Fungal Biology and Biotechnology最新文献

Background: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle.

Results: In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance.

Conclusions: Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

Background: Trichoderma atroviride is a competitive soil-borne mycoparasitic fungus with extensive applications as a biocontrol agent in plant protection. Despite its importance and application potential, reference genes for RT-qPCR analysis in T. atroviride have not been evaluated. Light exerts profound effects on physiology, such as growth, conidiation, secondary metabolism, and stress response in T. atroviride, as well as in other fungi. In this study, we aimed to address this gap by identifying stable reference genes for RT-qPCR experiments in T. atroviride under different light conditions, thereby enhancing accurate and reliable gene expression analysis in this model mycoparasite. We measured and compared candidate reference genes using commonly applied statistical algorithms.

Results: Under cyclic light-dark cultivation conditions, tbp and rho were identified as the most stably expressed genes, while act1, fis1, btl, and sar1 were found to be the least stable. Similar stability rankings were obtained for cultures grown under complete darkness, with tef1 and vma1 emerging as the most stable genes and act1, rho, fis1, and btl as the least stable genes. Combining the data from both cultivation conditions, gapdh and vma1 were identified as the most stable reference genes, while sar1 and fis1 were the least stable. The selection of different reference genes had a significant impact on the calculation of relative gene expression, as demonstrated by the expression patterns of target genes pks4 and lox1.

Conclusion: The data emphasize the importance of validating reference genes for different cultivation conditions in fungi to ensure accurate interpretation of gene expression data.

Background: Fungi are prolific producers of bioactive small molecules of pharmaceutical or agricultural interest. The secondary metabolism of higher fungi (Dikarya) has been well-investigated which led to > 39,000 described compounds. However, natural product researchers scarcely drew attention to early-diverging fungi (Mucoro- and Zoopagomycota) as they are considered to rarely produce secondary metabolites. Indeed, only 15 compounds have as yet been isolated from the entire phylum of the Zoopagomycota.

Results: Here, we showcase eight species of the order Kickxellales (phylum Zoopagomycota) as potent producers of the indole-3-acetic acid (IAA)-derived compounds lindolins A and B. The compounds are produced both under laboratory conditions and in the natural soil habitat suggesting a specialized ecological function. Indeed, lindolin A is a selective agent against plant-pathogenic oomycetes such as Phytophthora sp. Lindolin biosynthesis was reconstituted in vitro and relies on the activity of two enzymes of dissimilar evolutionary origin: Whilst the IAA-CoA ligase LinA has evolved from fungal 4-coumaryl-CoA synthetases, the subsequently acting IAA-CoA:anthranilate N-indole-3-acetyltransferase LinB is a unique enzyme across all kingdoms of life.

Conclusions: This is the first report on bioactive secondary metabolites in the subphylum Kickxellomycotina and the first evidence for a non-clustered, two-step biosynthetic route of secondary metabolites in early-diverging fungi. Thus, the generally accepted "gene cluster hypothesis" for natural products needs to be reconsidered for early diverging fungi.

Background: The filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed.

Results: A gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), β-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, β-xylosidase, xylanase, β-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3.

Conclusions: Current work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited.

Background: Non-reducing polyketide synthases (NR-PKSs) account for a major share of natural product diversity produced by both Asco- and Basidiomycota. The present evolutionary diversification into eleven clades further underscores the relevance of these multi-domain enzymes. Following current knowledge, NR-PKSs initiate polyketide assembly by an N-terminal starter unit:acyl transferase (SAT) domain that catalyzes the transfer of an acetyl starter from the acetyl-CoA thioester onto the acyl carrier protein (ACP).

Results: A comprehensive phylogenetic analysis of NR-PKSs established a twelfth clade from which three representatives, enzymes CrPKS1-3 of the webcap mushroom Cortinarius rufoolivaceus, were biochemically characterized. These basidiomycete synthases lack a SAT domain yet are fully functional hepta- and octaketide synthases in vivo. Three members of the other clade of basidiomycete NR-PKSs (clade VIII) were produced as SAT-domainless versions and analyzed in vivo and in vitro. They retained full activity, thus corroborating the notion that the SAT domain is dispensable for many basidiomycete NR-PKSs. For comparison, the ascomycete octaketide synthase atrochrysone carboxylic acid synthase (ACAS) was produced as a SAT-domainless enzyme as well, but turned out completely inactive. However, a literature survey revealed that some NR-PKSs of ascomycetes carry mutations within the catalytic motif of the SAT domain. In these cases, the role of the domain and the origin of the formal acetate unit remains open.

Conclusions: The role of SAT domains differs between asco- and basidiomycete NR-PKSs. For the latter, it is not part of the minimal set of NR-PKS domains and not required for function. This knowledge may help engineer compact NR-PKSs for more resource-efficient routes. From the genomic standpoint, seemingly incomplete or corrupted genes encoding SAT-domainless NR-PKSs should not automatically be dismissed as non-functional pseudogenes, but considered during genome analysis to decipher the potential arsenal of natural products of a given fungus.

Background: Endophytic Aspergillus species produce countless valuable bioactive secondary metabolites. In the current study, Aspergillus flavus an endophyte from the soft coral Sarcophyton ehrenbergi was chemically explored and the extracted phytoconstituents were subsequently evaluated for antimicrobial activity. This is accomplished by employing nuclear magnetic resonance (NMR) spectroscopy and computational techniques. Additionally, An in vitro anticancer analysis of A. flavus total extract against breast cancer cells (MCF-7) was investigated.

Result: Six compounds were separated from the crude alcohol extract of the endophytic Aspergillus flavus out of which anhydro-mevalonolactone was reported for the first time. The anti-fungal and anti-Helicobacter pylori properties of two distinct compounds (Scopularides A and B) were assessed. Additionally, computational research was done to identify the binding mechanisms for all compounds. Both the compounds were found to be active against H. pylori with minimum inhibitory concentration (MIC) values ranging from 7.81 to 15.63 µg/ mL as compared with clarithromycin 1.95 µg/ mL. Scopularides A was potent against both Candida albicans and Aspergillus niger with MIC values ranging from 3.9 to 31.25 µg/ mL, while scopularides B only inhibits Candida albicans with MIC value of 15.63 µg/ mL and weak inhibitory activity against A. niger (MIC = 125 µg/ mL). Furthermore, cytotoxic activity showed a significant effect (IC₅₀: 30.46 mg/mL) against MCF-7 cells.

Conclusion: Our findings report that cytotoxic activity and molecular docking support the antimicrobial activity of Aspergillus flavus, which could be a promising alternative source as a potential antimicrobial agent.

Background: Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals.

Results: In this study we developed a rapid and robust technique called "Squash-PCR" to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains.

Conclusion: The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.

Background: The terphenylquinones represent an ecologically remarkable class of basidiomycete natural products as they serve as central precursors of pigments and compounds that impact on microbial consortia by modulating bacterial biofilms and motility. This study addressed the phylogenetic origin of the quinone synthetases that assemble the key terphenylquinones polyporic acid and atromentin.

Results: The activity of the Hapalopilus rutilans synthetases HapA1, HapA2 and of Psilocybe cubensis PpaA1 were reconstituted in Aspergilli. Liquid chromatography and mass spectrometry of the culture extracts identified all three enzymes as polyporic acid synthetases. PpaA1 is unique in that it features a C-terminal, yet catalytically inactive dioxygenase domain. Combined with bioinformatics to reconstruct the phylogeny, our results demonstrate that basidiomycete polyporic acid and atromentin synthetases evolved independently, although they share an identical catalytic mechanism and release structurally very closely related products. A targeted amino acid replacement in the substrate binding pocket of the adenylation domains resulted in bifunctional synthetases producing both polyporic acid and atromentin.

Conclusions: Our results imply that quinone synthetases evolved twice independently in basidiomycetes, depending on the aromatic α-keto acid substrate. Furthermore, key amino acid residues for substrate specificity were identified and changed which led to a relaxed substrate profile. Therefore, our work lays the foundation for future targeted enzyme engineering.

Background: Fungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolated Cephalotrichum gorgonifer (strain NG_p51). In the course of strain characterisation work we found that this strain is able to naturally produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and showing anti-tumor activity in KRAS-dependent cancer cells.

Results: In order to elucidate the biosynthetic pathway of rasfonin, the strain was genome sequenced, annotated, submitted to transcriptome analysis and genetic transformation was established. Biosynthetic gene cluster (BGC) prediction revealed the existence of 22 BGCs of which the majority was not expressed under our experimental conditions. In silico prediction revealed two BGCs with a suite of enzymes possibly involved in rasfonin biosynthesis. Experimental verification by gene-knock out of the key enzyme genes showed that one of the predicted BGCs is indeed responsible for rasfonin biosynthesis.

Conclusions: This study identified a biosynthetic gene cluster containing a key-gene responsible for rasfonin production. Additionally, molecular tools were established for the non-model fungus Cephalotrichum gorgonifer which allows strain engineering and heterologous expression of the BGC for high rasfonin producing strains and the biosynthesis of rasfonin derivates for diverse applications.

Background: The use of microbial biomasses, such as fungal biomass, to catalyze the transesterification of triglycerides (TG) for biodiesel production provides a sustainable, economical alternative while still having the main advantages of expensive immobilized enzymes.

Results: Biomasses of Aspergillus flavus and Rhizopus stolonifera were used to catalyze the transesterification of TG in waste frying oil (WFO). Isopropanol as an acyl-acceptor reduced the catalytic capability of the biomasses, while methanol was the most potent acyl-acceptor with a final fatty acid methyl ester (FAME) concentration of 85.5 and 89.7%, w/w, for R. stolonifer and A. flavus, respectively. Different mixtures of the fungal biomasses were tested, and higher proportions of A. flavus biomass improved the mixture's catalytic capability. C. sorokiniana cultivated in synthetic wastewater was used as feedstock to cultivate A. flavus. The biomass produced had the same catalytic capability as the biomass produced in the control culture medium. Response surface methodology (RSM) was adopted using central composite design (CCD) to optimize the A. flavus biomass catalytic transesterification reaction, where temperature, methanol concentration, and biomass concentration were selected for optimization. The significance of the model was verified, and the suggested optimum reaction conditions were 25.5 °C, 250 RPM agitation with 14%, w/w, biomass, 3 mol/L methanol, and a reaction duration of 24 h. The suggested optimum conditions were tested to validate the model and a final FAME concentration of 95.53%. w/w was detected.

Conclusion: Biomasses cocktails might be a legitimate possibility to provide a cheaper technical solution for industrial applications than immobilized enzymes. The use of fungal biomass cultivated on the microalgae recovered from wastewater treatment for the catalysis of transesterification reaction provides an additional piece of the puzzle of biorefinery. Optimizing the transesterification reaction led to a valid prediction model with a final FAME concentration of 95.53%, w/w.