Modeling Studies of the Mechanism of Context-Dependent Bidirectional Movements of Kinesin-14 Motors.

Kinesin-14s, a subfamily of the large superfamily of kinesin motor proteins, function mainly in spindle assembly and maintenance during mitosis and meiosis. KlpA from Aspergillus nidulans and GiKIN14a from Giardia intestinalis are two types of kinesin-14s. Available experimental results puzzlingly showed that while KlpA moves preferentially toward the minus end in microtubule-gliding setups and inside parallel microtubule overlaps, it moves preferentially toward the plus end on single microtubules. More puzzlingly, the insertion of an extra polypeptide linker in the central region of the neck stalk switches the motility direction of KlpA on single microtubules to the minus end. Prior experimental results showed that GiKIN14a moves preferentially toward the minus end on single microtubules in either tailless or full-length forms. The tail not only greatly enhances the processivity but also accelerates the ATPase rate and velocity of GiKIN14a. The insertion of an extra polypeptide linker in the central region of the neck stalk reduces the ATPase rate of GiKIN14a. However, the underlying mechanism of these puzzling dynamical features for KlpA and GiKIN14a is unclear. Here, to understand this mechanism, the dynamics of KlpA and GiKIN14a were studied theoretically on the basis of the proposed model, incorporating potential changes between the kinesin head and microtubule, as well as the potential between the tail and microtubule. The theoretical results quantitatively explain the available experimental results and provide predicted results. It was found that the elasticity of the neck stalk determines the directionality of KlpA on single microtubules and affects the ATPase rate and velocity of GiKIN14a on single microtubules.

Detailed characterisation of invasive aspergillosis in a murine model of X-linked chronic granulomatous disease shows new insights in infections caused by Aspergillus fumigatus versus Aspergillus nidulans.

Introduction: Invasive aspergillosis (IA) is the most prevalent infectious complication in patients with chronic granulomatous disease (CGD). Yet, understanding of fungal pathogenesis in the CGD host remains limited, particularly with regards to A. nidulans infection. Methods: We have used a murine model of X-linked CGD to investigate how the pathogenesis of IA varies between A. fumigatus and A. nidulans, comparing infection in both X-linked CGD (gp91-/-) mice and their parent C57BL/6 (WT) mice. A 14-colour flow cytometry panel was used to assess the cell dynamics over the course of infection, with parallel assessment of pulmonary cytokine production and lung histology. Results: We observed a lack of association between pulmonary pathology and infection outcome in gp91-/- mice, with no significant mortality in A. nidulans infected mice. An overwhelming and persistent neutrophil recruitment and IL-1 release in gp91-/- mice following both A. fumigatus and A. nidulans infection was observed, with divergent macrophage, dendritic cell and eosinophil responses and distinct cytokine profiles between the two infections. Conclusion: We have provided an in-depth characterisation of the immune response to pulmonary aspergillosis in an X-linked CGD murine model. This provides the first description of distinct pulmonary inflammatory environments in A. fumigatus and A. nidulans infection in X-linked CGD and identifies several new avenues for further research.

Heterologous production of bioactive xenoacremone analogs in Aspergillus nidulans.

Tyrosine-decahydrofluorene derivatives are a class of hybrid compounds that integrate the properties of polyketides and nonribosomal peptides. These compounds feature a [6.5.6] tricarbocyclic core and a para-cyclophane ether moiety in their structures and exhibit anti-tumor and anti-microbial activities. In this study, we constructed the biosynthetic pathway of xenoacremones from Xenoacremonium sinensis ML-31 in the Aspergillus nidulans host, resulting in the identification of four novel tyrosine-decahydrofluorene analogs, xenoacremones I-L (1-4), along with two known analogs, xenoacremones A and B. Remarkably, compounds 3 and 4 contained a 12-membered para-cyclophane ring system, which is unprecedented among tyrosine-decahydrofluorene analogs in X. sinensis. The successful reconstruction of the biosynthetic pathway and the discovery of novel analogs demonstrate the utility of heterologous expression strategy for the generation of structurally diverse natural products with potential biological activities.

The role of peroxiredoxins PrxA and PrxB in the antioxidant response, carbon utilization and development in Aspergillus nidulans.

In addition to their role in the breakdown of H2O2, some peroxiredoxins (Prxs) have chaperone and H2O2 sensing functions. Acting as an H2O2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA, ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H2O2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H2O2, indicating its critical role in H2O2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development.

Mining an O-methyltransferase for de novo biosynthesis of physcion in Aspergillus nidulans.

Physcion is one of natural anthraquinones, registered as a novel plant-derived fungicide due to its excellent prevention of plant disease. However, the current production of physcion via plant extraction limits its yield promotion and application. Here, a pair of polyketide synthases (PKS) in emodin biosynthesis were used as probes to mining the potential O-methyltransferase (OMT) responsible for physcion biosynthesis. Further refinement using the phylogenetic analysis of the mined OMTs revealed a distinct OMT (AcOMT) with the ability of transferring a methyl group to C-6 hydroxyl of emodin to form physcion. Through introducing AcOMT, we successfully obtained the de novo production of physcion in Aspergillus nidulans. The physcion biosynthetic pathway was further rationally engineered by expressing the decarboxylase genes from different fungi. Finally, the titer of physcion reached to 64.6 mg/L in shake-flask fermentation through enhancing S-adenosylmethionine supply. Our work provides a native O-methyltransferase for physcion biosynthesis and lays the foundation for further improving the production of physcion via a sustainable route. KEY POINTS: • Genome mining of the native O-methyltransferase responsible for physcion biosynthesis • De novo biosynthesis of physcion in the engineered Aspergillus nidulans • Providing an alternative way to produce plant-derived fungicide physcion.

Live-Cell Imaging of Dynein-Mediated Cargo Transport in Aspergillus nidulans.

Filamentous fungi have been used for studying long-distance transport of cargoes driven by cytoplasmic dynein. Aspergillus nidulans is a well-established genetic model organism used for studying dynein function and regulation in vivo. Here, we describe how we grow A. nidulans strains for live-cell imaging and how we observe the dynein-mediated distribution of early endosomes and secretory vesicles. Using an on-stage incubator and culture chambers for inverted microscopes, we can image fungal hyphae that naturally attach to the bottom of the chambers, using wide-field epifluorescence microscopes or the new Zeiss LSM 980 (with Airyscan 2) microscope. In addition to methods for preparing cells for imaging, a procedure for A. nidulans transformation is also described.

Kinesin-1 autoinhibition facilitates the initiation of dynein cargo transport.

The functional significance of Kinesin-1 autoinhibition has been unclear. Kinesin-1 transports multiple cargoes including cytoplasmic dynein to microtubule plus ends. From a genetic screen for Aspergillus mutants defective in dynein-mediated early endosome transport, we identified a kinesin-1 mutation kinAK895* at the C-terminal IAK motif involved in autoinhibition. The kinA∆IAK and kinAK895E mutants exhibited a similar defect in dynein-mediated early endosome transport, verifying the importance of kinesin-1 autoinhibition in dynein-mediated transport. Kinesin-1 autoinhibition is not critical for dynein accumulation at microtubule plus ends or for the secretory vesicle cargoes of kinesin-1 to reach the hyphal tip. However, it facilitates dynein to initiate early endosome transport. This is unrelated to a direct competition between dynein and kinesin-1 on early endosomes because kinesin-3 rather than kinesin-1 drives the plus-end-directed early endosome movement. This effect of kinesin-1 autoinhibition on dynein-mediated early endosome transport is related to cargo adapter-mediated dynein activation but at a step beyond the switching of dynein from its autoinhibited conformation.

Antibacterial diphenyl ether production induced by co-culture of Aspergillus nidulans and Aspergillus fumigatus.

Fungi are a rich source of secondary metabolites with potent biological activities. Co-culturing a fungus with another microorganism has drawn much attention as a practical method for stimulating fungal secondary metabolism. However, in most cases, the molecular mechanisms underlying the activation of secondary metabolite production in co-culture are poorly understood. To elucidate such a mechanism, in this study, we established a model fungal-fungal co-culture system, composed of Aspergillus nidulans and Aspergillus fumigatus. In the co-culture of A. nidulans and A. fumigatus, production of antibacterial diphenyl ethers was enhanced. Transcriptome analysis by RNA-sequencing showed that the co-culture activated expression of siderophore biosynthesis genes in A. fumigatus and two polyketide biosynthetic gene clusters (the ors and cic clusters) in A. nidulans. Gene disruption experiments revealed that the ors cluster is responsible for diphenyl ether production in the co-culture. Interestingly, the ors cluster was previously reported to be upregulated by co-culture of A. nidulans with the bacterium Streptomyces rapamycinicus; orsellinic acid was the main product of the cluster in that co-culture. In other words, the main product of the ors cluster was different in fungal-fungal and bacterial-fungal co-culture. The genes responsible for biosynthesis of the bacterial- and fungal-induced polyketides were deduced using a heterologous expression system in Aspergillus oryzae. The molecular genetic mechanisms that trigger biosynthesis of two different types of compounds in A. nidulans in response to the fungus and the bacterium were demonstrated, which provides an insight into complex secondary metabolic response of fungi to microorganisms. KEY POINTS: • Co-culture of two fungal species triggered antibiotic diphenyl ether production. • The co-culture affected expression levels of several genes for secondary metabolism. • Gene cluster essential for induction of the antibiotics production was determined.

Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain.

Cytoplasmic dynein is activated by the dynactin complex, cargo adapters and LIS1 (Lissencephaly 1). How this process is regulated in vivo remains unclear. The dynein motor ring contains six AAA+ (ATPases associated with diverse cellular activities) domains. Here, we used the filamentous fungus Aspergillus nidulans to examine whether ATP hydrolysis at AAA3 regulates dynein activation in the context of other regulators. In fungal hyphae, early endosomes undergo dynein-mediated movement away from the microtubule plus ends near the hyphal tip. Dynein normally accumulates at the microtubule plus ends. The early endosomal adaptor Hook protein, together with dynactin, drives dynein activation to cause its relocation to the microtubule minus ends. This activation process depends on LIS1, but LIS1 tends to dissociate from dynein after its activation. In this study, we found that dynein containing a mutation-blocking ATP hydrolysis at AAA3 can undergo LIS1-independent activation, consistent with our genetic data that the same mutation suppresses the growth defect of the A. nidulans LIS1-deletion mutant. Our data also suggest that blocking AAA3 ATP hydrolysis allows dynein activation by dynactin without the early endosomal adaptor. As a consequence, dynein accumulates at microtubule minus ends whereas early endosomes stay near the plus ends. Dynein containing a mutation-blocking ATP binding at AAA3 largely depends on LIS1 for activation, but this mutation abnormally prevents LIS1 dissociation upon dynein activation. Together, our data suggest that the AAA3 ATPase cycle regulates the coordination between dynein activation and cargo binding as well as the dynamic dynein-LIS1 interaction.

The DUG Pathway Governs Degradation of Intracellular Glutathione in Aspergillus nidulans.

Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance for deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigated functions of the dugB and dugC genes in the model fungus Aspergillus nidulans These genes are orthologues of dug2 and dug3, which are involved in cytosolic GSH degradation in Saccharomyces cerevisiae The deletion of dugB, dugC, or both resulted in a moderate increase in the GSH content in mycelia grown on glucose, reduced conidium production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding mitogen-activated protein (MAP) kinase pathway elements (e.g., steC, sskB, hogA, and mkkA) or regulatory proteins of conidiogenesis and sexual differentiation (e.g., flbA, flbC, flbE, nosA, rosA, nsdC, and nsdD) were downregulated in the ΔdugB ΔdugC mutant. Deletion of dugB and/or dugC slowed the depletion of GSH pools during carbon starvation. It also reduced accumulation of reactive oxygen species and decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions-in contrast to mycotoxin production-were controlled at the posttranscriptional level. We suggest that GSH connects starvation and redox regulation to each other: cells utilize GSH as a stored carbon source during starvation. The reduction of GSH content alters the redox state, activating regulatory pathways responsible for carbon starvation stress responses.IMPORTANCE Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character, and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, and secondary metabolite production (including mycotoxin formation), as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH-producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity, or oxidative stress tolerance of a fungal strain is manipulated.

Histone mRNA is subject to 3' uridylation and re-adenylation in Aspergillus nidulans.

The role of post-transcriptional RNA modification is of growing interest. One example is the addition of non-templated uridine residues to the 3' end of transcripts. In mammalian systems, uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense-mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3' uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3' tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3' end degraded transcripts are also subject to re-adenylation. Both mRNA pyrimidine tagging and re-adenylation are dependent on the same terminal-nucleotidyltransferases, CutA, and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3' tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation.

Tolerance to alkaline ambient pH in Aspergillus nidulans depends on the activity of ENA proteins.

Tolerance of microorganisms to abiotic stress is enabled by regulatory mechanisms that coordinate the expression and activity of resistance genes. Alkalinity and high salt concentrations are major environmental physicochemical stresses. Here, we analyzed the roles of sodium-extrusion family (ENA) transporters EnaA, EnaB and EnaC in the response to these stress conditions in the filamentous fungus Aspergillus nidulans. While EnaC has a minor role, EnaB is a key element for tolerance to Na+ and Li+ toxicity. Adaptation to alkaline pH requires the concerted action of EnaB with EnaA. Accordingly, expression of enaA and enaB was induced by Na+, Li+ and pH 8. These expression patterns are altered in a sltAΔ background and completely inhibited in a mutant expressing non-functional PacC protein (palH72). However, a constitutively active PacC form was not sufficient to restore maximum enaA expression. In agreement with their predicted role as membrane ATPases, EnaA localized to the plasma membrane while EnaB accumulated at structures resembling the endoplasmic reticulum. Overall, results suggest different PacC- and SltA-dependent roles for EnaB in pH and salt homeostasis, acting in coordination with EnaA at pH 8 but independently under salt stress.

Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine.

Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3'-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3' CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

Localization of NPFxD motif-containing proteins in Aspergillus nidulans.

During growth, filamentous fungi produce polarized cells called hyphae. It is generally presumed that polarization of hyphae is dependent upon secretion through the Spitzenkörper, as well as a mechanism called apical recycling, which maintains a balance between the tightly coupled processes of endocytosis and exocytosis. Endocytosis predominates in an annular domain called the sub-apical endocytic collar, which is located in the region of plasma membrane 1-5 µm distal to the Spitzenkörper. It has previously been proposed that one function of the sub-apical endocytic collar is to maintain the apical localization of polarization proteins. These proteins mark areas of polarization at the apices of hyphae. However, as hyphae grow, these proteins are displaced along the membrane and some must then be removed at the sub-apical endocytic collar in order to maintain the hyphoid shape. While endocytosis is fairly well characterized in yeast, comparatively little is known about the process in filamentous fungi. Here, a bioinformatics approach was utilized to identify 39 Aspergillus nidulans proteins that are predicted to be cargo of endocytosis based on the presence of an NPFxD peptide motif. This motif is a necessary endocytic signal sequence first established in Saccharomyces cerevisiae, where it marks proteins for endocytosis through an interaction with the adapter protein Sla1p. It is hypothesized that some proteins that contain this NPFxD peptide sequence in A. nidulans will be potential targets for endocytosis, and therefore will localize either to the endocytic collar or to more proximal polarized regions of the cell, e.g. the apical dome or the Spitzenkörper. To test this, a subset of the motif-containing proteins in A. nidulans was tagged with GFP and the dynamic localization was evaluated. The documented localization patterns support the hypothesis that the motif marks proteins for localization to the polarized cell apex in growing hyphae.

Functional Characterization of Clinical Isolates of the Opportunistic Fungal Pathogen Aspergillus nidulans.

Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulanssensu stricto clinical isolates. Just as in Aspergillus fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity.IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans, are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.

The splicing-factor Prp40 affects dynein-dynactin function in Aspergillus nidulans.

The multi-component cytoplasmic dynein transports cellular cargoes with the help of another multi-component complex dynactin, but we do not know enough about factors that may affect the assembly and functions of these proteins. From a genetic screen for mutations affecting early-endosome distribution in Aspergillus nidulans, we identified the prp40AL438* mutation in Prp40A, a homologue of Prp40, an essential RNA-splicing factor in the budding yeast. Prp40A is not essential for splicing, although it associates with the nuclear splicing machinery. The prp40AL438* mutant is much healthier than the ∆prp40A mutant, but both mutants exhibit similar defects in dynein-mediated early-endosome transport and nuclear distribution. In the prp40AL438* mutant, the frequency but not the speed of dynein-mediated early-endosome transport is decreased, which correlates with a decrease in the microtubule plus-end accumulations of dynein and dynactin. Within the dynactin complex, the actin-related protein Arp1 forms a mini-filament. In a pull-down assay, the amount of Arp1 pulled down with its pointed-end protein Arp11 is lowered in the prp40AL438* mutant. In addition, we found from published interactome data that a mammalian Prp40 homologue PRPF40A interacts with Arp1. Thus, Prp40 homologues may regulate the assembly or function of dynein-dynactin and their mechanisms deserve to be further studied.

Heterologous and Engineered Biosynthesis of Nematocidal Polyketide-Nonribosomal Peptide Hybrid Macrolactone from Extreme Thermophilic Fungi.

Fungal polyketide-nonribosomal peptide (PK-NRP) hybrid macrolactones are a growing family of natural products with biomedical and agricultural activities. One of the most important families is the thermolides, which are produced by extreme thermophilic fungi and exhibit strong nematocidal activity. We show here that the genes ThmABCE from Talaromyces thermophilus NRRL 2155 are critical for thermolide synthesis. Two separate single-module hrPKS (ThmA) and NRPS (ThmB) enzymes collaborate to synthesize the core macrolactone backbone (6 or 7), and the NRPS ThmB-CT domain catalyzes the key macrocyclization step in PK-NRP intermediate release via ester bond formation, representing a novel function of fungal NRPS C domains. We also show that heterologous and engineered expression of the Thm genes in the type strains of Aspergillus nidulans and Escherichia coli not only dramatically enhances the yields of thermolides but also affords different esterified analogues, such as butyryl- (thermolides J and K, 15 and 16), hexanoyl-, and octanyl- derivatives or mixed thermolides. Thermolides L and M (18 and 19), discovered via genome mining-based combinatorial biosynthesis, represent the first l-phenylalanine-based thermolides. Our work shows a unique biosynthetic mechanism of PK-NRP hybrid macrolactones from extremophiles, which led to the discovery of novel compounds and furthers our biosynthetic knowledge.

Overexpression of an LaeA-like Methyltransferase Upregulates Secondary Metabolite Production in Aspergillus nidulans.

Fungal secondary metabolites (SMs) include medically valuable compounds as well as compounds that are toxic, carcinogenic, and/or contributors to fungal pathogenesis. It is consequently important to understand the regulation of fungal secondary metabolism. McrA is a recently discovered transcription factor that negatively regulates fungal secondary metabolism. Deletion of mcrA (mcrAΔ), the gene encoding McrA, results in upregulation of many SMs and alters the expression of more than 1000 genes. One gene strongly upregulated by the deletion of mcrA is llmG, a putative methyl transferase related to LaeA, a major regulator of secondary metabolism. We artificially upregulated llmG by replacing its promoter with strong constitutive promoters in strains carrying either wild-type mcrA or mcrAΔ. Upregulation of llmG on various media resulted in increased production of the important toxin sterigmatocystin and compounds from at least six major SM pathways. llmG is, thus, a master SM regulator. mcrAΔ generally resulted in greater upregulation of SMs than upregulation of llmG, indicating that the full effects of mcrA on secondary metabolism involve genes in addition to llmG. However, the combination of mcrAΔ and upregulation of llmG generally resulted in greater compound production than mcrAΔ alone (in one case more than 460 times greater than the control). This result indicates that deletion of mcrA and/or upregulation of llmG can likely be combined with other strategies for eliciting SM production to greater levels than can be obtained with any single strategy.

Novel peroxiredoxin-based sensor for sensitive detection of hydrogen peroxide.

A series of genetically encoded sensors have been developed to detect the important signaling molecule H2O2 in living cells. However, more responsive and sensitive biosensors need to be developed. To address these demands, we used E. coli as a platform to develop a novel fluorescent H2O2 sensor, which we refer to as TScGP. This sensor employs a circularly permuted YFP (cpYFP) and is based on a redox relay between peroxiredoxin (Prx) and thioredoxin (Trx). Structurally, cpYFP is sandwiched between a fungal PrxA and a C-terminal cysteine mutated TrxA that can form a stabilized disulfide bond between PrxA and TrxA in response to H2O2. We confirmed that TScGP can be used for detecting exogenous H2O2 in the range of 0.5-5 µM with high selectivity and rapidly detecting H2O2 within 30 s in E. coli. To demonstrate an application, cellular H2O2 production by menadione was detected directly by TScGP. Our results demonstrated that using Prx-Trx combination as a sensing moiety is another strategy in designing H2O2 sensor with high performance.

Fungal Dirigent Protein Controls the Stereoselectivity of Multicopper Oxidase-Catalyzed Phenol Coupling in Viriditoxin Biosynthesis.

Paecilomyces variotii produces the antibacterial and cytotoxic ( M)-viriditoxin (1) together with a trace amount of its atropisomer ( P)-viriditoxin 1'. Elucidation of the biosynthesis by heterologous pathway reconstruction in Aspergillus nidulans identified the multicopper oxidase (MCO) VdtB responsible for the regioselective 6,6'-coupling of semiviriditoxin (10), which yielded 1 and 1' at a ratio of 1:2. We further uncovered that VdtD, an α/ß hydrolase-like protein lacking the catalytic serine, directs the axial chirality of the products. Using recombinant VdtB and VdtD as cell-free extracts from A. nidulans, we demonstrated that VdtD acts like a dirigent protein to control the stereoselectivity of the coupling catalyzed by VdtB to yield 1 and 1' at a ratio of 20:1. Furthermore, we uncovered a unique Baeyer-Villiger monooxygenase (BVMO) VdtE that could transform the alkyl methylketone side chain to methyl ester against the migratory aptitude.