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Black apples are the result of late-stage microbial decomposition after falling to the ground. This phenomenon is highly comparable from year to year, with the filamentous fungus Monilinia fructigena most commonly being the first invader, followed by Penicillium expansum . Motivated by the fact that only little chemistry has been reported from apple microbiomes, we set out to investigate the chemical diversity and potential ecological roles of secondary metabolites (SMs) in a total of 38 black apples. Metabolomics analyses were conducted on either whole apples or small excisions of fungal biomass derived from black apples. Annotation of fungal SMs in black apple extracts was aided by the cultivation of 15 recently isolated fungal strains on 9 different substrates in a One Strain Many Compounds (OSMAC) approach, leading to the identification of 3,319 unique chemical features. Only 6.4% were attributable to known compounds based on analysis of high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS/MS) data using spectral library matching tools. Of the 1,606 features detected in the black apple extracts, 32% could be assigned as fungal-derived, due to their presence in the OSMAC-based training data set. Notably, the detection of several antifungal compounds indicates the importance of such compounds for the invasion of and control of other microbial competitors on apples. In conclusion, the diversity and abundance of microbial SMs on black apples were found to be much higher than that typically observed for other environmental microbiomes. Detection of SMs known to be produced by the six fungal species tested also highlights a succession of fungal growth following the initial invader M. fructigena .IMPORTANCEMicrobial secondary metabolites constitute a significant reservoir of biologically potent and clinically valuable chemical scaffolds. However, their usefulness is hampered by rapidly developing resistance, resulting in reduced profitability of such research endeavors. Hence, the ecological role of such microbial secondary metabolites must be considered to understand how best to utilize such compounds as chemotherapeutics. Here, we explore an under-investigated environmental microbiome in the case of black apples; a veritable "low-hanging fruit," with relatively high abundances and diversity of microbially produced secondary metabolites. Using both a targeted and untargeted metabolomics approach, the interplay between metabolites, other microbes, and the apple host itself was investigated. This study highlights the surprisingly low incidence of known secondary metabolites in such a system, highlighting the need to study the functionality of secondary metabolites in microbial interactions and complex microbiomes., Competing Interests: The authors declare no conflict of interest.

Synthetic biology dictates the data-driven engineering of biocatalysis, cellular functions, and organism behavior. Integral to synthetic biology is the aspiration to efficiently find, access, interoperate, and reuse high-quality data on genotype-phenotype relationships of native and engineered biosystems under FAIR principles, and from this facilitate forward-engineering strategies. However, biology is complex at the regulatory level, and noisy at the operational level, thus necessitating systematic and diligent data handling at all levels of the design, build, and test phases in order to maximize learning in the iterative design-build-test-learn engineering cycle. To enable user-friendly simulation, organization, and guidance for the engineering of biosystems, we have developed an open-source python-based computer-aided design and analysis platform operating under a literate programming user-interface hosted on Github. The platform is called teemi and is fully compliant with FAIR principles. In this study we apply teemi for i) designing and simulating bioengineering, ii) integrating and analyzing multivariate datasets, and iii) machine-learning for predictive engineering of metabolic pathway designs for production of a key precursor to medicinal alkaloids in yeast. The teemi platform is publicly available at PyPi and GitHub., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: M.K.J., L.G.H. J.D.K. and J.Z. are inventors on pending patent applications. M.K.J., L.G.H., J.D.K., and J.Z. have financial interest in Biomia Aps. J.D.K. also has a financial interest in Amyris, Lygos, Demetrix, Napigen, Apertor Pharmaceuticals, Maple Bio, Ansa Biotechnologies, Berkeley Yeast, and Zero Acre Farms. The remaining authors declare no competing interests., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Filamentous fungi are prolific producers of secondary metabolites (SecMets), including compounds with antibiotic properties, like penicillin, that allows the producing fungus to combat competitors in a shared niche. However, the biological function of the majority of these small complex metabolites for the producing fungi remains unclear (Macheleidt et al., 2016). In an effort to address this lack of knowledge, we have chosen to study the microbial community of moldy apples in the hope of shedding more light on the role of SecMets for the dynamics of the microbial community. Penicillium expansum is one of the prevalent fungal species in this system, and in co-culture experiments with other apple fungal pathogens, we have observed up- and downregulation of several SecMets when compared to monocultures. However, molecular genetic dissection of the observed changes is challenging, and new methodologies for targeted genetic engineering in P. expansum are needed. In the current study, we have established a CRISPR-Cas9 dependent genetic engineering toolbox for the targeted genetic manipulation of P. expansum to allow for single-step construction of marker-free strains. The method and effect of different combinations of a Cas9-sgRNA expressing plasmids and repair template substrates in the NHEJ-proficient WT strain is tested by targeted deletion of melA, encoding a PKS responsible for pigment formation, which upon deletion resulted in white mutants. Co-transformation with a linear double-stranded DNA fragment consisting of two 2 kb homology arms flanking the PKS gene proved to be the most efficient strategy with 100% confirmed deletions by diagnostic PCR. Shorter homology arms (500-1000 bp) resulted in 20-30% deletion efficiency. Furthermore, we demonstrate the application of the CRISPR-Cas9 method for targeted deletion of biosynthetic genes without a visible phenotype, insertion of a visual reporter-encoding gene (mRFP), and overexpression of biosynthetic genes. Combined, these tools will advance in enabling the deciphering of SecMet biosynthetic pathways, provide in situ insight into when and where SecMets are produced, and provide an avenue to study the role of P. expansum SecMets in shaping the microbial community development on moldy apples via marker-free targeted genetic engineering of P. expansum., (Copyright © 2022. Published by Elsevier Inc.)

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora , with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium , and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium , as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast.

This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous mold Fusarium Fusarium species can cause localized and life-threating infections in humans. Of the 70 Fusarium species that have been reported to cause infections, close to one-third are members of the Fusarium solani species complex (FSSC), and they collectively account for approximately two-thirds of all reported Fusarium infections. Many of these species were recently given scientific names for the first time by a research group in the Netherlands, but they were misplaced in the genus Neocosmospora In this paper, we present genetic arguments that strongly support inclusion of the FSSC in Fusarium There are potentially serious consequences associated with using the name Neocosmospora for Fusarium species because clinicians need to be aware that fusaria are broadly resistant to the spectrum of antifungals that are currently available.

Fusarium graminearum is one of the most destructive plant pathogens worldwide, causing fusarium head blight (FHB) on cereals. F. graminearum colonizes wheat plant surfaces with specialized unbranched hyphae called runner hyphae (RH), which develop multicelled complex appressoria called infection cushions (IC). IC generate multiple penetration sites, allowing the fungus to enter the plant cuticle. Complex infection structures are typical for several economically important plant pathogens, yet with unknown molecular basis. In this study, RH and IC formed on the surface of wheat paleae were isolated by laser capture microdissection. RNA-Seq-based transcriptomic analyses were performed on RH and IC and compared to mycelium grown in complete medium (MY). Both RH and IC displayed a high number of infection up-regulated genes (982), encoding, among others, carbohydrate-active enzymes (CAZymes: 140), putative effectors (PE: 88), or secondary metabolism gene clusters (SMC: 12 of 67 clusters). RH specifically up-regulated one SMC corresponding to aurofusarin biosynthesis, a broad activity antibiotic. IC specifically up-regulated 248 genes encoding mostly putative virulence factors such as 7 SMC, including the mycotoxin deoxynivalenol and the newly identified fusaoctaxin A, 33 PE, and 42 CAZymes. Furthermore, we studied selected candidate virulence factors using cellular biology and reverse genetics. Hence, our results demonstrate that IC accumulate an arsenal of proven and putative virulence factors to facilitate the invasion of epidermal cells., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Worldwide, 463 million people are affected by diabetes of which the majority is diagnosed with Type 2 Diabetes (T2D). T2D can ultimately lead to retinopathy, nephropathy, nerve damage, and amputation of the lower extremities. α-Glucosidase, responsible for converting starch to monosaccharides, is a key therapeutic target for the management of T2D. However, due to substantial side effects of currently marketed drugs, there is an urgent need for the discovery of new α-glucosidase inhibitors. In our ongoing efforts to identify novel α-glucosidase inhibitors from Nature, we are investigating the potential of endophytic filamentous fungi as sustainable sources of hits and/or leads for future antihyperglycemic drugs. Here we report one previously unreported xanthone (5) and two known xanthones (7 and 11) as α-glucosidase inhibitors, isolated from an endophytic Penicillium canescens, recovered from fruits of Juniperus polycarpos. The three xanthones 5, 7, and 11 showed inhibitory activities against α-glucosidase with IC 50 values of 38.80 ± 1.01 μM, 32.32 ± 1.01 μM, and 75.20 ± 1.02 μM, respectively. Further pharmacological characterization revealed a mixed-mode inhibition for 5, a competitive inhibition for 7, while 11 acted as a non-competitive inhibitor., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast-based platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host., Competing Interests: Jay D. Keasling has commercial interests in Amyris, Lygos, Demetrix, Napigen, Apertor Labs, Berkeley Brewing Sciences, and Ansa Biosciences., (© 2020 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

We staged the transfer of the aurofusarin and bikaverin biosynthetic gene clusters (BGCs) to Aspergillus nidulans with the aim of gaining functional insights into dynamics immediately following a horizontal gene transfer (HGT) event. While the introduction of both BGCs resulted in the production of detectable pathway metabolites in A. nidulans, the transferred aurofusarin BGC formed dimeric shunt products instead of aurofusarin. This was linked to low transcription of the cluster activator and insufficient activity of tailoring enzymes, demonstrating how a shift of the pathway bottleneck after HGT can result in metabolic innovation. The transferred bikaverin BGC readily produced bikaverin, providing a model system for studying the conservation of regulatory responses to environmental cues. Conserved PacC-mediated pH regulation of the bikaverin BGC was observed between original host Fusarium fujikuroi and A. nidulans. Contrary to strong nitrogen responses described in other hosts, the BGC appeared unresponsive to environmental nitrogen in A. nidulans. While F. fujikuroi and A. nidulans both form chlamydospore-like structures when exposed to ralsolamycin, specific induction of the bikaverin BGC was not observed in A. nidulans. We propose that the presence of compatible cis-regulatory elements in BGCs facilitates regulatory conservation after transfer, without which the chromosomal context would dictate expression., (© 2019 John Wiley & Sons Ltd.)

Background: Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor -related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol., Results: A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor , resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster ( calA to calM ). Implementation of the CRISPR/Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase ( calA ), a major facilitator pump ( calB ) and a binuclear zinc cluster transcription factor ( calC ). Detailed metabolic profiling, using UHPLC-MS, of the ∆ calA (PKS) and ∆ calC (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆ calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B., Conclusion: Here we report the discovery of a gene cluster ( calA - M ) involved in the biosynthesis of the polyketide calbistrin in P. decumbens . Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal -cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.

The natural red food colorants carmine (E120) and carminic acid are currently produced from scale insects. The access to raw material is limited and current production is sensitive to fluctuation in weather conditions. A cheaper and more stable supply is therefore desirable. Here we present the first proof-of-concept of heterologous microbial production of carminic acid in Aspergillus nidulans by developing a semi-natural biosynthetic pathway. Formation of the tricyclic core of carminic acid is achieved via a two-step process wherein a plant type III polyketide synthase (PKS) forms a non-reduced linear octaketide, which subsequently is folded into the desired flavokermesic acid anthrone (FKA) structure by a cyclase and a aromatase from a bacterial type II PKS system. The formed FKA is oxidized to flavokermesic acid and kermesic acid, catalyzed by endogenous A. nidulans monooxygenases, and further converted to dcII and carminic acid by the Dactylopius coccus C-glucosyltransferase DcUGT2. The establishment of a functional biosynthetic carminic acid pathway in A. nidulans serves as an important step towards industrial-scale production of carminic acid via liquid-state fermentation using a microbial cell factory.

The chemical composition of the scale insect Dactylopius coccus was analyzed with the aim to discover new possible intermediates in the biosynthesis of carminic acid. UPLC-DAD/HRMS analyses of fresh and dried insects resulted in the identification of three novel carminic acid analogues and the verification of several previously described intermediates. Structural elucidation revealed that the three novel compounds were desoxyerythrolaccin-O-glucosyl (DE-O-Glcp), 5,6-didehydroxyerythrolaccin 3-O-β-D-glucopyranoside (DDE-3-O-Glcp), and flavokermesic acid anthrone (FKA). The finding of FKA in D. coccus provides solid evidence of a polyketide, rather than a shikimate, origin of coccid pigments. Based on the newly identified compounds, we present a detailed biosynthetic scheme that accounts for the formation of carminic acid (CA) in D. coccus and all described coccid pigments which share a flavokermesic acid (FK) core. Detection of coccid pigment intermediates in members of the Planococcus (mealybugs) and Pseudaulacaspis genera shows that the ability to form these pigments is taxonomically more widely spread than previously documented. The shared core-FK-biosynthetic pathway and wider taxonomic distribution suggests a common evolutionary origin for the trait in all coccid dye producing insect species., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, containing a cleavable N-terminal signal peptide and a C-terminal transmembrane helix that anchors the protein to the ER, followed by a short cytoplasmic tail. DcUGT2 is found to be heavily glycosylated. Truncated DcUGT2 proteins synthesized in yeast indicate the presence of an internal ER-targeting signal. The cleavable N-terminal signal peptide is shown to be essential for the activity of DcUGT2, whereas the transmembrane helix/cytoplasmic domains, although important, are not crucial for its catalytic function.

Background: Penicillium species are important producers of bioactive secondary metabolites. However, the immense diversity of the fungal kingdom is only scarcely represented in industrial bioprocesses and the upscaling of compound production remains a costly and labor intensive challenge. In order to facilitate the development of novel secondary metabolite producing processes, two routes are typically explored: optimization of the native producer or transferring the enzymatic pathway into a heterologous host. Recent genome sequencing of ten Penicillium species showed the vast amount of secondary metabolite gene clusters present in their genomes, and makes them accessible for rational strain improvement. In this study, we aimed to characterize the potential of these ten Penicillium species as native producing cell factories by testing their growth performance and secondary metabolite production in submerged cultivations., Results: Cultivation of the fungal species in controlled submerged bioreactors showed that the ten wild type Penicillium species had promising, highly reproducible growth characteristics in two different media. Analysis of the secondary metabolite production using liquid chromatography coupled with high resolution mass spectrometry proved that the species produced a broad range of secondary metabolites, at different stages of the fermentations. Metabolite profiling for identification of the known compounds resulted in identification of 34 metabolites; which included several with bioactive properties such as antibacterial, antifungal and anti-cancer activities. Additionally, several novel species-metabolite relationships were found., Conclusions: This study demonstrates that the fermentation characteristics and the highly reproducible performance in bioreactors of ten recently genome sequenced Penicillium species should be considered as very encouraging for the application of native hosts for production via submerged fermentation. The results are particularly promising for the potential development of the ten analysed Penicillium species for production of novel bioactive compounds via submerged fermentations.

Carminic acid is a C-glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane-bound enzymes., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For plant-eating insects, we still have only a nascent understanding of the genetic basis of host-use promiscuity. Here, to improve that situation, we investigated host-induced gene expression plasticity in the invasive lobate lac scale insect, Paratachardina pseudolobata (Hemiptera: Keriidae). We were particularly interested in the differential expression of detoxification and effector genes, which are thought to be critical for overcoming a plant's chemical defenses. We collected RNA samples from P. pseudolobata on three different host plant species, assembled transcriptomes de novo, and identified transcripts with significant host-induced gene expression changes. Gene expression plasticity was pervasive, but the expression of most detoxification and effector genes was insensitive to the host environment. Nevertheless, some types of detoxification genes were more differentially expressed than expected by chance. Moreover, we found evidence of a trade-off between expression of genes involved in primary and secondary metabolism; hosts that induced lower expression of genes for detoxification induced higher expression of genes for growth. Our findings are largely consonant with those of several recently published studies of other plant-eating insect species. Thus, across plant-eating insect species, there may be a common set of gene expression changes that enable host-use promiscuity.

Fusarium langsethiae is a widespread pathogen of small grain cereals, causing problems with T-2 and HT-2 toxin contamination in grains every year. In an effort to better understand the biology of this fungus, we present a draft genome sequence of F. langsethiae Fl201059 isolated from oats in Norway. The assembly was fragmented, but reveals a genome of approximately 37.5 Mb, with a GC content around 48%, and 12,232 predicted protein-coding genes. Focusing on secondary metabolism we identified candidate genes for 12 polyketide synthases, 13 non-ribosomal peptide synthetases, and 22 genes for terpene/isoprenoid biosynthesis. Some of these were found to be unique compared to sequence databases. The identified putative Tri5 cluster was highly syntenic to the cluster reported in F. sporotrichioides. Fusarium langsethiae Fl201059 produces a high number of secondary metabolites on Yeast Extract Sucrose (YES) agar medium, dominated by type A trichothecenes. Interestingly we found production of glucosylated HT-2 toxin (Glu-HT-2), previously suggested to be formed by the host plant and not by the fungus itself. In greenhouse inoculations of F. langsethiae Fl201059 on barley and oats, we detected the type A trichothecenes: neosolaniol, HT-2 toxin, T-2 toxin, Glu-HT-2 and numerous derivatives of these., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key enzyme in cholesterol biosynthesis. Their extensive use in treatment and prevention of cardiovascular diseases placed statins among the best selling drugs. Construction of Saccharomyces cerevisiae cell factory for the production of high concentrations of natural statins will require establishment of a non-destructive self-resistance mechanism to overcome the undesirable growth inhibition effects of statins. To establish active export of statins from yeast, and thereby detoxification, we integrated a putative efflux pump-encoding gene mlcE from the mevastatin-producing Penicillium citrinum into the S. cerevisiae genome. The resulting strain showed increased resistance to both natural statins (mevastatin and lovastatin) and semi-synthetic statin (simvastatin) when compared to the wild type strain. Expression of RFP-tagged mlcE showed that MlcE is localized to the yeast plasma and vacuolar membranes. We provide a possible engineering strategy for improvement of future yeast based production of natural and semi-synthetic statins., (© 2015 The Authors.)