Absence of repellents in Ustilago maydis induces genes encoding small secreted proteins

The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae

Fungal based biocomposite for habitat structures on the Moon and Mars

One of the key capabilities for long term human exploration missions beyond low Earth orbit is a suitable technology for in-situ resource utilization (ISRU). Indigenous or locally cultivated resources lower the mass and volume of payload that needs to be brought from Earth, and as a result decrease the costs. Therefore, an efficient trade-off space has to be created between robotic ISRU systems brought from Earth, and type of materials used in the process to increase the long term mission sustainability. The objective of the current study is to investigate the production process and feasibility of fungal based biocomposite material for habitat structures on the Moon and Mars, using automated additive construction technology. Previous studies have shown that certain types of fungi are able to survive in extreme environmental conditions. Experiments with γ-radiation in Co-60 facility at ESTEC showed that the chosen model organism, Schizophyllum commune, is able to survive in 20 Gy and 200 Gy dose levels, with the ~30% of colony forming units at 200 Gy. The lower levels of gravity will also not affect the growth of the fungus. Experiments with the random positioning machine (RPM) showed that Schizophyllum commune 227 grows even faster in simulated microgravity conditions than at 1G. The proposed production process of the biocomposite on the Moon or Mars would require cultivating the mycelium of Schizophyllum commune (SC) in-situ from a minimum amount of starter culture brought from Earth. This would be combined with locally grown Azolla filiculoides (AF), an aquatic fern, with the ability to rapidly increase its biomass growing on water, while fixing nitrogen and carbon directly from atmosphere. For an additive construction experiment with a 6-axis robotic arm a mixture of SC mycelium, AF, water and psyllium was used to generate an extractable paste through a nozzle system to fabricate a number of prototypes with different print parameters

Hydrophobin gene deletion and environmental growth conditions impact mechanical properties of mycelium by affecting the density of the material

Filamentous fungi colonize substrates by forming a mycelium. This network of hyphae can be used as a bio-based material. Here, we assessed the impact of environmental growth conditions and deletion of the hydrophobin gene sc3 on material properties of the mycelium of the mushroom forming fungus Schizophyllum commune. Thermogravimetric analysis showed that Δsc3 mycelium retained more water with increasing temperature when compared to the wild type. The Young's modulus (E) of the mycelium ranged between 438 and 913 MPa when the wild type strain was grown in the dark or in the light at low or high CO2levels. This was accompanied by a maximum tensile strength (σ) of 5.1-9.6 MPa. In contrast, E and σ of the Δsc3 strain were 3-4- fold higher with values of 1237-2727 MPa and 15.6-40.4 MPa, respectively. These values correlated with mycelium density, while no differences in chemical composition of the mycelia were observed as shown by ATR-FTIR. Together, genetic modification and environmental growth conditions impact mechanical properties of the mycelium by affecting the density of the mycelium. As a result, mechanical properties of wild type mycelium were similar to those of natural materials, while those of Δsc3 were more similar to thermoplastics

FluG affects secretion in colonies of Aspergillus niger

Colonies of Aspergillus niger are characterized by zonal heterogeneity in growth, sporulation, gene expression and secretion. For instance, the glucoamylase gene glaA is more highly expressed at the periphery of colonies when compared to the center. As a consequence, its encoded protein GlaA is mainly secreted at the outer part of the colony. Here, multiple copies of amyR were introduced in A. niger. Most transformants over-expressing this regulatory gene of amylolytic genes still displayed heterogeneous glaA expression and GlaA secretion. However, heterogeneity was abolished in transformant UU-A001.13 by expressing glaA and secreting GlaA throughout the mycelium. Sequencing the genome of UU-A001.13 revealed that transformation had been accompanied by deletion of part of the fluG gene and disrupting its 3' end by integration of a transformation vector. Inactivation of fluG in the wild-type background of A. niger also resulted in breakdown of starch under the whole colony. Asexual development of the ∆fluG strain was not affected, unlike what was previously shown in Aspergillus nidulans. Genes encoding proteins with a signal sequence for secretion, including part of the amylolytic genes, were more often downregulated in the central zone of maltose-grown ∆fluG colonies and upregulated in the intermediate part and periphery when compared to the wild-type. Together, these data indicate that FluG of A. niger is a repressor of secretion

Fungal based biocomposite for habitat structures on the Moon and Mars

One of the key capabilities for long term human exploration missions beyond low Earth orbit is a suitable technology for in-situ resource utilization (ISRU). Indigenous or locally cultivated resources lower the mass and volume of payload that needs to be brought from Earth, and as a result decrease the costs. Therefore, an efficient trade-off space has to be created between robotic ISRU systems brought from Earth, and type of materials used in the process to increase the long term mission sustainability. The objective of the current study is to investigate the production process and feasibility of fungal based biocomposite material for habitat structures on the Moon and Mars, using automated additive construction technology. Previous studies have shown that certain types of fungi are able to survive in extreme environmental conditions. Experiments with γ-radiation in Co-60 facility at ESTEC showed that the chosen model organism, Schizophyllum commune, is able to survive in 20 Gy and 200 Gy dose levels, with the ~30% of colony forming units at 200 Gy. The lower levels of gravity will also not affect the growth of the fungus. Experiments with the random positioning machine (RPM) showed that Schizophyllum commune 227 grows even faster in simulated microgravity conditions than at 1G. The proposed production process of the biocomposite on the Moon or Mars would require cultivating the mycelium of Schizophyllum commune (SC) in-situ from a minimum amount of starter culture brought from Earth. This would be combined with locally grown Azolla filiculoides (AF), an aquatic fern, with the ability to rapidly increase its biomass growing on water, while fixing nitrogen and carbon directly from atmosphere. For an additive construction experiment with a 6-axis robotic arm a mixture of SC mycelium, AF, water and psyllium was used to generate an extractable paste through a nozzle system to fabricate a number of prototypes with different print parameters

Hydrophobin gene deletion and environmental growth conditions impact mechanical properties of mycelium by affecting the density of the material

Filamentous fungi colonize substrates by forming a mycelium. This network of hyphae can be used as a bio-based material. Here, we assessed the impact of environmental growth conditions and deletion of the hydrophobin gene sc3 on material properties of the mycelium of the mushroom forming fungus Schizophyllum commune. Thermogravimetric analysis showed that Δsc3 mycelium retained more water with increasing temperature when compared to the wild type. The Young's modulus (E) of the mycelium ranged between 438 and 913 MPa when the wild type strain was grown in the dark or in the light at low or high CO2 levels. This was accompanied by a maximum tensile strength (σ) of 5.1-9.6 MPa. In contrast, E and σ of the Δsc3 strain were 3-4- fold higher with values of 1237-2727 MPa and 15.6-40.4 MPa, respectively. These values correlated with mycelium density, while no differences in chemical composition of the mycelia were observed as shown by ATR-FTIR. Together, genetic modification and environmental growth conditions impact mechanical properties of the mycelium by affecting the density of the mycelium. As a result, mechanical properties of wild type mycelium were similar to those of natural materials, while those of Δsc3 were more similar to thermoplastics.Emerging Material

FluG affects secretion in colonies of Aspergillus niger

Colonies of Aspergillus niger are characterized by zonal heterogeneity in growth, sporulation, gene expression and secretion. For instance, the glucoamylase gene glaA is more highly expressed at the periphery of colonies when compared to the center. As a consequence, its encoded protein GlaA is mainly secreted at the outer part of the colony. Here, multiple copies of amyR were introduced in A. niger. Most transformants over-expressing this regulatory gene of amylolytic genes still displayed heterogeneous glaA expression and GlaA secretion. However, heterogeneity was abolished in transformant UU-A001.13 by expressing glaA and secreting GlaA throughout the mycelium. Sequencing the genome of UU-A001.13 revealed that transformation had been accompanied by deletion of part of the fluG gene and disrupting its 3' end by integration of a transformation vector. Inactivation of fluG in the wild-type background of A. niger also resulted in breakdown of starch under the whole colony. Asexual development of the ∆fluG strain was not affected, unlike what was previously shown in Aspergillus nidulans. Genes encoding proteins with a signal sequence for secretion, including part of the amylolytic genes, were more often downregulated in the central zone of maltose-grown ∆fluG colonies and upregulated in the intermediate part and periphery when compared to the wild-type. Together, these data indicate that FluG of A. niger is a repressor of secretion

Hydrophobin gene deletion and environmental growth conditions impact mechanical properties of mycelium by affecting the density of the material

Filamentous fungi colonize substrates by forming a mycelium. This network of hyphae can be used as a bio-based material. Here, we assessed the impact of environmental growth conditions and deletion of the hydrophobin gene sc3 on material properties of the mycelium of the mushroom forming fungus Schizophyllum commune. Thermogravimetric analysis showed that Δsc3 mycelium retained more water with increasing temperature when compared to the wild type. The Young's modulus (E) of the mycelium ranged between 438 and 913 MPa when the wild type strain was grown in the dark or in the light at low or high CO2levels. This was accompanied by a maximum tensile strength (σ) of 5.1-9.6 MPa. In contrast, E and σ of the Δsc3 strain were 3-4- fold higher with values of 1237-2727 MPa and 15.6-40.4 MPa, respectively. These values correlated with mycelium density, while no differences in chemical composition of the mycelia were observed as shown by ATR-FTIR. Together, genetic modification and environmental growth conditions impact mechanical properties of the mycelium by affecting the density of the mycelium. As a result, mechanical properties of wild type mycelium were similar to those of natural materials, while those of Δsc3 were more similar to thermoplastics

Fabrication factors influencing mechanical, moisture- and water-related properties of mycelium-based composites

Mycelium-based composites result from the growth of filamentous fungi on organic materials such as agricultural waste streams. These novel biomaterials represent a promising alternative for product design and manufacturing both in terms of sustainable manufacturing processes and circular lifespan. This study shows that their morphology, density, tensile and flexural strength, as well as their moisture- and water-uptake properties can be tuned by varying type of substrate (straw, sawdust, cotton), fungal species (Pleurotus ostreatus vs. Trametes multicolor) and processing technique (no pressing or cold or heat pressing). The fungal species impacts colonization level and the thickness of the air-exposed mycelium called fungal skin. Colonization level and skin thickness as well as the type of substrate determine the stiffness and water resistance of the materials. Moreover, it is shown that heat pressing improves homogeneity, strength and stiffness of the materials shifting their performance from foam-like to cork- and wood-like. Together, these results demonstrate that by changing the fabrication process, differences in performance of mycelium materials can be achieved. This highlights the possibility to produce a range of mycelium-based composites. In fact, it is the first time mycelium composites have been described with natural material properties.Emerging Material

Spatially Resolving the Secretome within the Mycelium of the Cell Factory <i>Aspergillus niger</i>

<i>Aspergillus niger</i> is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of <i>A. niger</i> that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in <i>A. niger</i>, which is instrumental in understanding how fungi degrade complex substrates in nature