mrflbA, encoding a putative FlbA, is involved in aerial hyphal development and secondary metabolite production in Monascus ruber M-7 (original) (raw)
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Heterologous gene expression in filamentous fungi
Trends in Biotechnology, 1989
Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi.
Regulation of Secondary Metabolism in Filamentous Fungi
Annual Review of Phytopathology, 2005
■ Abstract Fungal secondary metabolites are of intense interest to humankind due to their pharmaceutical (antibiotics) and/or toxic (mycotoxins) properties. In the past decade, tremendous progress has been made in understanding the genes that are associated with production of various fungal secondary metabolites. Moreover, the regulatory mechanisms controlling biosynthesis of diverse groups of secondary metabolites have been unveiled. In this review, we present the current understanding of the genetic regulation of secondary metabolism from clustering of biosynthetic genes to global regulators balancing growth, sporulation, and secondary metabolite production in selected fungi with emphasis on regulation of metabolites of agricultural concern. Particularly, the roles of G protein signaling components and developmental regulators in the mycotoxin sterigmatocystin biosynthesis in the model fungus Aspergillus nidulans are discussed in depth.
MHP1, aMagnaporthe griseahydrophobin gene, is required for fungal development and plant colonization
Molecular Microbiology, 2005
Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re-introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infectionrelated fungal development, and is required for pathogenicity of M. grisea .
2012 Cellular & Molecular Fungal Biology Gordon Research Conference, June 17 - 22, 2012
2012
The Gordon Research Conference on CELLULAR & MOLECULAR FUNGAL BIOLOGY was held at Holderness School, Holderness New Hampshire, June 17 - 22, 2012. The 2012 Gordon Conference on Cellular and Molecular Fungal Biology (CMFB) will present the latest, cutting-edge research on the exciting and growing field of molecular and cellular aspects of fungal biology. Topics will range from yeast to filamentous fungi, from model systems to economically important organisms, and from saprophytes and commensals to pathogens of plants and animals. The CMFB conference will feature a wide range of topics including systems biology, cell biology and morphogenesis, organismal interactions, genome organisation and regulation, pathogenesis, energy metabolism, biomass production and population genomics. The Conference was well-attended with 136 participants. Gordon Research Conferences does not permit publication of meeting proceedings.
Eukaryotic Cell, 2007
The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The ⌬rasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasB⌬113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited ϳ65% survival compared to ϳ10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.
FEMS Microbiology Reviews, 2012
Filamentous fungi produce a number of small bioactive molecules as part of their secondary metabolism ranging from benign antibiotics such as penicillin to threatening mycotoxins such as aflatoxin. Secondary metabolism can be linked to fungal developmental programs in response to various abiotic or biotic external triggers. The velvet family of regulatory proteins plays a key role in coordinating secondary metabolism and differentiation processes such as asexual or sexual sporulation and sclerotia or fruiting body formation. The velvet family shares a protein domain that is present in most parts of the fungal kingdom from chytrids to basidiomycetes. Most of the current knowledge derives from the model Aspergillus nidulans where VeA, the founding member of the protein family, was discovered almost half a century ago. Different members of the velvet protein family interact with each other and the nonvelvet protein LaeA, primarily in the nucleus. LaeA is a methyltransferase-domain protein that functions as a regulator of secondary metabolism and development. A comprehensive picture of the molecular interplay between the velvet domain protein family, LaeA and other nuclear regulatory proteins in response to various signal transduction pathway starts to emerge from a jigsaw puzzle of several recent studies.
Genetics, 1999
We showed previously that two genes, flbA and fadA, have a major role in determining the balance between growth, sporulation, and mycotoxin (sterigmatocystin; ST) production by the filamentous fungus Aspergillus nidulans. fadA encodes the α subunit for a heterotrimeric G-protein, and continuous activation of FadA blocks sporulation and ST production while stimulating growth. flbA encodes an A. nidulans regulator of G-protein signaling (RGS) domain protein that antagonizes FadA-mediated signaling to allow development. To better understand FlbA function and other aspects of FadA-mediated growth control, we have isolated and characterized mutations in four previously undefined genes designated as sfaA, sfaC, sfaD, and sfaE (suppressors of flbA), and a new allele of fadA (fadAR205H), all of which suppress a flbA loss-of-function mutation (flbA98). These suppressors overcome flbA losses of function in both sporulation and ST biosynthesis. fadAR205H, sfaC67, sfaD82, and sfaE83 mutations a...
G3: Genes, Genomes, Genetics, 2015
G protein2coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization. KEYWORDS filamentous fungi G protein2 coupled receptors functional genomics signal transduction Heterotrimeric G proteins G protein2coupled receptors (GPCRs) are responsible for a diversity of cell functions, including environmental sensing, metabolism, immunity, growth, and development in eukaryotic cells (Bock et al. 2014; Chini et al. 2013). GPCRs are seven alpha-helical transmembrane (TM) proteins anchored in the plasma membrane, with an intracellular carboxyl-and extracellular amino-terminus (Shukla et al. 2014; Chini et al. 2013). The heterotrimeric G protein2signaling cascade initiates with the GPCR sensing the stimulus (ligand) and then transducing this signal to the inside of the cell via heterotrimeric G proteins (Tesmer 2010). Heterotrimeric G proteins are composed of three subunits (a, b, and g) that are associated with the GPCR in the inactive state. Ligand binding to the GPCR causes a conformational change within the receptor that leads to exchange of GDP for GTP on the Ga subunit and dissociation of the Ga from the Gbg heterodimer and the GPCR (Jastrzebska 2013; Tesmer 2010). Both the Ga-GTP and Gbg can then regulate downstream effectors, which include mitogen-activated protein kinase cascades, ion channels, adenylyl cyclases, phosphodiesterases and phopholipases (Chini et al. 2013; Neves et al. 2002; Mende et al. 1998). The Ga subunit has native GTPase activity, hydrolyzing GTP to GDP. In addition, Regulators of G protein Signaling (RGS) proteins, also known as GTPase-Activating Proteins, can accelerate the hydrolysis process by 2000-fold (Ross and Wilkie 2000). Subsequently, the Ga-GDP and Gbg reassociate to form the inactive heterotrimeric complex at the GPCR on the membrane. Neurospora crassa has served as a eukaryotic model for human and plant pathogenic fungi and has been the most-studied filamentous fungal species for the past hundred years [reviewed in (Davis and Perkins 2002; Borkovich et al. 2004; Selker 2011)]. N. crassa has a more complex life cycle than budding or fission yeasts and grows through extension, branching, and fusion of tube-like structures called hyphae [reviewed in (Springer 1993; Borkovich et al. 2004; Glass et al. 2004)]. N. crassa hyphae contain incomplete cross-walls (septa) that delineate cell compartments but still allow flow of organelles and cytoplasmic materials throughout the intertwined hyphal structure (mycelium).
…, 2005
Nematode-trapping fungi enter the parasitic stage by developing specific morphological structures called traps. The global patterns of gene expression in traps and mycelium of the fungus Monacrosporium haptotylum were compared. The trap of this fungus is a unicellular spherical structure called the knob, which develops on the apex of a hyphal branch. RNA was isolated from knobs and mycelium and hybridized to a cDNA array containing probes of 2822 EST clones of M. haptotylum. Despite the fact that the knobs and mycelium were grown in the same medium, there were substantial differences in the patterns of genes expressed in the two cell types. In total, 23?3 % (657 of 2822) of the putative genes were differentially expressed in knobs versus mycelium. Several of these genes displayed sequence similarities to genes known to be involved in regulating morphogenesis and cell polarity in fungi. Among them were several putative homologues for small GTPases, such as rho1, rac1 and ras1, and a rho GDP dissociation inhibitor (rdi1). Several homologues to genes involved in stress response, protein synthesis and protein degradation, transcription, and carbon metabolism were also differentially expressed. In the last category, a glycogen phosphorylase (gph1) gene homologue, one of the most upregulated genes in the knobs as compared to mycelium, was characterized. A number of the genes that were differentially expressed in trap cells are also known to be regulated during the development of infection structures in plant-pathogenic fungi. Among them, a gas1 (mas3) gene homologue (designated gks1), which is specifically expressed in appressoria of the rice blast fungus, was characterized.
Bioscience, Biotechnology, and Biochemistry
In order to increase secondary metabolite production in filamentous fungi, a transcription factor gene in the biosynthetic gene cluster and global regulator genes such as laeA are considered plausible as targets for overexpression by genetic modification. In this study, we examined these overexpression effect in fungal sp. No. 14919 that produces FR901512, an HMG-CoA reductase inhibitor. Resultantly, the productivity was improved at 1.7–1.8 fold by overexpressing frlE, a transcription factor gene in the biosynthetic gene cluster, whereas productivity did not change by overexpression of laeA and veA. Furthermore, we searched for extra transcription factors affecting the productivity by transcriptome analysis between wild-type strain and highly productive UV mutants. After verifying productivity decrease by overexpression, Drf1, a novel transcription factor encoded by drf1 was identified as the negative regulator. Because each frlE product (FrlE) and Drf1 worked on the same cluster in...