Targeted Discovery of Glycosylated Natural Products by Tailoring Enzyme-Guided Genome Mining and MS-Based Metabolome Analysis.

Glycosides make up a biomedically important class of secondary metabolites. Most naturally occurring glycosides were isolated from plants and bacteria; however, the chemical diversity of glycosylated natural products in fungi remains largely unexplored. Herein, we present a paradigm to specifically discover diverse and bioactive glycosylated natural products from fungi by combining tailoring enzyme-guided genome mining with mass spectrometry (MS)-based metabolome analysis. Through in vivo genes deletion and heterologous expression, the first fungal C-glycosyltransferase AuCGT involved in the biosynthesis of stromemycin was identified from Aspergillus ustus. Subsequent homology-based genome mining for fungal glycosyltransferases by using AuCGT as a probe revealed a variety of biosynthetic gene clusters (BGCs) containing its homologues in diverse fungi, of which the glycoside-producing capability was corroborated by high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. Consequently, 28 fungal aromatic polyketide C/O-glycosides, including 20 new compounds, were efficiently discovered and isolated from the three selected fungi. Moreover, several novel fungal C/O-glycosyltransferases, especially three novel α-pyrone C-glycosyltransferases, were functionally characterized and verified in the biosynthesis of these glycosides. In addition, a proof of principle for combinatorial biosynthesis was applied to design the production of unnatural glycosides in Aspergillus nidulans. Notably, the newly discovered glycosides exhibited significant antiviral, antibacterial, and antidiabetic activities. Our work demonstrates the promise of tailoring enzyme-guided genome-mining approach for the targeted discovery of fungal glycosides and promotes the exploration of a broader chemical space for natural products with a target structural motif in microbial genomes.

Coordination of two regulators SscA and VosA in Aspergillus nidulans conidia.

Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in A. nidulans.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Extending the Structural Diversity of Labdane Diterpenoids from Marine-Derived Fungus Talaromyces sp. HDN151403 Using Heterologous Expression.

Heterologous biosynthesis has become an effective means to activate fungal silent biosynthetic gene clusters (BGCs) and efficiently utilize fungal genetic resources. Herein, thirteen labdane diterpene derivatives, including five undescribed ones named talarobicins A-E (3-7), were discovered via heterologous expression of a silent BGC (labd) in Aspergillus nidulans. Their structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as electronic circular dichroism (ECD) calculations. These labdanes belong to four skeleton types, and talarobicin B (4) is the first 3,18-dinor-2,3:4,18-diseco-labdane diterpene with the cleavage of the C2-C3 bond in ring A and the decarboxylation at C-3 and C-18. Talarobicin B (4) represents the key intermediate in the biosynthesis of penioxalicin and compound 13. The combinatorial heterologous expression and feeding experiments revealed that the cytochrome P450 enzymes LabdC, LabdE, and LabdF were responsible for catalyzing various chemical reactions, such as oxidation, decarboxylation, and methylation. All of the compounds are noncytotoxic, and compounds 2 and 8 displayed inhibitory effects against methicillin-resistant coagulase-negative staphylococci (MRCNS) and Bacillus cereus.

Mutagenic effects of a commercial glyphosate-based herbicide formulation on the soil filamentous fungus Aspergillus nidulans depending on the mode of exposure.

Glyphosate-based herbicides (GBH) are the most used pesticides worldwide. This widespread dissemination raises the question of non-target effects on a wide range of organisms, including soil micro-organisms. Despite a large body of scientific studies reporting the harmful effects of GBHs, the health and environmental safety of glyphosate and its commercial formulations remains controversial. In particular, contradictory results have been obtained on the possible genotoxicity of these herbicides depending on the organisms or biological systems tested, the modes and durations of exposure and the sensitivity of the detection technique used. We previously showed that the well-characterized soil filamentous fungus Aspergillus nidulans was highly affected by a commercial GBH formulation containing 450 g/L of glyphosate (R450), even when used at doses far below the agricultural application rate. In the present study, we analysed the possible mutagenicity of R450 in A. nidulans by screening for specific mutants after different modes of exposure to the herbicide. R450 was found to exert a mutagenic effect only after repeated exposure during growth on agar-medium, and depending on the metabolic status of the tested strain. The nature of some mutants and their ability to tolerate the herbicide better than did the wild-type strain suggested that their emergence may reflect an adaptive response of the fungus to offset the herbicide effects. The use of a non-selective molecular approach, the quantitative random amplified polymorphic DNA (RAPD-qPCR), showed that R450 could also exert a mutagenic effect after a one-shot overnight exposure during growth in liquid culture. However, this effect was subtle and no longer detectable when the fungus had previously been repeatedly exposed to the herbicide on a solid medium. This indicated an elevation of the sensitivity threshold of A. nidulans to the R450 mutagenicity, and thus confirmed the adaptive capacity of the fungus to the herbicide.

The bZIP-type transcription factors NapA and RsmA modulate the volumetric ratio and the relative superoxide ratio of mitochondria in Aspergillus nidulans.

Basic leucine zipper (bZIP) transcription factors are crucial components of differentiation, cellular homeostasis and the environmental stress defense of eukaryotes. In this work, we further studied the consequence of gene deletion and overexpression of two bZIP transcription factors, NapA and RsmA, on superoxide production, mitochondrial morphology and hyphal diameter of Aspergillus nidulans. We have found that reactive oxygen species production was influenced by both gene deletion and overexpression of napA under tert-butylhydroperoxide (tBOOH) elicited oxidative stress. Furthermore, gene expression of napA negatively correlated with mitochondrial volumetric ratio as well as sterigmatocystin production of A. nidulans. High rsmA expression was accompanied with elevated relative superoxide ratio in the second hyphal compartment. A negative correlation between the expression of rsmA and catalase enzyme activity or mitochondrial volumetric ratio was also confirmed by statistical analysis. Hyphal diameter was independent on either rsmA and napA expression as well as 0.2 mM tBOOH treatment.

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.

Comprehensive analysis of the composition of the major phospholipids during the asexual life cycle of the filamentous fungus Aspergillus nidulans.

Filamentous fungi undergo significant cellular morphological changes during their life cycle. It has recently been reported that deletions of genes that are involved in phospholipid synthesis led to abnormal hyphal morphology and differentiation in filamentous fungi. Although these results suggest the importance of phospholipid balance in their life cycle, comprehensive analyses of cellular phospholipids are limited. Here, we performed lipidomic analysis of A. nidulans during morphological changes in a liquid medium and of colonies on a solid medium. We observed that the phospholipid composition and transcription of the genes involved in phospholipid synthesis changed dynamically during the life cycle. Specifically, the levels of phosphatidylethanolamine, and highly unsaturated phospholipids increased during the establishment of polarity. Furthermore, we demonstrated that the phospholipid composition in the hyphae at colony margins is similar to that during conidial germination. Furthermore, we demonstrated that common and characteristic phospholipid changes occurred during germination in A. nidulans and A. oryzae, and that species-specific changes also occurred. These results suggest that the exquisite regulation of phospholipid composition is crucial for the growth and differentiation of filamentous fungi.

The novel spore-specific regulator SscA controls Aspergillus conidiogenesis.

IMPORTANCE: Filamentous fungi produce myriads of asexual spores, which are the main reproductive particles that act as infectious or allergenic agents. Although the serial of asexual sporogenesis is coordinated by various genetic regulators, there remain uncharacterized transcription factors in Aspergillus. To understand the underlying mechanism of spore formation, integrity, and viability, we have performed comparative transcriptomic analyses on three Aspergillus species and found a spore-specific transcription factor, SscA. SscA has a major role in conidial formation, maturation and dormancy, and germination in Aspergillus nidulans. Functional studies indicate that SscA coordinates conidial wall integrity, amino acid production, and secondary metabolism in A. nidulans conidia. Furthermore, the roles of SscA are conserved in other Aspergillus species. Our findings that the SscA has broad functions in Aspergillus conidia will help to understand the conidiogenesis of Aspergillus species.

Fungal COP9 signalosome assembly requires connection of two trimeric intermediates for integration of intrinsic deneddylase.

The conserved eight-subunit COP9 signalosome (CSN) is required for multicellular fungal development. The CSN deneddylase cooperates with the Cand1 exchange factor to control replacements of E3 ubiquitin cullin RING ligase receptors, providing specificity to eukaryotic protein degradation. Aspergillus nidulans CSN assembles through a heptameric pre-CSN, which is activated by integration of the catalytic CsnE deneddylase. Combined genetic and biochemical approaches provided the assembly choreography within a eukaryotic cell for native fungal CSN. Interactomes of functional GFP-Csn subunit fusions in pre-CSN deficient fungal strains were compared by affinity purifications and mass spectrometry. Two distinct heterotrimeric CSN subcomplexes were identified as pre-CSN assembly intermediates. CsnA-C-H and CsnD-F-G form independently of CsnB, which connects the heterotrimers to a heptamer and enables subsequent integration of CsnE to form the enzymatically active CSN complex. Surveillance mechanisms control accurate Csn subunit amounts and correct cellular localization for sequential assembly since deprivation of Csn subunits changes the abundance and location of remaining Csn subunits.

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.

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.

Streptomyces polyketides mediate bacteria-fungi interactions across soil environments.

Although the interaction between prokaryotic and eukaryotic microorganisms is crucial for the functioning of ecosystems, information about the processes driving microbial interactions within communities remains scarce. Here we show that arginine-derived polyketides (arginoketides) produced by Streptomyces species mediate cross-kingdom microbial interactions with fungi of the genera Aspergillus and Penicillium, and trigger the production of natural products. Arginoketides can be cyclic or linear, and a prominent example is azalomycin F produced by Streptomyces iranensis, which induces the cryptic orsellinic acid gene cluster in Aspergillus nidulans. Bacteria that synthesize arginoketides and fungi that decode and respond to this signal were co-isolated from the same soil sample. Genome analyses and a literature search indicate that arginoketide producers are found worldwide. Because, in addition to their direct impact, arginoketides induce a secondary wave of fungal natural products, they probably contribute to the wider structure and functioning of entire soil microbial communities.

A Flavin-Dependent Oxygenase Catalyzes Hydroxylation and Simultaneous Pyrrolidine Ring Formation in Protubonine Biosynthesis in Aspergillus ustus.

The hydroxylated and diacetylated cyclo-l-Trp-l-Leu derivative (-)-protubonine B was isolated from a culture of Aspergillus ustus 3.3904. Genome mining led to the identification of a putative biosynthetic gene cluster coding for a bimodular nonribosomal peptide synthetase, a flavin-dependent monooxygenase, and two acetyltransferases. Heterologous expression of the pbo cluster in Aspergillus nidulans showed that it is responsible for the formation of the isolated metabolite. Gene deletion experiments and structural elucidation of the isolated intermediates confirmed the biosynthetic steps. In vitro experiments with the recombinant protein proved that the flavin-dependent oxygenase is responsible for stereospecific hydroxylation at the indole ring accompanied by pyrrolidine ring formation.

Detection and quantification of the histone code in the fungal genus Aspergillus.

In eukaryotes, the combination of different histone post-translational modifications (PTMs) - the histone code - impacts the chromatin organization as compact and transcriptionally silent heterochromatin or accessible and transcriptionally active euchromatin. Although specific histone PTMs have been studied in fungi, an overview of histone PTMs and their relative abundance is still lacking. Here, we used mass spectrometry to detect and quantify histone PTMs in three fungal species belonging to three distinct taxonomic sections of the genus Aspergillus (Aspergillus niger, Aspergillus nidulans (two strains), and Aspergillus fumigatus). We overall detected 23 different histone PTMs, including a majority of lysine methylations and acetylations, and 23 co-occurrence patterns of multiple histone PTMs. Among those, we report for the first time the detection of H3K79me1, H3K79me2, and H4K31ac in Aspergilli. Although all three species harbour the same PTMs, we found significant differences in the relative abundance of H3K9me1/2/3, H3K14ac, H3K36me1 and H3K79me1, as well as the co-occurrence of acetylation on both K18 and K23 of histone H3 in a strain-specific manner. Our results provide novel insights about the underexplored complexity of the histone code in filamentous fungi, and its functional implications on genome architecture and gene regulation.

Disruption of Dcp1 leads to a Dcp2-dependent aberrant ribosome profiles in Aspergillus nidulans.

There are multiple RNA degradation mechanisms in eukaryotes, key among these is mRNA decapping, which requires the Dcp1-Dcp2 complex. Decapping is involved in various processes including nonsense-mediated decay (NMD), a process by which aberrant transcripts with a premature termination codon are targeted for translational repression and rapid decay. NMD is ubiquitous throughout eukaryotes and the key factors involved are highly conserved, although many differences have evolved. We investigated the role of Aspergillus nidulans decapping factors in NMD and found that they are not required, unlike Saccharomyces cerevisiae. Intriguingly, we also observed that the disruption of one of the decapping factors, Dcp1, leads to an aberrant ribosome profile. Importantly this was not shared by mutations disrupting Dcp2, the catalytic component of the decapping complex. The aberrant profile is associated with the accumulation of a high proportion of 25S rRNA degradation intermediates. We identified the location of three rRNA cleavage sites and show that a mutation targeted to disrupt the catalytic domain of Dcp2 partially suppresses the aberrant profile of Δdcp1 strains. This suggests that in the absence of Dcp1, cleaved ribosomal components accumulate and Dcp2 may be directly involved in mediating these cleavage events. We discuss the implications of this.

The N-terminal disordered region of ChsB regulates its efficient transport to the hyphal apical surface in Aspergillus nidulans.

In fungi, the cell wall plays a crucial role in morphogenesis and response to stress from the external environment. Chitin is one of the main cell wall components in many filamentous fungi. In Aspergillus nidulans, a class III chitin synthase ChsB plays a pivotal role in hyphal extension and morphogenesis. However, little is known about post-translational modifications of ChsB and their functional impacts. In this study, we showed that ChsB is phosphorylated in vivo. We characterized strains that produce ChsB using stepwise truncations of its N-terminal disordered region or deletions of some residues in that region and demonstrated its involvement in ChsB abundance on the hyphal apical surface and in hyphal tip localization. Furthermore, we showed that some deletions in this region affected the phosphorylation states of ChsB, raising the possibility that these states are important for the localization of ChsB to the hyphal surface and the growth of A. nidulans. Our findings indicate that ChsB transport is regulated by its N-terminal disordered region.

srdA mutations suppress the rseA/cpsA deletion mutant conidiation defect in Aspergillus nidulans.

Conidiation is an important reproductive process in Aspergillus. We previously reported, in A. nidulans, that the deletion of a putative glycosyltransferase gene, rseA/cpsA, causes an increase in the production of extracellular hydrolases and a severe reduction in conidiation. The aim of this study was to obtain novel genetic factors involved in the repression of conidiation in the rseA deletion mutant. We isolated mutants in which the rseA deletion mutant conidiation defect is suppressed and performed a comparative genomic analysis of these mutants. A gene encoding a putative transcription factor was identified as the associated candidate causative gene. The candidate gene was designated as srdA (suppressor gene for the conidiation defect of the rseA deletion mutant). The conidiation efficiency of the rseAsrdA double-deletion mutant was increased. Introduction of wild-type srdA into the suppressor mutants caused a conidiation defect similar to that of the rseA deletion mutant. Notably, the conidiation efficiencies of the rseAsrdA double-deletion and srdA single-deletion mutants were higher than that of the wild-type strain. These results indicate that srdA is a novel genetic factor that strongly represses conidiation of the rseA deletion mutant, and a putative transcriptional regulator, SrdA is a negative regulator of conidiation in A. nidulans.

Unnatural tetradeoxy echinocandins produced by gene cluster design and heterologous expression.

The hydroxyl groups in the amino acid residues of echinocandin B were related to the biological activity, the instability, and the drug resistance. The modification of hydroxyl groups was expected to obtain the new lead compounds for next generation of echinocandin drug development. In this work one method for heterologous production of the tetradeoxy echinocandin was achieved. A reconstructed biosynthetic gene cluster for tetradeoxy echinocandins composed of ecdA/I/K and htyE was designed and successfully hetero-expressed in Aspergillus nidulans. The target product of echinocandin E (1) together with one unexpected derivative echinocandin F (2), were isolated from the fermentation culture of engineered strain. Both of compounds were unreported echinocandin derivatives and the structures were identified on the basis of mass and NMR spectral data analysis. Compared with echinocandin B, echinocandin E demonstrated superior stability and comparable antifungal activity.

Genome-Wide Gene Expression Analyses of the AtfA/AtfB-Mediated Menadione Stress Response in Aspergillus nidulans.

The bZIP transcription factors (TFs) govern regulation of development, secondary metabolism, and various stress responses in filamentous fungi. In this work, we carried out genome-wide expression studies employing Illumina RNAseq to understand the roles of the two bZIP transcription factors AtfA and AtfB in Aspergillus nidulans. Comparative analyses of transcriptomes of control, ΔatfA, ΔatfB, and ΔatfAΔatfB mutant strains were performed. Dependence of a gene on AtfA (AtfB) was decided by its differential downregulation both between the reference and ΔatfA (ΔatfB) strains and between the ΔatfB (ΔatfA) and the ΔatfAΔatfB strains in vegetatively grown cells (mycelia) and asexual spores (conidia) of menadione sodium bisulfite (MSB)-treated or untreated cultures. As AtfA is the primary bZIP TF governing stress-response in A. nidulans, the number of differentially expressed genes for ΔatfA was significantly higher than for ΔatfB in both mycelial and conidial samples, and most of the AtfB-dependent genes showed AtfA dependence, too. Moreover, the low number of genes depending on AtfB but not on AtfA can be a consequence of ΔatfA leading to downregulation of atfB expression. Conidial samples showed much higher abundance of atfA and atfB mRNAs and more AtfA- and AtfB-affected genes than mycelial samples. In the presence of MSB, the number of AtfB- (but not of AtfA-) affected genes decreased markedly, which was accompanied with decreased mRNA levels of atfB in MSB-treated mycelial (reference strain) and conidial (ΔatfA mutant) samples. In mycelia, the overlap between the AtfA-dependent genes in MSB-treated and in untreated samples was low, demonstrating that distinct genes can be under AtfA control under different conditions. Carbohydrate metabolism genes were enriched in the set of AtfA-dependent genes. Among them, AtfA-dependence of glycolytic genes in conidial samples was the most notable. Levels of transcripts of certain secondary metabolitic gene clusters, such as the Emericellamide cluster, also showed AtfA-dependent regulation. Genes encoding catalase and histidine-containing phosphotransfer proteins showed AtfA-dependence under all experimental conditions. There were 23 AtfB-dependent genes that did not depend on AtfA under any of our experimental conditions. These included a putative α-glucosidase (agdB), a putative α-amylase, calA, which is involved in early conidial germination, and an alternative oxidase. In summary, in A. nidulans there is a complex interaction between the two bZIP transcription factors, where AtfA plays the primary regulatory role.