Experimentally Validated Reconstruction and Analysis of a Genome-Scale Metabolic Model of an Anaerobic Neocallimastigomycota Fungus

ABSTRACT Anaerobic gut fungi in the phylum Neocallimastigomycota typically inhabit the digestive tracts of large mammalian herbivores, where they play an integral role in the decomposition of raw lignocellulose into its constitutive sugar monomers. However, quantitative tools to study their physiology are lacking, partially due to their complex and unresolved metabolism that includes the largely uncharacterized fungal hydrogenosome. Modern omics approaches combined with metabolic modeling can be used to establish an understanding of gut fungal metabolism and develop targeted engineering strategies to harness their degradation capabilities for lignocellulosic bioprocessing. Here, we introduce a high-quality genome of the anaerobic fungus Neocallimastix lanati from which we constructed the first genome-scale metabolic model of an anaerobic fungus. Relative to its size (200 Mbp, sequenced at 62× depth), it is the least fragmented publicly available gut fungal genome to date. Of the 1,788 lignocellulolytic enzymes annotated in the genome, 585 are associated with the fungal cellulosome, underscoring the powerful lignocellulolytic potential of N. lanati. The genome-scale metabolic model captures the primary metabolism of N. lanati and accurately predicts experimentally validated substrate utilization requirements. Additionally, metabolic flux predictions are verified by 13C metabolic flux analysis, demonstrating that the model faithfully describes the underlying fungal metabolism. Furthermore, the model clarifies key aspects of the hydrogenosomal metabolism and can be used as a platform to quantitatively study these biotechnologically important yet poorly understood early-branching fungi. IMPORTANCE Recent genomic analyses have revealed that anaerobic gut fungi possess both the largest number and highest diversity of lignocellulolytic enzymes of all sequenced fungi, explaining their ability to decompose lignocellulosic substrates, e.g., agricultural waste, into fermentable sugars. Despite their potential, the development of engineering methods for these organisms has been slow due to their complex life cycle, understudied metabolism, and challenging anaerobic culture requirements. Currently, there is no framework that can be used to combine multi-omic data sets to understand their physiology. Here, we introduce a high-quality PacBio-sequenced genome of the anaerobic gut fungus Neocallimastix lanati. Beyond identifying a trove of lignocellulolytic enzymes, we use this genome to construct the first genome-scale metabolic model of an anaerobic gut fungus. The model is experimentally validated and sheds light on unresolved metabolic features common to gut fungi. Model-guided analysis will pave the way for deepening our understanding of anaerobic gut fungi and provides a systematic framework to guide strain engineering efforts of these organisms for biotechnological use.

Biodegradation of atrazine and ligninolytic enzyme production by basidiomycete strains

Background Atrazine is one of the most widespread chlorinated herbicides, leaving large bulks in soils and groundwater. The biodegradation of atrazine by bacteria is well described, but many aspects of the fungal metabolism of this compound remain unclear. Thus, we investigated the toxicity and degradation of atrazine by 13 rainforest basidiomycete strains. Results In liquid medium, Pluteus cubensis SXS320, Gloelophyllum striatum MCA7, and Agaricales MCA17 removed 30, 37, and 38%, respectively, of initial 25 mg L-1 of the herbicide within 20 days. Deficiency of nitrogen drove atrazine degradation by Pluteus cubensis SXS320; this strain removed 30% of atrazine within 20 days in a culture medium with 2.5 mM of N, raising three metabolites; in a medium with 25 mM of N, only 21% of initial atrazine were removed after 40 days, and two metabolites appeared in culture extracts. This is the first report of such different outcomes linked to nitrogen availability during the biodegradation of atrazine by basidiomycetes. The herbicide also induced synthesis and secretion of extracellular laccases by Datronia caperata MCA5, Pycnoporus sanguineus MCA16, and Polyporus tenuiculus MCA11. Laccase levels produced by of P. tenuiculus MCA11 were 13.3-fold superior in the contaminated medium than in control; the possible role of this enzyme on atrazine biodegradation was evaluated, considering the strong induction and the removal of 13.9% of the herbicide in vivo. Although 88% of initial laccase activity remained after 6 h, no evidence of in vitro degradation was observed, even though ABTS was present as mediator. Conclusions This study revealed a high potential for atrazine biodegradation among tropical basidiomycete strains. Further investigations, focusing on less explored ligninolytic enzymes and cell-bound mechanisms, could enlighten key aspects of the atrazine fungal metabolism and the role of the nitrogen in the process.

Biodegradation of Atrazine and Ligninolytic Enzyme Production by Basidiomycete Strains

BackgroundAtrazine is one of the most widespread chlorinated herbicides, leaving large bulks in soils and groundwater. The biodegradation of atrazine by bacteria is well described, but many aspects of the fungal metabolism of this compound remain unclear. Thus, we investigated the toxicity and degradation of atrazine by 13 rainforest basidiomycete strains.ResultsIn liquid medium, Pluteus cubensis SXS320, Gloelophyllum striatum MCA7, and Agaricales MCA17 removed 30, 37, and 38%, respectively, of initial 25 mg/l of the herbicide within 20 days. Deficiency of nitrogen drove atrazine degradation by Pluteus cubensis SXS320; this strain removed 30% of atrazine within 20 days in a culture medium with 2.5 mM of N, raising three metabolites; in a medium with 25 mM of N, only 21% of initial atrazine were removed after 40 days, and two metabolites appeared in culture extracts. This is the first report of such different outcomes linked to nitrogen availability during the biodegradation of atrazine by basidiomycetes. The herbicide also induced synthesis and secretion of extracellular laccases by Datronia caperata MCA5, Pycnoporus sanguineus MCA16, and Polyporus tenuiculus MCA11. Laccase levels produced by of P. tenuiculus MCA11 were 13.3-fold superior in the contaminated medium than in control; the possible role on this enzyme on atrazine biodegradation was evaluated, considering the strong induction and the removal of 13.9% of the herbicide in vivo . Although 88% of initial laccase activity remained after 6 h, no evidence of in vitro degradation was observed, even though ABTS was present as mediator.ConclusionsThis study revealed a high potential for atrazine biodegradation among tropical basidiomycete strains. Further investigations, focusing on less explored ligninolytic enzymes and cell-bound mechanisms, could enlightens key aspects of the atrazine fungal metabolism and the role of the nitrogen in the process.

Uji Antagonis Jamur Gliocladium sp dalam Menghambat Pertumbuhan Jamur Fusarium sp Penyebab Penyakit Layu Pada Tanaman Pisang (Musa paradisiaca L.)

The fungus Gliocladium sp. is the microbial soil that can be isolated from the root zone of banana plant (Musa Paradica L.) Those fungi are included in the group of potentially antagonistic microbes in the growth inhibiting of Fusarium sp fungus which infects the plants and it cause disease on banana crops withered. This research aim was to know the difference of growth inhibiting percentage of Gliocladium sp, to the Fusarium sp. fungus at pH 5.5 and pH 7. This research uses experiment methods. The results of the growth inhibiting percentage in the last day was higher at pH 5.5 of 35.2% whereas at pH 7 of 14%. This indicates that the growth of the fungus is more suitable in pH acidic approach because of its nature which is able to adjust to the fungal metabolism. The inhibition mechanism of the Gliocladium sp. on fungus inhibiting of Fusarium sp. has the stages of the competition, lysis and mycoparasit. Keywords: Gliocladium sp., Fusarium sp, banana plants, pH ABSTRAK Jamur Gliocladium sp. merupakan mikroba tanah yang dapat diisolasi dari daerah perakaran tanaman pisang (Musa Paradica L.) Jamur tersebut termasuk dalam kelompok mikroba antagonis yang berpotensi dalam menghambat pertumbuhan jamur Fusarium sp yang menginfeksi tanaman yang menyebabkan penyakit layu pada tanaman pisang. Penelitian ini bertujuan untuk mengetahui perbedaan persentase daya hambat jamur Gliocladium sp. dalam menghambat pertumbuhan jamur Fusarium sp. pada pH 5,5 dan pH 7. Penelitian ini menggunakan metode deskriptif dan eksperimen. Hasil dari persentase daya hambat hari terakhir lebih tinggi pada pH 5,5 sebesar 35,2% sedangkan pada pH 7 sebesar 14%. Hal ini menandakan bahwa pertumbuhan jamur lebih cocok di pH mendekati asam karena sifatnya yang mampu menyesuaikan dengan metabolisme jamur. Mekanisme Penghambatan jamur Gliocladium sp. kepada jamur Fusarium sp. mempunyai tahap kompetisi, lisis dan mikoparasit. Kata kunci: Gliocladium sp., Fusarium sp., tanaman pisang, pH

Clinostat Rotation Affects Metabolite Transportation and Increases Organic Acid Production by Aspergillus carbonarius, as Revealed by Differential Metabolomic Analysis

ABSTRACT Contamination by fungi may pose a threat to the long-term operation of the International Space Station because fungi produce organic acids that corrode equipment and mycotoxins that harm human health. Microgravity is an unavoidable and special condition in the space station. However, the influence of microgravity on fungal metabolism has not been well studied. Clinostat rotation is widely used to simulate the microgravity condition in studies carried out on Earth. Here, we used metabolomics differential analysis to study the influence of clinostat rotation on the accumulation of organic acids and related biosynthetic pathways in ochratoxin A (OTA)-producing Aspergillus carbonarius. As a result, clinostat rotation did not affect fungal cell growth or colony appearance but significantly increased the accumulation of organic acids, particularly isocitric acid, citric acid, and oxalic acid, and OTA both inside cells and in the medium, as well as resulted in a much higher level of accumulation of some products inside than outside cells, indicating that the transport of these metabolites from the cell to the medium was inhibited. This finding corresponded to the change in the fatty acid composition of cell membranes and the reduced thickness of the cell walls and cell membranes. Amino acid and energy metabolic pathways, particularly the tricarboxylic acid cycle, were influenced the most during clinostat rotation compared to the effects of normal gravity on these pathways. IMPORTANCE Fungi are ubiquitous in nature and have the ability to corrode various materials by producing metabolites. Research on how the space station environment, especially microgravity, affects fungal metabolism is helpful to understand the role of fungi in the space station. This work provides insights into the mechanisms involved in the metabolism of the corrosive fungus Aspergillus carbonarius under simulated microgravity conditions. Our findings have significance not only for preventing material corrosion but also for ensuring food safety, especially in the space environment.

Comparative Transcriptome Analysis Shows Conserved Metabolic Regulation during Production of Secondary Metabolites in Filamentous Fungi

ABSTRACTFilamentous fungi possess great potential as sources of medicinal bioactive compounds, such as antibiotics, but efficient production is hampered by a limited understanding of how their metabolism is regulated. We investigated the metabolism of six secondary metabolite-producing fungi of thePenicilliumgenus during nutrient depletion in the stationary phase of batch fermentations and assessed conserved metabolic responses across species using genome-wide transcriptional profiling. A coexpression analysis revealed that expression of biosynthetic genes correlates with expression of genes associated with pathways responsible for the generation of precursor metabolites for secondary metabolism. Our results highlight the main metabolic routes for the supply of precursors for secondary metabolism and suggest that the regulation of fungal metabolism is tailored to meet the demands for secondary metabolite production. These findings can aid in identifying fungal species that are optimized for the production of specific secondary metabolites and in designing metabolic engineering strategies to develop high-yielding fungal cell factories for production of secondary metabolites.IMPORTANCESecondary metabolites are a major source of pharmaceuticals, especially antibiotics. However, the development of efficient processes of production of secondary metabolites has proved troublesome due to a limited understanding of the metabolic regulations governing secondary metabolism. By analyzing the conservation in gene expression across secondary metabolite-producing fungal species, we identified a metabolic signature that links primary and secondary metabolism and that demonstrates that fungal metabolism is tailored for the efficient production of secondary metabolites. The insight that we provide can be used to develop high-yielding fungal cell factories that are optimized for the production of specific secondary metabolites of pharmaceutical interest.