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Activation of secondary metabolism in Aspergillus and related filamentous fungi through regulatory engineering.

Filamentous fungi are major contributors to diverse secondary metabolites with broad applications to medicine, agriculture, and biotechnology. Advances in genome sequencing and bioinformatic tools have revealed that fungal genomes encode far more biosynthetic gene clusters (BGCs) than are expressed under normal laboratory conditions, leaving much biosynthetic potential transcriptionally silent. Overcoming this gap between predicted and observed secondary metabolism has become a major challenge in fungal natural product discovery. In this review, we summarize current strategies for activating silent or weakly expressed fungal BGCs through regulatory engineering, with an emphasis on approaches validated in Aspergillus, Penicillium, Monascus, and related filamentous fungi. We focus on genetic and chemical manipulations that enable coordinated activation of multiple biosynthetic pathways through chromatin-level modifiers, global transcriptional regulators, and developmental regulators. By framing these regulators as practical tools rather than solely biological components, we demonstrate their strengths, limitations, and applications in Aspergillus and related filamentous fungi. We further discuss emerging combinatorial and integrative approaches that use regulatory engineering alongside omics technologies and predictive tools, outlining alternatives and future directions for improving the interpretability of silent pathway activation.

Journal Article

Absence of spermine in filamentous fungi.

Polyamines were examined in several yeasts and filamentous fungi. Whereas putrescine, spermidine, and spermine were present in the yeasts, spermine was not detected in any of the filamentous fungi.

Ascomycota

Lytic enzymes in the autolysis of filamentous fungi.

The degrees of autolysis attained by five different genera of filamentous fungi during an incubation period of 60 days, under the same culture conditions were: 87.3% for Penicillium oxalicum; 65.9% for Neurospora crassa; 62.7% for Polystictus versicolor; 51.7% for Aspergillus niger and 23.5% for Nectria galligena. N. crassa, A. niger and P. versicolor reached the end of the autolysis during this incubation period (60 days), whereas P. oxalicum and N. galligena did not. The excretion of the lytic enzymes beta-N-acetylglucosaminidase, beta -1-3 glucanase, chitinase, invertase and acid phosphatase into the culture medium during growth and autolysis was investigated. The excretion of these enzymes was consistent with the degree of autolysis reached, the maximum excretion belonging to P. oxalicum and the minimum to N. galligena. The N. crassa invertase was excreted into the culture liquid at levels very much higher than the other enzymes studied, and at levels very much higher than the invertases excreted by the other fungi.

Acetylglucosaminidase

Subchronic toxicity test for two thermotolerant filamentous fungi used for single cell protein production.

Safety evaluations of two thermotolerant filamentous fungi, Cephalosporium eichhorniae 152 (C. 152) and Rhizopus chinensis 180 (R. 180), grown on a sugar-salts medium were carried out through feeding the biomases to rats at 20% or 40% dietary levels for 90 days. There was a control group fed soybean meal. Weight gain and feed consumption for rats fed 20% C. 152 were equal to those for the control animals, but were depressed in the other three groups, especially the rats fed R. 180. All animals appeared normal and healthy except that transient alopecia was found for a short duration in the fungi-treated rats in the initial period. The cause of this lesion is not clear. At the end of the feeding trial, clinical determinations of constituents in blood and urine samples were conducted. The animals were autopsied and weights for four organs were taken. Histopathological examinations for 26 different tissues were carried out. Mild changes were found in both C. 152 and R. 180-treated rats but most of the these were not considered to be related to treatment.

Acremonium

Mixed cultures of different yeasts species and yeasts with filamentous fungi in the SCP production. I. Production of single cell protein by mixed cultures Candida lipolytica and Candida tropicalis.

The aim of this study was to determine the application of mixed cultures Candida lipolytica and Candida tropicalis in the SCP production. N-paraffin fraction of crude oil and individual n-alkanes C:7--C:17 and glucose were used as carbon sources. The cultures were grown on laboratory scale in shaking flasks and in a 7 1 fermentor. It was found that the mixed cultures gave about 18% higher yield of biomass than the individual cultures.

Alkanes

Barcoded mutant library enables high-throughput functional genomics in a filamentous fungus.

Advances in sequencing technology enabling rapid and inexpensive whole-genome sequencing highlight how few genes are functionally characterized. This problem is particularly acute in filamentous fungi, where even in the best studied organisms upward of half of genes are poorly characterized or unannotated. High-throughput tools to identify gene function exist for single-celled organisms, like yeast and bacteria. However, filamentous fungi present challenges to high-throughput gene characterization, including low transformation efficiency and multinucleate cells. Filamentous fungi are critical components of nutrient cycling in ecosystems, form symbioses with plants that improve nutrient uptake, and are devastating human, plant, and animal pathogens causing millions of deaths and substantial crop loss each year. Thus, it is critical to overcome challenges to rapid gene characterization in filamentous fungi. We generated a library of hundreds of millions of uniquely barcoded plasmids containing a broad host-range drug resistance marker for ectopic insertion into filamentous fungal genomes by Agrobacterium tumefaciens. We then optimized A. tumefaciens mediated transformation of the biocontrol agent Trichoderma atroviride and made an insertional mutagenesis library containing 83,311 barcoded insertions, disrupting 5,331 of 11,863 predicted genes. This library enables high-throughput screens to rapidly connect genotype to phenotype. Quantifying relative barcode abundance in the pooled library before and after exposure to experimental conditions identified candidate genes and recovered known pathway components in amino acid biosynthetic, fructose utilization, and xylose utilization pathways. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling investigations of fungal biology to improve medical outcomes, biotechnology, and sustainable agriculture.

Genomics

An automated radiometric microassay of fungal growth: quantitation of growth of T. mentagrophytes.

An automated radiometric microassay of the growth of Trichophyton mentagrophytes and other filamentous fungi is described. The assay is based upon the incorporation of 14C(U) glucose into the organism. Fractionation studies indicate that 73% of the label is found in trichloroacetic acid-insoluble macromolecular components of the mycelium. Incorporation of label directly correlated with growth as estimated by visual scoring of turbidity and as recorded in photomicrographs. Incorporation of 14C(U) glucose delineated a lag, exponential and stationary or plateau phase of growth. These phases could be completely inhibited by the antifungal agent tolnaftate. It was concluded that the growth of filamentous fungi can be successfully monitored by the radiometric method described. Moreover, this method is sensitive, accurate, reproducible, rapid and free of the variability inherent in many traditional estimates growth.

Radiometry

Studies on the fungal flora in the lung of human necropsy cases. A critical survey in connection with the pathogenesis of opportunistic fungus infections.

An ecological study was carried out on the fungal flora in the human lung of 159 autopsy cases. Fungi were isolated from 129 cases (81.1%). Filamentous fungi consisting of 918 strains were isolated from 113 cases, and the dominant genera were Aspergillus and Penicillium. Yeasts were isolated from 58 cases, and the dominant genus appeared to be Candida. With morbid anatomical study, the incidence of the fungus isolation was examined from various points of view. It was concluded that even a healthy respiratory parenchyma of the human lung cannot be assumed as aseptic. However, only a few numbers of a few genera of air-born fungi were isolated. Special stress was laid on the exposure of the respiratory parenchyma of the human lung to air-born fungi in connection with the pathogenesis of opportunistic fungus infections.

Adolescent

Mycological profile of crew during 56-day simulated orbital flight.

Mycological analyses were made on specimens obtained from the skin, oral rinse, urine, and feces of three subjects participating in a 56 day Skylab simulation test. Samples were taken over a 175 day period divided into three phases consisting of a prechamber, intrachamber, and postchamber period. A total of 69 species of filamentous fungi and 22 species of yeast and yeast-like fungi were recovered and identified. There was a marked decrease in the isolation incidence of both the filamentous and yeast and yeast-like fungi during the intrachamber period. This, taken with the fact that 71% of the filamentous species and 55% of the yeast and yeast-like species were isolated no more than twice, reflects the transitory nature of the relationship between the human body and most fungi. However, there was a relative increase in the potential pathogens Candida albicans and Candida parapsilosis during the intrachamber period. Over 50% of all fungi recovered were isolated from the nasal and oral cavities. There was no evidence of intercrew transfer of a particular species during the intrachamber period nor was there any evidence of microbial shock during the postchamber period.

Adult

Mycoflora of activated sewage sludge.

Thirty-eight species of fungi were identified in pure culture after isolation from activated sewage sludge by serial dilution. Nine species and genera were identified that had not been previously reported. In 1963, Cooke (1) published an excellent laboratory guide on the identification of fungi from polluted water, sewage, and sewage treatment systems; of approximately 30 papers cited only one (2) dealt with fungi from activated sewage sludge. Later (1970), Cooke & Pipes (3) enumerated 47 fungi consisting of 4 genera of yeasts and 33 genera of filamentous fungi that had been isolated from activated sludge. This paper reports the mycoflora of anaerobically digested sludge from a residential area in Auburn, Alabama.

Alabama

Xy-31 and Lat-1 regulate the induction of polysaccharide disintegrating genes by arabinose in Neurospora crassa OR74A.

BACKGROUND: Arabinose has been identified as a potent inducer of the cellulase regulon in filamentous fungi. However, the precise molecular mechanisms that drive this induction remain largely unknown. This study aimed to elucidate the mechanisms underlying the arabinose-induced activation of cellulase and hemicellulase enzyme clusters in the model filamentous fungus Neurospora crassa OR74A. RESULTS: Arabinose stimulates the secretion of cellulases and hemicellulases in N. crassa. While genome-wide scrutiny predicted several sugar transporters in N. crassa, expression studies under avicel and arabinan conditions indicated a notable upregulation of pentose transporters encoded by the lat-1 and xy-31 genes. The lat-1 and xy-31 deleted strains showed reduced arabinose uptake and diminished growth on arabinose media, highlighting their role in arabinose transport. Additionally, these transporter-deficient strains exhibited a significant decrease in endoglucanase and arabinase activity, which correlated with lower expression of cellulase-encoding transcripts, indicating the crucial roles of Lat-1 and Xy-31 in promoting the secretion of enzymes that degrade biomass. Consistent with these findings, overexpression of lat-1 and xy-31 in N. crassa resulted in a significant increase in cellulase secretion. CONCLUSION: We demonstrated that N. crassa has multiple pentose transporters. Furthermore, we validated that the Lat-1 and Xy-31 transporters play crucial roles in arabinose-mediated induction of polysaccharide-disintegrating enzymes in N. crassa. Both transporters are required for efficient arabinose uptake and for the subsequent arabinose-dependent induction of polysaccharide-disintegrating enzymes. Collectively, these findings significantly advance the molecular understanding of fungal hemicellulose utilization and identify key transporters that represent promising targets for enhancing fungal biomass bioconversion.

arabinose

Microbial aggregate contamination of water lines in dental equipment and its control.

Water from some dental clinics has been examined and found to be discoloured, badly tasting and with a foul odour. Moreover, brown or black flakes were often present in tap water, as well as in the water lines of dental equipment. Examination by phase-contrast and electron microscope showed the flakes to consist of aggregated fungi and bacteria, and similar structures were found in a layer on the inner surfaces of the clinics water tubes and pipes. The ultrastructure of some aggregating microorganisms, including fungal hyphae and sheath-forming and stalked bacteria, was studied in detail, and several modes of aggregation were suggested. Cultivation of contaminated water samples revealed the presence of filamentous fungi, including Cladosporium and Cephalosporium, and of non-fluorescent Pseudomonas, Aeromonas, Acinetobacter, Alcaligenes, Flavobacterium, and Moraxella (?). Removal of microorganisms from the walls of the tubing was effectively accomplished by rinsing with the non-corrosive solution of 4 per cent Tween 80, coloured with Ponceau 4 R.

Bacteria

Multistrategy metabolic engineering of Talaromyces pinophilus for α-amylase production from lignocellulosic biomass.

Filamentous fungi are important hosts for industrial enzyme production. Growing demand for α-amylase has increased reliance on food-derived carbon substrates, necessitating fungal strains that efficiently utilize nongrain biomass. In this study, Talaromyces pinophilus Y117 was metabolically engineered to produce α-amylase from lignocellulosic biomass. A strong cellobiohydrolase I gene (cbh1) promoter (Pcbh1Tru) was identified to drive expression. Multiple rounds of multilocus integration of the α-amylase gene were performed using homologous multicopy genomic sequences as recombination arms with a Cre/loxP-based recyclable selection system, yielding the multicopy strain Tp4, which achieved 4124.5 U/mL α-amylase activity in shake-flask fermentation with corncob powder as the sole carbon source. To minimize enzyme degradation, the protease gene 8538 was deleted using the Cre/lox2272 system, generating Tp4Δp. This strain showed a 50% increase in shake-flask α-amylase activity (6208.4 U/mL). In 3-L bioreactor cultivation, Tp4Δp exhibited excellent production performance, achieving 26 712.2 U/mL α-amylase activity. When corncob powder was used as the sole substrate, the cellulose and hemicellulose degradation rates reached 90.00% and 70.01%, respectively, and the enzyme yield reached 213 697.5 U per gram of corncob powder. This engineered strain demonstrates strong potential for industrial applications. The synthesis-degradation synergistic optimization strategy provides a practical approach for engineering filamentous fungal cell factories to produce enzymes directly from lignocellulosic biomass. One sentence summary Metabolic engineering of Talaromyces pinophilus through promoter optimization, multicopy integration, and protease deletion enables efficient α-amylase production from lignocellulosic biomass, achieving 26 712 U/mL in bioreactor fermentation.

Talaromyces

A simple device for stationary cultivation of microorganisms.

A new device is designed that is suitable for both laboratory- and industrial-scale cultivation of microorganisms, particularly filamentous fungi, on the surface of liquid media. The device reliably ensures the sterility of cultivation, regardless of its duration. Its installation costs are relatively low and maximum production is reached per unit of installation space. In comparison with submerged cultures in fermentors, the proposed mode does not use a high consumption of energy for stirring and aeration (consequently, the duration of cultivation is not an economically relevant factor) and does not require maintenance of complicated equipment. Due to the building and construction of the technological equipment, the production plant is capable of very fast expansion and, when the need arises of very fast and economical liquidation.

Economics

Acid phosphatase localization in the fungus Whetzelinia sclerotiorum.

Acid phosphatase was localized by light and electron microscopy in chains of vacuoles in hyphal tip cells of Whetzelinia sclerotiorum. The enzyme was present in these vacuoles whether or not conditions favored extracellular acid phosphatase secretion. Apical vesicles, microbodies, Woronin bodies, and lipid bodies did not contain acid phosphatase. The implications regarding terminology of organelles in filamentous fungi are discussed with special reference to the fungal spherosome concept.

Acid Phosphatase

Mycotoxicoses of animals.

Mycotoxicoses are intoxications caused by ingestions of foodstuffs contaminated with mycotoxins, i.e. toxic secondary metabolites of microscopic filamentous fungi (moulds). By field observations and by experimental testing, toxins or toxic strains of more than 100 species of fungi have been encountered. However, causal associations have so far only been established for a small number of mycotoxicoses in farm animals, and the more important mycotoxicoses are aflatoxicosis, facial eczema, mycotoxic nephropathy, and estrogenic syndrome. A full assessment of the impact of mycotoxins on the health of farm animals can hardly be made at present, because the amount of surveillance data from mycotoxicoses is very limited, due mainly to inadequate diagnostic criteria. A new set of criteria for the diagnosis of mycotoxicosis is proposed, which has been successfully applied in the causative study of mycotoxic porcine nephropathy.

Animal Feed