RESUMEN
Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two mechanisms in filamentous fungi to repair DNA damages. NHEJ is the dominant response pathway to rapidly join DNA double-strand breaks, but often leads to insertions or deletions. On the other hand, HDR is more precise and utilizes a homologous DNA template to restore the damaged sequence. Both types are exploited in genetic engineering approaches ranging from knock-out mutations to precise sequence modifications.In this study, we evaluated the efficiency of an HDR based gene integration system designed for the pyrG locus of Aspergillus niger. While gene integration was achieved at a rate of 91.4%, we also discovered a mixed-type repair (MTR) mechanism with simultaneous repair of a Cas9-mediated double-strand break by both NHEJ and HDR. In 20.3% of the analyzed transformants the donor DNA was integrated by NHEJ at the 3' end and by HDR at the 5' end of the double-strand break. Furthermore, sequencing of the locus revealed different DNA repair mechanisms at the site of the NHEJ event.Together, the results support the applicability of the genome integration system and a novel DNA repair type with implication on the diversity of genetic modifications in filamentous fungi.
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The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell. Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in Aspergillus niger. In addition, growth and budding of Aureobasidium pullulans cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of A. niger expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in Saccharomyces cerevisiae and Trichoderma reesei are challenging due to cell size restrictions. Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.
Asunto(s)
Aspergillus niger , Hifa , Imagenología Tridimensional , Hifa/crecimiento & desarrollo , Imagenología Tridimensional/métodos , Aspergillus niger/crecimiento & desarrollo , Microscopía Fluorescente/métodos , Esporas Fúngicas/crecimiento & desarrollo , Pared Celular , Hongos , Núcleo CelularRESUMEN
Aspergillus niger is an important filamentous fungus used for the industrial production of citric acid. One of the most important factors that affect citric acid production is the concentration of manganese(II) ions present in the culture broth. Under manganese(II)-limiting conditions, the fungus develops a pellet-like morphology that is crucial for high citric acid accumulation. The impact of manganese(II) ions on the transcription of the major citrate exporter encoding gene cexA was studied under manganese(II)-deficient and -sufficient conditions. Furthermore, citric acid production was analyzed in overexpression mutant strains of cexA in the presence and absence of manganese(II) ions, and the influence of CexA on fungal morphology was investigated by microscopy. Transcriptional upregulation of cexA in the absence of manganese(II) ions was observed and, by decoupling cexA expression from the native promoter system, it was possible to secrete more citric acid even in the presence of manganese. This effect was shown for both an inducible and a constitutive overexpression of cexA. Furthermore, it was found that the presence of CexA influences fungal morphology and promotes a more branched phenotype. According to this study, manganese(II) ions suppress transcription of the citrate exporter cexA in Aspergillus niger, causing citric acid secretion to decrease.
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BACKGROUND: Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. RESULTS: Here, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. CONCLUSION: The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.
RESUMEN
Lytic polysaccharide monooxygenases (LPMOs) have emerged as key proteins for depolymerization of cellulose. These copper-containing enzymes oxidize C-1 and/or C-4 bonds in cellulose, promoting increased hydrolysis of the oxidized cellulose chains. The LPMO from Thermoascus aurantiacus, a thermophilic ascomycete fungus, has been extensively studied and has served as a model LPMO. A method was developed to purify the LPMO from culture filtrates of T. aurantiacus along with its native cellobiohydrolase and endoglucanase. The activity of the purified LPMO was measured with a colorimetric assay that established the Topt of the native LPMO at 60 °C. Purification of the components of the T. aurantiacus cellulase mixture also enabled quantification of the amounts of cellobiohydrolase, endoglucanase and LPMO present in the T. aurantiacus culture filtrate, establishing that the LPMO was the most abundant protein in the culture supernatants. The importance of the LPMO to activity of the mixture was demonstrated by saccharifications with Avicel and acid-pretreated corn stover.
Asunto(s)
Proteínas Fúngicas , Oxigenasas de Función Mixta , Thermoascus/enzimología , Biomasa , Celulasas/química , Celulasas/aislamiento & purificación , Celulasas/metabolismo , Celulosa/análisis , Celulosa/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Hidrólisis , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/aislamiento & purificación , Oxigenasas de Función Mixta/metabolismoRESUMEN
Fragrance and flavor industries could not imagine business without aldehydes. Processes for their commercial production raise environmental and ecological concerns. The chemical reduction of organic acids to aldehydes is challenging. To fulfill the demand of a mild and selective reduction of carboxylic acids to aldehydes, carboxylic acid reductases (CARs) are gaining importance. We identified two new subtype IV fungal CARs from Dichomitus squalens CAR (DsCAR) and Trametes versicolor CAR (Tv2CAR) in addition to literature known Trametes versicolor CAR (TvCAR). Expression levels were improved by the co-expression of GroEL-GroES with either the trigger factor or the DnaJ-DnaK-GrpE system. Investigation of the substrate scope of the three enzymes revealed overlapping substrate-specificities. Tv2CAR and DsCAR showed a preferred pH range of 7.0 to 8.0 in bicine buffer. TvCAR showed highest activity at pHâ 6.5 to 7.5 in MES buffer and slightly reduced activity at pHâ 6.0 or 8.0. TvCAR appeared to tolerate a wider pH range without significant loss of activity. Type IV fungal CARs optimal temperature was in the range of 25-35 °C. TvCAR showed a melting temperature (Tm) of 55 °C indicating higher stability compared to type III and the other type IV fungal CARs (Tm 51-52 °C). Finally, TvCAR was used as the key enzyme for the bioreduction of 3,4-dihydroxyphenylacetic acid to the antioxidant 3-hydroxytyrosol (3-HT) and gave 58â mM of 3-HT after 24â h, which correlates to a productivity of 0.37â g L-1 h-1.
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A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30⯰C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40⯰C was determined to be 8.25â¯h and its melting temperature (Tm) was 56⯰C. In vitro reactions with TtCAR reduced 95.2% of 10â¯mM vanillic acid, which correlated to a titer of 1.4â¯gâ¯L-1 of vanillin. The space time yield of 0.029â¯gâ¯L-1â¯h-1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30â¯gâ¯L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive.