most of the SMs analyzed in Fusarium spp. Notably, KdmB could not rescue the demethylase function in ∆fgkdm5 but restored all demethylase-independent phenotypes.

Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum.

Filamentous fungi such as species from the genus Fusarium are capable of producing a wide palette of interesting metabolites relevant to health, agriculture and biotechnology. Secondary metabolites are formed from large synthase/synthetase enzymes often encoded in gene clusters containing additional enzymes cooperating in the metabolite's biosynthesis. The true potential of fungal metabolomes remain untapped as the majority of secondary metabolite gene clusters are silent under standard laboratory growth conditions. One way to achieve expression of biosynthetic pathways is to clone the responsible genes and express them in a well-suited heterologous host, which poses a challenge since Fusarium polyketide synthase and non-ribosomal peptide synthetase gene clusters can be large (e.g. as large as 80 kb) and comprise several genes necessary for product formation. The major challenge associated with heterologous expression of fungal biosynthesis pathways is thus handling and cloning large DNA sequences. In this paper we present the successful workflow for cloning, reconstruction and heterologous production of two previously characterized Fusarium pseudograminearum natural product pathways in Fusarium graminearum. In vivo yeast recombination enabled rapid assembly of the W493 (NRPS32-PKS40) and the Fusarium Cytokinin gene clusters. F. graminearum transformants were obtained through protoplast-mediated and Agrobacterium tumefaciens-mediated transformation. Whole genome sequencing revealed isolation of transformants carrying intact copies the gene clusters was possible. Known Fusarium cytokinin metabolites; fusatin, 8-oxo-fusatin, 8-oxo-isopentenyladenine, fusatinic acid together with cis- and trans-zeatin were detected by liquid chromatography and mass spectrometry, which confirmed gene functionality in F. graminearum. In addition the non-ribosomal lipopeptide products W493 A and B was heterologously produced in similar amounts to that observed in the F. pseudograminearum doner. The Fusarium pan-genome comprises more than 60 uncharacterized putative secondary metabolite gene clusters. We nominate the well-characterized F. graminearum as a heterologous expression platform for Fusarium secondary metabolite gene clusters, and present our experience cloning and introducing gene clusters into this species. We expect the presented methods will inspire future endevours in heterologous production of Fusarium metabolites and potentially aid the production and characterization of novel natural products.

Advances in linking polyketides and non-ribosomal peptides to their biosynthetic gene clusters in Fusarium.

The eukaryotic ascomycete genus Fusarium comprises many species capable of producing secondary metabolites important for agriculture, health, and biotechnology. Filamentous fungi share common physiological features, but even within Fusarium, there are significant differences that affect the success of biotechnological methods used to unravel biosynthetic pathways. The aim of this review is to describe the different methods that have successfully been used throughout the genus Fusarium to identify the products of novel biosynthetic pathways. The results are presented in tables to give the reader an overview and thereby enable the selection of the most appropriate method to the problem, regarding both species and target products. Significant work has gone into characterization of the underlying molecular genetics of secondary metabolites, but still, the products of only 25-30% of predicted gene clusters have been identified. In this review, we highlight existing knowledge and encourage the development of new techniques and strategies to provide access to the many unknown polyketide and non-ribosomal peptide products that await discovery in Fusarium.

Fusaoctaxin A, an Example of a Two-Step Mechanism for Non-Ribosomal Peptide Assembly and Maturation in Fungi.

Fungal non-ribosomal peptide synthetase (NRPS) clusters are spread across the chromosomes, where several modifying enzyme-encoding genes typically flank one NRPS. However, a recent study showed that the octapeptide fusaoctaxin A is tandemly synthesized by two NRPSs in Fusarium graminearum. Here, we illuminate parts of the biosynthetic route of fusaoctaxin A, which is cleaved into the tripeptide fusatrixin A and the pentapeptide fusapentaxin A during transport by a cluster-specific ABC transporter with peptidase activity. Further, we deleted the histone H3K27 methyltransferase kmt6, which induced the production of fusaoctaxin A.

There it is! Fusarium pseudograminearum did not lose the fusaristatin gene cluster after all.

Fusarium pseudograminearum is a significant pathogen of cereals in arid regions worldwide and has the ability to produce numerous bioactive secondary metabolites. The genome sequences of seven F. pseudograminearum strains have been published and in one of these strains, C5834, we identified an intact gene cluster responsible for biosynthesis of the cyclic lipopeptide fusaristatin A. The high level of sequence identity of the fusaristatin cluster remnant in strains that do not produce fusaristatin suggests that the absence of the cluster evolved once, and subsequently the resulting locus with the cluster fragments became widely dispersed among strains of F. pseudograminearum in Australia. We examined a selection of 99 Australian F. pseudograminearum isolates to determine how widespread the ability to produce fusaristatin A is in F. pseudograminearum. We identified 15 fusaristatin producing strains, all originating from Western Australia. Phylogenetic analyses could not support a division of F. pseudograminearum into fusaristatin producing and nonproducing populations, which could indicate the loss has occurred relatively recent.

A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani.

BACKGROUND: Besides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation. RESULTS: To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)2Cys6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin. CONCLUSION: By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (PKS3: fsr) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094.

Enhancing the Production of the Fungal Pigment Aurofusarin in Fusarium graminearum.

There is an increasing demand for products from natural sources, which includes a growing market for naturally-produced colorants. Filamentous fungi produce a vast number of chemically diverse pigments and are therefore explored as an easily accessible source. In this study we examine the positive regulatory effect of the transcription factor AurR1 on the aurofusarin gene cluster in Fusarium graminearum. Proteomic analyses showed that overexpression of AurR1 resulted in a significant increase of five of the eleven proteins belonging to the aurofusarin biosynthetic pathway. Further, the production of aurofusarin was increased more than threefold in the overexpression mutant compared to the wild type, reaching levels of 270 mg/L. In addition to biosynthesis of aurofusarin, several yet undescribed putative naphthoquinone/anthraquinone analogue compounds were observed in the overexpression mutant. Our results suggest that it is possible to enhance the aurofusarin production through genetic engineering.