Jin Woo Bok

Relationship between Secondary Metabolism and Fungal Development

SUMMARY Filamentous fungi are unique organisms—rivaled only by actinomycetes and plants—in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway.

LaeA, a Regulator of Secondary Metabolism in Aspergillus spp.

ABSTRACT Secondary metabolites, or biochemical indicators of fungal development, are of intense interest to humankind due to their pharmaceutical and/or toxic properties. We present here a novel Aspergillus nuclear protein, LaeA, as a global regulator of secondary metabolism in this genus. Deletion of laeA (ΔlaeA) blocks the expression of metabolic gene clusters, including the sterigmatocystin (carcinogen), penicillin (antibiotic), and lovastatin (antihypercholesterolemic agent) gene clusters. Conversely, overexpression of laeA triggers increased penicillin and lovastatin gene transcription and subsequent product formation. laeA expression is negatively regulated by AflR, a sterigmatocystin Zn2Cys6 transcription factor, in a unique feedback loop, as well as by two signal transduction elements, protein kinase A and RasA. Although these last two proteins also negatively regulate sporulation, ΔlaeA strains show little difference in spore production compared to the wild type, indicating that the primary role of LaeA is to regulate metabolic gene clusters.

VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism.

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans, light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites.

Transcriptional Regulation of Chemical Diversity in Aspergillus fumigatus by LaeA

Secondary metabolites, including toxins and melanins, have been implicated as virulence attributes in invasive aspergillosis. Although not definitively proved, this supposition is supported by the decreased virulence of an Aspergillus fumigatus strain, ΔlaeA, that is crippled in the production of numerous secondary metabolites. However, loss of a single LaeA-regulated toxin, gliotoxin, did not recapitulate the hypovirulent ΔlaeA pathotype, thus implicating other toxins whose production is governed by LaeA. Toward this end, a whole-genome comparison of the transcriptional profile of wild-type, ΔlaeA, and complemented control strains showed that genes in 13 of 22 secondary metabolite gene clusters, including several A. fumigatus–specific mycotoxin clusters, were expressed at significantly lower levels in the ΔlaeA mutant. LaeA influences the expression of at least 9.5% of the genome (943 of 9,626 genes in A. fumigatus) but positively controls expression of 20% to 40% of major classes of secondary metabolite biosynthesis genes such as nonribosomal peptide synthetases (NRPSs), polyketide synthases, and P450 monooxygenases. Tight regulation of NRPS-encoding genes was highlighted by quantitative real-time reverse-transcription PCR analysis. In addition, expression of a putative siderophore biosynthesis NRPS (NRPS2/sidE) was greatly reduced in the ΔlaeA mutant in comparison to controls under inducing iron-deficient conditions. Comparative genomic analysis showed that A. fumigatus secondary metabolite gene clusters constitute evolutionarily diverse regions that may be important for niche adaptation and virulence attributes. Our findings suggest that LaeA is a novel target for comprehensive modification of chemical diversity and pathogenicity.

Histone deacetylase activity regulates chemical diversity in Aspergillus.

ABSTRACT Bioactive small molecules are critical in Aspergillus species during their development and interaction with other organisms. Genes dedicated to their production are encoded in clusters that can be located throughout the genome. We show that deletion of hdaA, encoding an Aspergillus nidulans histone deacetylase (HDAC), causes transcriptional activation of two telomere-proximal gene clusters—and subsequent increased levels of the corresponding molecules (toxin and antibiotic)—but not of a telomere-distal cluster. Introduction of two additional HDAC mutant alleles in a ΔhdaA background had minimal effects on expression of the two HdaA-regulated clusters. Treatment of other fungal genera with HDAC inhibitors resulted in overproduction of several metabolites, suggesting a conserved mechanism of HDAC repression of some secondary-metabolite gene clusters. Chromatin regulation of small-molecule gene clusters may enable filamentous fungi to successfully exploit environmental resources by modifying chemical diversity.

Chromatin-level regulation of biosynthetic gene clusters

Loss-of-function Aspergillus nidulans CclA, a Bre2 ortholog involved in histone H3 lysine 4 methylation, activated the expression of cryptic secondary metabolite clusters in A. nidulans. One new cluster generated monodictyphenone, emodin and emodin derivatives, whereas a second encoded two anti-osteoporosis polyketides, F9775A and F9775B. Modification of the chromatin landscape in fungal secondary metabolite clusters allows for a simple technological means to express silent fungal secondary metabolite gene clusters.

LaeA, a Regulator of Morphogenetic Fungal Virulence Factors

ABSTRACT Opportunistic animal and plant pathogens, well represented by the genus Aspergillus, have evolved unique mechanisms to adapt to and avoid host defenses. Aspergillus fumigatus, an increasingly serious pathogen owing to expanding numbers of immunocompromised patients, causes the majority of human infections; however, an inability to identify bona fide virulence factors has impeded therapeutic advances. We show that an A. fumigatus mutation in a developmentally expressed transcriptional regulator (ΔlaeA) coordinating morphological and chemical differentiation reduces virulence in a murine model; impaired virulence is associated with decreased levels of pulmonary gliotoxin and multiple changes in conidial and hyphal susceptibility to host phagocytes ex vivo. LaeA, a conserved protein in filamentous fungi, is a developmental regulator of virulence genes and, possibly, the first antimicrobial target specific to filamentous fungi that are pathogenic to plants and animals.

FfVel1 and FfLae1, components of a velvet‐like complex in Fusarium fujikuroi, affect differentiation, secondary metabolism and virulence

Besides industrially produced gibberellins (GAs), Fusarium fujikuroi is able to produce additional secondary metabolites such as the pigments bikaverin and neurosporaxanthin and the mycotoxins fumonisins and fusarin C. The global regulation of these biosynthetic pathways is only poorly understood. Recently, the velvet complex containing VeA and several other regulatory proteins was shown to be involved in global regulation of secondary metabolism and differentiation in Aspergillus nidulans. Here, we report on the characterization of two components of the F. fujikuroi velvet‐like complex, FfVel1 and FfLae1. The gene encoding this first reported LaeA orthologue outside the class of Eurotiomycetidae is upregulated in ΔFfvel1 microarray‐studies and FfLae1 interacts with FfVel1 in the nucleus. Deletion of Ffvel1 and Fflae1 revealed for the first time that velvet can simultaneously act as positive (GAs, fumonisins and fusarin C) and negative (bikaverin) regulator of secondary metabolism, and that both components affect conidiation and virulence of F. fujikuroi. Furthermore, the velvet‐like protein FfVel2 revealed similar functions regarding conidiation, secondary metabolism and virulence as FfVel1. Cross‐genus complementation studies of velvet complex component mutants between Fusarium, Aspergillus and Penicillium support an ancient origin for this complex, which has undergone a divergence in specific functions mediating development and secondary metabolism.

Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans.

Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM‐gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de‐repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein‐1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM‐specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.

Secondary metabolic gene cluster silencing in Aspergillus nidulans

In contrast to most primary metabolism genes, the genes involved in secondary metabolism and certain nutrient utilization pathways are clustered in fungi. Recently a nuclear protein, LaeA, was found to be required for the transcription of several secondary metabolite gene clusters in Aspergillus nidulans. Here we show that LaeA regulation does not extend to nutrient utilization or the spoC1 sporulation clusters. One of the secondary metabolite clusters regulated by LaeA contains the positive regulatory (i.e. aflR) and biosynthetic genes required for biosynthesis of sterigmatocystin (ST), a carcinogenic toxin. Analysis of ST gene cluster expression indicates LaeA regulation of the cluster is location specific as transcription of genes bordering the ST cluster are unaffected in a ΔlaeA mutant and placement of a primary metabolic gene, argB, in the ST cluster resulted in argB silencing in the ΔlaeA background. ST cluster gene expression was remediated when an additional copy of aflR was placed outside of the cluster but not when placed in the cluster. Site‐specific mutation of an s‐adenosyl methionine (AdoMet) binding site in LaeA generated a ΔlaeA phenotype suggesting the protein to be a methyltransferase.