Potential Protein Production from Lignocellulosic Materials Using Edible Mushroom Forming Fungi.

There is a need for new protein sources to feed the world in a sustainable way. Converting non-food-grade "woody" side streams into food containing proteins will contribute to this mission. Mushroom forming fungi are unique in their capability to convert lignocellulosic substances into edible biomass containing protein. Especially if substrate mycelium can be used instead of mushrooms, this technology could be a serious contribution to addressing the protein challenge. In this Perspective, we discuss challenges toward production, purification, and market introduction of mushroom mycelium based foods.

Cytoplasmic Mixing, Not Nuclear Coexistence, Can Explain Somatic Incompatibility in Basidiomycetes.

Nonself recognition leading to somatic incompatibility (SI) is commonly used by mycologists to distinguish fungal individuals. Despite this, the process remains poorly understood in basidiomycetes as all current models of SI are based on genetic and molecular research in ascomycete fungi. Ascomycete fungi are mainly found in a monokaryotic stage, with a single type of haploid nuclei, and only briefly during mating do two genomes coexist in heterokaryotic cells. The sister phylum, Basidiomycota, differs in several relevant aspects. Basidiomycete fungi have an extended heterokaryotic stage, and SI is generally observed between heterokaryons instead of between homokaryons. Additionally, considerable nuclear migration occurs during a basidiomycete mating reaction, introducing a nucleus into a resident homokaryon with cytoplasmic mixing limited to the fused or neighboring cells. To accommodate these differences, we describe a basidiomycete model for nonself recognition using post-translational modification, based on a reader-writer system as found in other organisms. This post-translational modification combined with nuclear migration allows for the coexistence of two genomes in one individual while maintaining nonself recognition during all life stages. Somewhat surprisingly, this model predicts localized cell death during mating, which is consistent with previous observations but differs from the general assumptions of basidiomycete mating. This model will help guide future research into the mechanisms behind basidiomycete nonself recognition.

Mapping Recombination Landscape and Basidial Spore Number in the Button Mushroom Agaricus bisporus.

The button mushroom Agaricus bisporus is represented mainly by two varieties, a secondarily homothallic variety with predominantly two heterokaryotic spores per basidia and a heterothallic variety with predominantly four homokaryotic spored basidium. Both varieties also differ in their recombination landscape with the former showing crossovers (CO) predominantly at chromosome ends whereas the latter has a more evenly distribution of CO over the chromosomes. The two varieties are compatible, and this has been used to study segregation of the basidial spore number (BSN) and the genomic positions of recombination, i.e., the CO landscape, in order to find the underlying genetic determinants. Knowledge on genes controlling CO positions might facilitate either the conservation of favorable allele combinations or the disruption of unwanted allele combinations to reduce linkage drag. For BSN, in total seven QTL were found with the major QTL on chromosome 1 explaining ca. 55% of the phenotypic variation. It appeared, however, difficult to map the recombination landscape. This phenotype can only be assessed in the meiotic offspring of an intervarietal hybrid which is a laborious and difficult task. Nevertheless, this was done, and we were able to map three QTLs for this trait, two on chromosome 1 and one on chromosome 2 not overlapping with the QTL for BSN. The hurdles encountered are discussed and a new strategy is proposed that can solves these. We propose to use two genetically unrelated mapping populations both offspring of a cross between a var. bisporus and a var. burnettii homokaryon and thus segregating both for CO and BSN. Homokaryotic offspring of both populations can be intercrossed without limitation of mating incompatibility and marker homozygosity and the hybrid mushrooms directly used to map BSN. Homokaryotic offspring of these hybrid mushrooms can be genotypes to assess CO positions using next generation sequencing technologies that will solve marker problems encountered, especially for genotyping chromosome ends. This new approach can be a useful strategy for a more efficient breeding strategy for mushrooms in general.

Cycling in degradation of organic polymers and uptake of nutrients by a litter-degrading fungus.

Wood and litter degrading fungi are the main decomposers of lignocellulose and thus play a key role in carbon cycling in nature. Here, we provide evidence for a novel lignocellulose degradation strategy employed by the litter degrading fungus Agaricus bisporus (known as the white button mushroom). Fusion of hyphae allows this fungus to synchronize the activity of its mycelium over large distances (50 cm). The synchronized activity has a 13-h interval that increases to 20 h before becoming irregular and it is associated with a 3.5-fold increase in respiration, while compost temperature increases up to 2°C. Transcriptomic analysis of this burst-like phenomenon supports a cyclic degradation of lignin, deconstruction of (hemi-) cellulose and microbial cell wall polymers, and uptake of degradation products during vegetative growth of A. bisporus. Cycling in expression of the ligninolytic system, of enzymes involved in saccharification, and of proteins involved in nutrient uptake is proposed to provide an efficient way for degradation of substrates such as litter.

Interruption of an MSH4 homolog blocks meiosis in metaphase I and eliminates spore formation in Pleurotus ostreatus.

Pleurotus ostreatus, one of the most widely cultivated edible mushrooms, produces high numbers of spores causing severe respiratory health problems for people, clogging of filters and spoilage of produce. A non-sporulating commercial variety (SPOPPO) has been successfully introduced into the market in 2006. This variety was generated by introgression breeding of a natural mutation into a commercial variety. Our cytological studies revealed that meiosis in the natural and derived sporeless strains was blocked in metaphase I, apparently resulting in a loss of spore formation. The gene(s) underlying this phenotype were mapped to an 80 kb region strongly linked to sporelessness and identified by transformation of wild type genes of this region into a sporeless strain. Sporulation was restored by re-introduction of the DNA sequence encoding the P. ostreatus meiotic recombination gene MSH4 homolog (poMSH4). Subsequent molecular analysis showed that poMSH4 in the sporeless P. ostreatus was interrupted by a DNA fragment containing a region encoding a CxC5/CxC6 cysteine cluster associated with Copia-type retrotransposons. The block of meiosis in metaphase I by a poMSH4 null mutant suggests that this protein plays an essential role in both Class I and II crossovers in mushrooms, similar to animals (mice), but unlike in plants. MSH4 was previously shown to be a target for breeding of sporeless varieties in P. pulmonarius, and the null mutant of the MSH4 homolog of S. commune (scMSH4) confers an extremely low level of spore formation. We propose that MSH4 homologs are likely to be a breeding target for sporeless strains both within Pleurotus sp. and in other Agaricales.

Telomere-to-telomere assembled and centromere annotated genomes of the two main subspecies of the button mushroom Agaricus bisporus reveal especially polymorphic chromosome ends.

Agaricus bisporus, the most cultivated edible mushroom worldwide, is represented mainly by the subspecies var. bisporus and var. burnettii. var. bisporus has a secondarily homothallic life cycle with recombination restricted to chromosome ends, while var. burnettii is heterothallic with recombination seemingly equally distributed over the chromosomes. To better understand the relationship between genomic make-up and different lifestyles, we have de novo sequenced a burnettii homokaryon and synchronised gene annotations with updated versions of the published genomes of var. bisporus. The genomes were assembled into telomere-to-telomere chromosomes and a consistent set of gene predictions was generated. The genomes of both subspecies were largely co-linear, and especially the chromosome ends differed in gene model content between the two subspecies. A single large cluster of repeats was found on each chromosome at the same respective position in all strains, harbouring nearly 50% of all repeats and likely representing centromeres. Repeats were all heavily methylated. Finally, a mapping population of var. burnettii confirmed an even distribution of crossovers in meiosis, contrasting the recombination landscape of var. bisporus. The new findings using the exceptionally complete and well annotated genomes of this basidiomycete demonstrate the importance for unravelling genetic components underlying the different life cycles.

Critical Factors Involved in Primordia Building in Agaricus bisporus: A Review.

The button mushroom Agaricus bisporus is an economically important crop worldwide. Many aspects of its cultivation are well known, except for the precise biological triggers for its fructification. By and large, for most basidiomycete species, nutrient availability, light and a drop in temperature are critical factors for fructification. A. bisporus deviates from this pattern in the sense that it does not require light for fructification. Furthermore its fructification seems to be inhibited by a self-generated factor which needs to be removed by microorganisms in order to initiate fruiting. This review explores what is known about the morphogenesis of fruiting initiation in A. bisporus, the microflora, the self-inhibitors for fruiting initiation and transcription factors involved. This information is subsequently contrasted with an overall model of the regulatory system involved in the initiation of the formation of primordia in basidiomycetes. The comparison reveals a number of the blank spots in our understanding of the fruiting process in A. bisporus.

The transcriptional regulator c2h2 accelerates mushroom formation in Agaricus bisporus.

The Cys2His2 zinc finger protein gene c2h2 of Schizophyllum commune is involved in mushroom formation. Its inactivation results in a strain that is arrested at the stage of aggregate formation. In this study, the c2h2 orthologue of Agaricus bisporus was over-expressed in this white button mushroom forming basidiomycete using Agrobacterium-mediated transformation. Morphology, cap expansion rate, and total number and biomass of mushrooms were not affected by over-expression of c2h2. However, yield per day of the c2h2 over-expression strains peaked 1 day earlier. These data and expression analysis indicate that C2H2 impacts timing of mushroom formation at an early stage of development, making its encoding gene a target for breeding of commercial mushroom strains.

Applications of hydrophobins: current state and perspectives.

Hydrophobins are proteins exclusively produced by filamentous fungi. They self-assemble at hydrophilic-hydrophobic interfaces into an amphipathic film. This protein film renders hydrophobic surfaces of gas bubbles, liquids, or solid materials wettable, while hydrophilic surfaces can be turned hydrophobic. These properties, among others, make hydrophobins of interest for medical and technical applications. For instance, hydrophobins can be used to disperse hydrophobic materials; to stabilize foam in food products; and to immobilize enzymes, peptides, antibodies, cells, and anorganic molecules on surfaces. At the same time, they may be used to prevent binding of molecules. Furthermore, hydrophobins have therapeutic value as immunomodulators and can been used to produce recombinant proteins.

Production of (+)-valencene in the mushroom-forming fungus S. commune.

Production of commercially interesting sesquiterpenes was previously examined in plants and microorganisms such as Escherichia coli and Saccharomyces cerevisiae. We here investigate the potential of the mushroom Schizophyllum commune for the production of sesquiterpenes. Genomic analysis of S. commune revealed that the mevalonate pathway required for the synthesis of the farnesyl diphosphate substrate for sesquiterpene production is operational. Introduction of a valencene synthase gene resulted in production of the sesquiterpene (+)-valencene, both in mycelium and in fruiting bodies. Levels of (+)-valencene in culture media of strains containing a mutated RGS regulatory protein gene (thn) were increased fourfold compared to those in wild-type transformants. Up to 16 mg L(-1) (+)-valencene was produced in these strains. In addition, the amount of (+)-valencene containing n-dodecane recovered from the culture medium increased sixfold to sevenfold in the thn mutant strains due to the absence of schizophyllan.

The antitumor activity of hydrophobin SC3, a fungal protein.

The use of mushroom extracts has been common practice in traditional medicine for centuries, including the treatment of cancer. Proteins called hydrophobins are very abundant in mushrooms. Here, it was examined whether they have antitumor activity. Hydrophobin SC3 of Schizophyllum commune was injected daily intraperitoneally starting 1 day after tumor induction in two tumor mouse models (sarcoma and melanoma). SC3 reduced the size and weight of the melanoma significantly, but the sarcoma seemed not affected. However, microscopic analysis of the tumors 12 days after induction revealed a strong antitumor effect of SC3 on both tumors. The mitotic activity of the tumor decreased 1.6- (melanoma) to 2.3-fold (sarcoma), while the vital mass decreased 2.3- (melanoma) to 4.3-fold (sarcoma) compared to the control. Treatment did not cause any signs of toxicity. Behavior, animal growth, and weight of organs were similar to animals injected with vehicle, and no histological abnormalities were found in the organs. In vitro cell culture studies revealed no direct cytotoxic effect of SC3 towards sarcoma cells, while cytotoxic activity was observed towards melanoma cells at a high SC3 concentration. Daily treatment with SC3 did not result in detectable levels of anti-SC3 antibodies in the plasma. Instead, a cellular immune response was observed. Incubation of spleen cells with SC3 resulted in a 1.5- to 2.5-fold increase in interleukin-10 and TNF-α mRNA levels. In conclusion, the nontoxic fungal hydrophobin SC3 showed tumor-suppressive activity possibly via immunomodulation and may be of benefit as adjuvant in combination with chemotherapy and radiation.

Creating Surface Properties Using a Palette of Hydrophobins.

Small secreted proteins called hydrophobins play diverse roles in the life cycle of filamentous fungi. For example, the hydrophobin SC3 of Schizophyllum commune is involved in aerial hyphae formation, cell-wall assembly and attachment to hydrophobic surfaces. Hydrophobins are capable of self-assembly at a hydrophilic-hydrophobic interface, resulting in the formation of an amphipathic film. This amphipathic film can make hydrophobic surfaces of a liquid or a solid material wettable, while a hydrophilic surface can be turned into a hydrophobic one. These properties, among others, make hydrophobins of interest for medical and technical applications. For instance, hydrophobins can be used to purify proteins from complex mixtures; to reduce the friction of materials; to increase the biocompatibility of medical implants; to increase the solubility of water insoluble drugs; and to immobilize enzymes, for example, biosensor surfaces.

Use of hydrophobins in formulation of water insoluble drugs for oral administration.

The poor water solubility of many drugs requires a specific formulation to achieve a sufficient bioavailability after oral administration. Suspensions of small drug particles can be used to improve the bioavailability. We here show that the fungal hydrophobin SC3 can be used to make suspensions of water insoluble drugs. Bioavailability of two of these drugs, nifedipine and cyclosporine A (CyA), was tested when administered as a SC3-based suspension. SC3 (in a 1:2 (w/w) drug:SC3 ratio) or 100% PEG400 increased the bioavailability of nifedipine to a similar degree (6+/-2- and 4+/-3-fold, respectively) compared to nifedipine powder without additives. Moreover, SC3 (in a 7:1 (w/w) drug:hydrophobin ratio) was as effective as a 20-fold diluted Neoral formulation by increasing bioavailability of CyA 2.3+/-0.3-fold compared to CyA in water. Interestingly, using SC3 in the CyA formulation resulted in a slower uptake (p<0.001 in T(max)) of the drug, with a lower peak concentration (C(max) 1.8 mg ml(-1)) at a later time point (T(max) 9+/-2 h) compared to Neoral (C(max) 2.2 mg ml(-1); T(max) 3.2+/-0.2). Consequently, SC3 will result in a more constant, longer lasting drug level in the body. Taken together, hydrophobins are attractive candidates to formulate hydrophobic drugs.

The use of mushroom-forming fungi for the production of N-glycosylated therapeutic proteins.

The market for N-glycosylated therapeutic proteins represents multi-billion dollars in sales and is growing more than 10% each year. This requires cost-effective production platforms that display correct and homogeneous N-glycosylation. Based on recent results, we propose to use mushroom-forming basidiomycetes for the production of N-glycosylated therapeutic proteins.

Assembly of the fungal SC3 hydrophobin into functional amyloid fibrils depends on its concentration and is promoted by cell wall polysaccharides.

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 microg ml(-1)) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (beta(1-3),beta(1-6)-glucan) and beta(1-3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.

Interaction and comparison of a class I hydrophobin from Schizophyllum commune and class II hydrophobins from Trichoderma reesei.

Hydrophobins fulfill a wide spectrum of functions in fungal growth and development. These proteins self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. Hydrophobins are divided into two classes based on their hydropathy patterns and solubility. We show here that the properties of the class II hydrophobins HFBI and HFBII of Trichoderma reesei differ from those of the class I hydrophobin SC3 of Schizophyllum commune. In contrast to SC3, self-assembly of HFBI and HFBII at the water-air interface was neither accompanied by a change in secondary structure nor by a change in ultrastructure. Moreover, maximal lowering of the water surface tension was obtained instantly or took several minutes in the case of HFBII and HFBI, respectively. In contrast, it took several hours in the case of SC3. Oil emulsions prepared with HFBI and SC3 were more stable than those of HFBII, and HFBI and SC3 also interacted more strongly with the hydrophobic Teflon surface making it wettable. Yet, the HFBI coating did not resist treatment with hot detergent, while that of SC3 remained unaffected. Interaction of all the hydrophobins with Teflon was accompanied with a change in the circular dichroism spectra, indicating the formation of an alpha-helical structure. HFBI and HFBII did not affect self-assembly of the class I hydrophobin SC3 of S. commune and vice versa. However, precipitation of SC3 was reduced by the class II hydrophobins, indicating interaction between the assemblies of both classes of hydrophobins.

Hydrophobins: proteins with potential.

Hydrophobins are self-assembling proteins of fungal origin. Their ability to self-assemble into an amphipathic membrane is of interest for many different applications, ranging from medical and technical coatings to the production of proteinaceous glue and cosmetics. Assembled hydrophobins can modify surface characteristics, thus controling the binding properties of the surface; for example, enzymes can be actively and non-covalently immobilized on electrode surfaces and medical coatings can be improved for biocompatibility. Over the past few years research on hydrophobins has contributed to a better understanding of the self-assembly process and is generating more handles to control and manipulate the process. This knowledge could have an immediate effect on production levels, which are not yet adequate, and provide the boost needed for hydrophobins to reach their full potential.

Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus.

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.

Surface modifications created by using engineered hydrophobins.

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.