Aspergillus Nidulans Research Articles

Gene expression screening and cell factory engineering for enhancing echinocandin B production in Aspergillus nidulans NRRL8112.

Echinocandin B (ECB) is a key precursor of the antifungal drug anidulafungin and its biosynthesis occurs via ani gene cluster in Aspergillus nidulans NRRL8112. Strain improvement for industrial ECB production has mainly relied on mutation breeding due to the lack of genetic tools. Here, a CRISPR-base-editing tool was developed in A. nidulans NRRL8112 for simultaneous inactivation of the nkuA gene and two marker genes, pryoA and riboB, which enabled efficient genetic manipulation. Then, in-vivo plasmid assembly was harnessed for ani gene expression screening, identifying the rate-limiting enzyme AniA and a pathway-specific transcription factor AniJ. Stepwise titer enhancement was achieved by overexpressing aniA and/or aniJ, and ECB production reached 1.5g/L during 5-L fed-batch fermentation, an increase of ~ 30-fold compared with the parent strain. This study, for the first time, revealed the regulatory mechanism of ECB biosynthesis and harnessed genetic engineering for the development of an efficient ECB-producing strain.

VezA/vezatin facilitates proper assembly of the dynactin complex in vivo

Cytoplasmic dynein-mediated intracellular transport needs the multi-component dynactin complex for cargo binding and motor activation. However, the cellular factors involved in dynactin assembly remain unexplored. Here, we found in Aspergillus nidulans that the vezatin homolog VezA is important for dynactin assembly. VezA affects the microtubule plus-end accumulation of dynein before cargo binding and cargo-adapter-mediated dynein activation, two processes that both need dynactin. The dynactin complex contains multiple components, including p150, p50, and an Arp1 (actin-related protein 1) mini-filament associated with a pointed-end sub-complex. VezA physically interacts with the Arp1mini-filament either directly or indirectly. Loss of VezA significantly decreases the amount of Arp1 pulled down with pointed-end proteins, as well as the protein levels of p50 and p150 in cell extract. Using various dynactin mutants, we further revealed that the dynactin assembly process must be highly coordinated. Together, these results shed light on dynactin assembly invivo.

Could the transcription factor AtnN coordinating the aspercryptin secondary metabolite gene cluster in Aspergillus nidulans be a global regulator?

Products of dormant secondary metabolite gene clusters of fungal genomes can be exploited for medical purposes as bioactive agents. These clusters can be switched on under oxidative stress and may endow fungi with a versatile chemical armory in a competitive niche. In Aspergillus nidulans, the aspercryptin gene cluster, including the synthase [atnA (AN7884)] and its transcription factor (atnN), was activated under menadione sodium bisulfite (MSB) treatment. In this study, we generated and phenotypically examined the gene deletion and overexpression mutants of atnN and studied the secondary metabolite production of the mutants. Overexpression of atnN significantly reduced the colony growth of surface cultures compared to the control. The ΔatnN gene deletion strain showed higher sensitivity to tert-butyl hydroperoxide (tBOOH), while the atnNOE strain was more resistant to MSB, Congo Red, and sorbitol. Interestingly, deletion of atnN decreased cleistothecia formation of A. nidulans. Manipulation of atnN affected the synthesis of several secondary metabolites, for example, the siderophore production of A. nidulans. The extracellular triacetylfusarinine C (TAFC) production decreased, while the intracellular ferricrocin (FC) concentration of the cultures increased in the atnNOE mutant cultivating A. nidulans in a complex medium containing 1% mycological peptone and 2% maltose. In Czapek-Dox Broth medium, increased asperthecin production was observed in the ΔatnN mutant. The mycotoxin sterigmatocystin synthesis elevated in the ΔatnN mutant, while reduced in the atnNOE mutant on minimal medium. Our study supports previous observations that secondary metabolite production is coordinated in a complex way, and the linkage of stress response, sexual reproduction, and secondary metabolite production can be governed by several transcription factors.

Optimized psilocybin production in tryptophan catabolism-repressed fungi.

The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy-refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a 'Breakthrough Therapy' by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an invivo psilocybin production chassis based on repression of l-tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3-dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l-tryptophan catabolism by genome mining and cross-complementation. The double deletion mutant of A. nidulans resulted in a 10-fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space-time-yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine-derived pharmaceuticals.

Screens for mutants defective in UapA trafficking highlight the importance of ER-exit as a primary control point in transporter biogenesis

Most transmembrane membrane proteins are thought to traffic to the plasma membrane (PM) via the conventional secretory pathway through sorting from the Golgi. However, our recent work has shown that in the filamentous fungus Aspergillus nidulans several nutrient transporters and other major membrane proteins traffic to the PM via Golgi-bypass and independently of known post-Golgi secretory mechanisms. Here in an effort to dissect the molecular mechanism underlying membrane cargo trafficking via Golgi-bypass we design and use unbiased genetic screens, based on the UapA uric acid-xanthine transporter, which allowed the isolation of mutants defective in UapA translocation to the plasma membrane. Analyses of these mutants highlight the importance of ER-exit as the primary control point in transporter trafficking via Golgi-bypass. Most mutants isolated concerned mutations within the uapA gene, albeit we also obtained uapA extragenetic mutants affecting secretion and growth pleiotropically or leading or apparent activation of an efflux transporter related to purine-detoxification. Our work paves the way to use genetic approaches targeting specifically trafficking mutations affecting Golgi-bypass.

Mutational analysis of Phanerochaete chrysosporium´s purine transporter.

We present here a mutational analysis of the purine transporter from Phanerochaete chrysosporium (PhZ), a member of the AzgA-like subfamily within the Nucleobase Ascorbate Transporters family. We identified key residues that determine its substrate specificity and transport efficiency. Thirteen PhZ mutants were generated and heterologously expressed in Aspergillus nidulans. The growth of mutant strains in the presence of purines and toxic analogues and the uptake rate of radiolabelled hypoxanthine were evaluated. Results revealed that ten mutants showed differences in transport compared to the wild-type PhZ: six mutants completely lost function, two exhibited decreased transport activity, and two showed increased hypoxanthine uptake. Subcellular localization and expression level analyses indicated that the differences in transport activity were not due to trafficking issues to the plasma membrane or protein stability. A three-dimensional model of PhZ, constructed with the artificial intelligence-based AlphaFold2 program, suggested that critical residues for transport are located in transmembrane segments and an internal helix. In the latter, the A418 residue was identified as playing a pivotal role in transport efficiency despite being far from the putative substrate binding site, as mutant A418V showed an increased initial uptake efficiency for the transporter´s physiological substrates. We also report that residue L124, which lies in the putative substrate binding site, plays a critical role in substrate transport, emerging as an additional determinant in the transport mechanism of this family of transporters. These findings underscore the importance of specific residues in AzgA-like transporters and enhance our understanding of the intricate mechanisms governing substrate specificity and transport efficiency within this family.

Evaluation of the Activity of Triazoles Against Non-fumigatus Aspergillus and Cryptic Aspergillus Species Causing Invasive Infections Tested in the SENTRY Program.

The activity of isavuconazole and other triazoles against non-fumigatus (non-AFM) Aspergillus causing invasive aspergillosis was evaluated. A total of 390 non-AFM isolates were collected (1/patient) in 2017-2021 from 41 hospitals. Isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and/or internal spacer region/β-tubulin sequencing and tested by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. CLSI epidemiological cutoff values were applied, where available. Isavuconazole showed activity against Aspergillus sections Flavi (n = 122; minimum inhibitory concentration [MIC]50/90, 0.5/1 mg/L), Terrei (n = 57; MIC50/90, 0.5/0.5 mg/L), Nidulantes (n = 34; MIC50/90, 0.12/0.25 mg/L), Versicolores (n = 7; MIC50, 1 mg/L), and Circumdati (n = 2; MIC range, 0.12-2 mg/L). Similar activity was displayed by other triazoles against those Aspergillus sections. Most of the isolates from Aspergillus sections Fumigati (n = 9), Nigri (n = 146), and Usti (n = 12) exhibited elevated MIC values to isavuconazole (MIC50/90, 2/-, 2/4, and 2/8 mg/L), voriconazole (MIC50/90, 2/-, 1/2, and 4/8 mg/L), itraconazole (MIC50/90, 2/-, 2/4, and 8/>8 mg/L), and posaconazole (MIC50/90, 0.5/-, 0.5/1, and >8/>8 mg/L), respectively. Isavuconazole was active (MIC values, ≤1 mg/L) against Aspergillus parasiticus, Aspergillus tamarii, Aspergillus nomius, Aspergillus nidulans, Aspergillus unguis, Aspergillus terreus, Aspergillus alabamensis, and Aspergillus hortai, while isavuconazole MIC values between 2 and 8 mg/L were observed against cryptic isolates from Aspergillus section Fumigati. Isavuconazole inhibited 96.1% of Aspergillus niger and 80.0% of Aspergillus tubingensis at ≤4 mg/L, the CLSI wild-type cutoff value for A niger. Voriconazole, itraconazole, and posaconazole showed similar activity to isavuconazole against most cryptic species. Isavuconazole exhibited potent in vitro activity against non-AFM; however, the activity of triazoles varies among and within cryptic species.

Quantitative Characterization of Gene Regulatory Circuits Associated With Fungal Secondary Metabolism to Discover Novel Natural Products.

Microbial genetic circuits are vital for regulating gene expression and synthesizing bioactive compounds. However, assessing their strength and timing, especially in multicellular fungi, remains challenging. Here, an advanced microfluidic platform is combined with a mathematical model enabling precise characterization of fungal gene regulatory circuits (GRCs) at the single-cell level. Utilizing this platform, the expression intensity and timing of 30 transcription factor-promoter combinations derived from two representative fungal GRCs, using the model fungus Aspergillus nidulans are determined. As a proof of concept, the selected GRC combination is utilized to successfully refactor the biosynthetic pathways of bioactive molecules, precisely control their production, and activate the expression of the silenced biosynthetic gene clusters (BGCs). This study provides insights into microbial gene regulation and highlights the potential of platform in fungal synthetic biology applications and the discovery of novel natural products.

Composite Recycling with Biocatalytic Thermoset Reforming.

Carbon fiber reinforced polymers (CFRPs, or composites) are increasingly replacing traditional manufacturing materials used in the automobile, aerospace, and energy sectors. With this shift, it is vital to develop end-of-life processes for CFRPs that retain the value of both the carbon fibers and the polymer matrix. Here we demonstrate a strategy to upcycle pre- and postconsumer polystyrene-containing CFRPs, cross-linked with unsaturated polyesters or vinyl esters, to benzoic acid. The thermoset matrix is upgraded via biocatalysis utilizing an engineered strain of the filamentous fungus Aspergillus nidulans, which gives access to valuable secondary metabolites in high yields, exemplified here by (2Z,4Z,6E)-octa-2,4,6-trienoic acid. Reactions are engineered to preserve the carbon fibers with much of their sizing so that the isolated carbon fiber plies are manufactured into new composite coupons that exhibit mechanical properties comparable to those of virgin manufacturing substrates. In sum, this represents the first system to reclaim a high value from both the fiber fabric and polymer matrix of a CFRP.

Modification of xylan in secondary walls alters cell wall biosynthesis and wood formation programs and improves saccharification.

Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. × tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.

Distinct trafficking routes of polarized and non-polarized membrane cargoes in Aspergillus nidulans.

Membrane proteins are sorted to the plasma membrane (PM) via Golgi-dependent trafficking. However, our recent studies challenged the essentiality of Golgi in the biogenesis of specific transporters. Here, we investigate the trafficking mechanisms of membrane proteins by following the localization of the polarized R-SNARE SynA versus the non-polarized transporter UapA, synchronously co-expressed in wild-type or isogenic genetic backgrounds repressible for conventional cargo secretion. In wild-type, the two cargoes dynamically label distinct secretory compartments, highlighted by the finding that, unlike SynA, UapA does not colocalize with the late-Golgi. In line with early partitioning into distinct secretory carriers, the two cargoes collapse in distinct ERES in a sec31ts background. Trafficking via distinct cargo-specific carriers is further supported by showing that repression of proteins essential for conventional cargo secretion does not affect UapA trafficking, while blocking SynA secretion. Overall, this work establishes the existence of distinct, cargo-dependent, trafficking mechanisms, initiating at ERES and being differently dependent on Golgi and SNARE interactions.

Improved gene editing and fluorescent-protein tagging in Aspergillus nidulans using a Golden Gate-based CRISPR-Cas9 plasmid system

CRISPR-Cas9 systems can be used for precise genome editing in filamentous fungi, including Aspergillus nidulans. However, current CRISPR-Cas9 systems for A. nidulans rely on relatively complex or multi-step cloning methods to build a plasmid expressing both Cas9 and an sgRNA targeting a genomic locus. In this study we improve on existing plasmid-based CRISPR-Cas9 systems for Aspergilli by creating an extremely simple-to-use CRISPR-Cas9 system for A. nidulans genome editing. In our system, a plasmid containing both Cas9 and an sgRNA is assembled in a one-step Golden Gate reaction. We demonstrate precise, scarless genome editing with nucleotide-level DNA substitutions, and we demonstrate markerless gene tagging by fusing fluorescent-protein coding sequences to the endogenous coding sequences of several A. nidulans genes. We also describe A. nidulans codon-adjusted versions of multiple recent-generation fluorescent proteins, which will be useful to the wider Aspergillus community.

Understanding fungal and plant active urea transport systems: Keys from Aspergillus nidulans and beyond

Urea is present in all ecosystems, as a result of the metabolism of different organisms and also of human activity, being the world's most common form of nitrogen fertilizer. Fungi and plants can use urea as a nitrogen source, taking it up from the environment through specialized active transport proteins. These proteins belong to a subfamily of urea/H+ symporters included in the Solute:Sodium Symporter (SSS) family of transporters. In this review we summarize the current knowledge on this group of transporters, based on our previous studies on Aspergillus nidulans UreA. We delve into its transcriptional and post-translational regulation, structure-function relationships, transport mechanism, and certain aspects of its biogenesis. Recent findings suggest that this urea transporter subfamily is more expanded than originally thought, with representatives found in organisms as diverse as Archaea and mollusks, which raises questions on evolutionary aspects. A. nidulans ureA knockout strains provide a valuable platform for expressing urea transporters from diverse sources, facilitating their characterization and functional analysis. In this context, given the close relationship between plant and fungal active urea transporters, this knowledge could serve to develop strategies to improve the efficiency of applied urea as fertilizer.

Chemical Survey and Antifungal Efficacy of Sargassum muticum's Alkaloids and Phenolic-Rich Fraction Against Airborne Toxigenic and Nosocomial Opportunistic Molds Isolates.

The Atlantic coastline of El-Jadida, Morocco, is renowned for its plentiful algae, especially brown seaweed, which is rich in active compounds known for their antifungal properties. This valuable resource offers an exciting opportunity to tackle the numerous challenges posed by invasive fungal infections, allergies, mycotoxin-related food poisoning, and drug-resistant strains. Underscoring the urgent need to explore alternative, sustainable, and environmentally friendly antifungal agents derived from algae. This study aimed to evaluate the antifungal activity of total alkaloids and phenolic-rich fractions derived from seven species of Pheophyceae: Sargassum muticum, Sargassum vulgare, Bifurcaria bifurcata, Cystoseira tamariscifolia, Cystoseira humilis, Laminaria ochroleuca, and Fucus spiralis against four fungi: airborne toxigenic isolates of Aspergillus westerdijkiae and Chaetomium globosum as well as nosocomial opportunistic isolates of Aspergillus nidulans and Scopulariopsis brevicaulis. The study also aimed to identify the most effective alga and its specific active compounds through LC-MS and GC-MS analysis. The invasive Sargassum muticum was chosen as the most potent alga in inhibiting the growth of mycelium. For the first time, the alkaloids palmatine and jatrorrhizine, along with caulerpin, have been identified. The chloroform fraction revealed the prevalence of phenolic compounds including, phenolic acids, flavonoids, and phlorotannins. The lowest minimum inhibitory concentrations (MICs), with a maximum fungal load of 108 colony-forming unit (CFU), recorded ranged from 3.12 to 6.25μg/mL by the phenolic-rich fraction against airborne toxigenic isolates, and from 100 to 200μg/mL against nosocomial opportunistic isolates by the total alkaloids. In comparison, the positive control, ketoconazole, showed higher MICs and resistance against A. nidulans. The valorization of Sargassum muticum is proposed as a green strategy to preserve the ecological balance, combat antifungal resistance, and address public health challenges.

Screening Proteins That Interact With AcHog1 and the Functional Analysis of AcSko1 in Aspergillus cristatus.

Aspergillus cristatus is a dominant fungus formed during the "flowering" process of Fuzhuan brick tea. Previous research has established that the sporulation of Aspergillus nidulans, a model organism of filamentous fungi, is regulated by light. However, the sporulation of A. cristatus is dependent on osmotic stress. In a previous study, we used pull-down and mass spectrometry to identify proteins that interacted with AcHog1 in A. cristatus when cultured under different conditions of osmotic stress. In the present study, we analyzed the proteins we identified previously to investigate their functional role. The AA1E3BER4 protein was located downstream of Hog1 in the HOG branch pathway and was identified that was regulated by AcHog1. Furthermore, yeast two-hybrid analysis showed that AA1E3BER4 interacted with AcHog1. In addition, we knocked out and complemented the Acsko1 gene encoding the AA1E3BER4 protein. We found that the number of sexual and asexual spores were downregulated by 3.81- and 4.57-fold, respectively, in the ΔAcsko1 strain. The sensitivity of the ΔAcsko1 strain to sorbitol and sucrose, as regulators of osmotic stress, increased, and the sensitivity to high sucrose was higher than that of sorbitol. Acsko1 also regulated the response of A. cristatus to oxidative stress, Congo red, and SDS (sodium dodecyl sulfate). In addition, the deletion of Acsko1 significantly increased the pigment of the ΔAcsko1 strain. This is the first study to report the role of the sko1 gene in oxidative stress, stress-induced damage to the cell wall, and pigment in Aspergillus cristatus.

Self-Resistance Gene-Guided Discovery of the Molecular Basis for Biosynthesis of the Fatty Acid Synthase Inhibitor Cerulenin.

Cerulenin (1) is the first reported natural fatty acid synthase inhibitor and has been intensively researched for its antifungal, anticancer and anti-obesity properties. However, the molecular basis for its biosynthesis has remained a mystery for six decades. Here, we have identified the polyketide biosynthetic gene cluster (cer) responsible for the biosynthesis of 1 from two Sarocladium species using a self-resistance gene mining approach, which we validated via heterologous reconstitution of cer cluster in an Aspergillus nidulans host. Expression of various combinations of cer genes uncovered key pathway intermediates, electrocyclisation products derived from PKS-encoded polyenoic acids, and a suite of 13 new analogues of 1. This enabled us to establish a biosynthetic pathway to 1 that starts with a C12 polyketide precursor containing both E and Z double bonds and involves a complex series of epoxidations, double bond shifts, E/Z isomerisation and epoxide reduction. Using in vitro assays, we further validated the roles of amidotransferase CerD in amidation, and oxidase CerF and reductase CerE in the final two-electron oxidation and enone reduction steps towards 1. These findings expand our understanding of complex tailoring modifications in highly reducing PKS pathways and pave the way for the engineered biosynthesis of cerulenin analogues.

Comparative analysis of polysaccharide and cell wall structure in Aspergillus nidulans and Aspergillus fumigatus by solid-state NMR

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13C, 15N, and 1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, α-glucan, and β-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.

Expanded Gene Regulatory Network Reveals Potential Light-Responsive Transcription Factors and Target Genes in Cordyceps militaris.

Cordyceps militaris, a fungus widely used in traditional Chinese medicine and pharmacology, is recognized for its abundant bioactive compounds, including cordycepin and carotenoids. The growth, development, and metabolite production in various fungi are influenced by the complex interactions between regulatory cascades and light-signaling pathways. However, the mechanisms of gene regulation in response to light exposure in C. militaris remain largely unexplored. This study aimed to identify light-responsive genes and potential transcription factors (TFs) in C. militaris through an integrative transcriptome analysis. To achieve this, we reconstructed an expanded gene regulatory network (eGRN) comprising 507 TFs and 8662 regulated genes using both interolog-based and homolog-based methods to build the protein-protein interaction network. Aspergillus nidulans and Neurospora crassa were chosen as templates due to their relevance as fungal models and the extensive study of their light-responsive mechanisms. By utilizing the eGRN as a framework for comparing transcriptomic responses between light-exposure and dark conditions, we identified five key TFs-homeobox TF (CCM_07504), FlbC (CCM_04849), FlbB (CCM_01128), C6 zinc finger TF (CCM_05172), and mcrA (CCM_06477)-along with ten regulated genes within the light-responsive subnetwork. These TFs and regulated genes are likely crucial for the growth, development, and secondary metabolite production in C. militaris. Moreover, molecular docking analysis revealed that two novel TFs, CCM_05727 and CCM_06992, exhibit strong binding affinities and favorable docking scores with the primary light-responsive protein CmWC-1, suggesting their potential roles in light signaling pathways. This information provides an important functional interactive network for future studies on global transcriptional regulation in C. militaris and related fungi.

Transcription Factor Engineering in Aspergillus nidulans Leads to the Discovery of an Orsellinaldehyde Derivative Produced via an Unlinked Polyketide Synthase Gene.

Secondary metabolites are generally produced by enzymes encoded by genes within a biosynthetic gene cluster. Transcription factor genes are frequently located within these gene clusters. These transcription factors often drive expression of the other genes of the biosynthetic gene cluster, and overexpression of the transcription factor provides a facile approach to express all genes within a gene cluster, resulting in production of downstream metabolite(s). Unfortunately this approach is not always successful, leading us to engineer more effective hybrid transcription factors. Herein, we attempted to activate a putative cryptic biosynthetic gene cluster in Aspergillus nidulans using a combination of transcription factor engineering and overexpression approaches. This resulted in the discovery of a novel secondary metabolite we term triorsellinaldehyde. Surprisingly, deletion of the polyketide synthase gene within the gene cluster did not prevent triorsellinaldehyde production. However, targeted deletion of a polyketide synthase gene elsewhere in the genome revealed its role in triorsellinaldehyde biosynthesis.

Using flux theory in dynamic omics data sets to identify differentially changing signals using DPoP

BackgroundDerivative profiling is a novel approach to identify differential signals from dynamic omics data sets. This approach applies variable step-size differentiation to time dynamic omics data. This work assumes that there is a general omics derivative that is a useful and descriptive feature of dynamic omics experiments. We assert that this omics derivative, or omics flux, is a valuable descriptor that can be used instead of, or with, fold change calculations.ResultsThe results of derivative profiling are compared to established methods such as Multivariate Adaptive Regression Splines, significance versus fold change analysis (Volcano), and an adjusted ratio over intensity (M/A) analysis to find that there is a statistically significant similarity between the results. This comparison is repeated for transcriptomic and phosphoproteomic expression profiles previously characterized in Aspergillus nidulans. This method has been packaged in an open-source, GUI-based MATLAB app, the Derivative Profiling omics Package (DPoP). Gene Ontology (GO) term enrichment has been included in the app so that a user can automatically/programmatically describe the over/under-represented GO terms in the derivative profiling results using domain specific knowledge found in their organism’s specific GO database file. The advantage of the DPoP analysis is that it is computationally inexpensive, it does not require fold change calculations, it describes both instantaneous as well as overall behavior, and it achieves statistical confidence with signal trajectories of a single bio-replicate over four or more points.ConclusionsWhile we apply this method to time dynamic transcriptomic and phosphoproteomic datasets, it is a numerically generalizable technique that can be applied to any organism and any field interested in time series data analysis. The app described in this work enables omics researchers with no computer science background to apply derivative profiling to their data sets, while also allowing multidisciplined users to build on the nascent idea of profiling derivatives in omics.