Only one of three hydrophobins (Hyd1-3) contributes to conidial hydrophobicity and insect pathogenicity of Metarhizium robertsii.

Class I/II hydrophobins constitute a family of small amphiphilic proteins that mediate cell hydrophobicity and adhesion to host or substrata and have pleiotropic effects in filamentous fungi. Here we report that only class I Hyd1 is essential for conidial hydrophobicity and insect pathogenicity among three hydrophobins (Hyd1-3) characterized in Metarhizium robertsii, an insect-pathogenic fungus. Aerial conidiation levels of three Δhyd1 mutants were much more reduced in 5-day-old cultures than in 7-day-old cultures, which were wettable (hydrophilic), but restored to a wild-type level in 15-day-old cultures. The Δhyd1 mutants were compromised in conidial quality, including significant decreases in hydrophobicity (58%), adhesion to insect cuticle (36%), insect pathogenicity via normal cuticle infection (37%), UVB resistance (20%), and heat tolerance (10%). In contrast, none of all examined phenotypes were affected in the null mutants of hyd2 and hyd3. Intriguingly, micromorphology and integrity of hydrophobin rodlet bundles on conidial coat were not affected in all mutant and wild-type strains, but the rodlet bundles were disordered in the absence of hyd1, suggesting a link of the disorder to the decreased hydrophobicity. Therefore, Hyd1 mediates the fungal hydrophobicity and plays an important role in conidial quality control and insect-pathogenic lifecycle. Class I Hyd2 and class II Hyd3 seem functionally redundant in M. robertsii.

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features.

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a ß-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.

A new hydrophobin candidate from Cladosporium macrocarpum with super-hydrophobic surface.

Hydrophobins are amphipathic proteins with small molecular weights produced in filamentous fungi. These proteins are highly stable due to the disulfide bonds formed between the protected cysteine residues. They have great potential for usage in many different fields such as surface modifications, tissue engineering, and drug transport systems because hydrophobins are surfactants and soluble in harsh mediums. In this study, it was aimed to determine the hydrophobin proteins responsible for the hydrophobicity of the super-hydrophobic fungi isolates in the culture medium and to carry out the molecular characterization of the hydrophobin producer species. As a result of measuring surface hydrophobicity by determining the water contact angle, five different fungi with the highest hydrophobicity were classified as Cladosporium by classical and molecular (ITS and D1-D2 regions) methods. Also, protein extraction according to the recommended method for obtaining hydrophobins from spores of these Cladosporium species indicated that the isolates have similar protein profiles. Ultimately, the isolate named A5 with the highest water contact angle was identified as Cladosporium macrocarpum, and the 7 kDa band was appointed as a hydrophobin since it was the most abundant protein in protein extraction for this species.

Functional roles of all five putative hydrophobin genes in growth, development, and secondary metabolism in Fusarium graminearum.

Fusarium graminearum is the causal agent of Fusarium head blight in cereal crops. As in other filamentous ascomycetes, F. graminearum contains genes encoding putative hydrophobins, which are small secreted amphiphilic proteins with eight conserved cysteine residues. Here, we investigated the roles of all five hydrophobin genes (designated FgHyd1, FgHyd2, FgHyd3, FgHyd4, and FgHyd5) in various mycological traits of F. graminearum. Gene expression analyses revealed that the five FgHyd genes, all of which were under the control of G protein signaling or velvet complex proteins, were differentially expressed under various developmental conditions. Three genes (FgHyd1, FgHyd2, and FgHyd3) were constitutively expressed in all aerial structures examined (hyphae, conidia, and perithecia), and two genes (FgHyd1 and FgHyd2) were also expressed in submerged hyphae. FgHyd3 was exclusively expressed in aerial hyphae on solid surfaces, including rice grains. These genes showed markedly reduced expression in F. asiaticum, which was a closely related to F. graminearum but exhibited different mycological traits from F. graminearum. Phenotypic analyses of various gene deletion strains, including the quintuple deletion (ΔFgHyd12345) strain, confirmed that in addition to their typical functions, all five FgHyd genes were involved in other traits, such as conidiation, pathogenicity, and secondary metabolism in F. graminearum. Both RNA-seq and chemical analyses confirmed that ΔFgHyd led to overproduction of specific terpenoid compounds (e.g., trichothecenes), which has not been reported previously. Nevertheless, the lack of complete phenotypic loss of any of the traits examined, even in the ΔFgHyd12345 strain, and little cumulative action of all five FgHyd genes strongly suggest that all five hydrophobins are redundant in function and are not absolutely essential for these fungal traits in F. graminearum.

Characterisation and comparative analysis of hydrophobin isolated from Pleurotus floridanus (PfH).

Hydrophobins are a class of small cysteine rich surface active proteins produced exclusively by filamentous fungi. It forms a nano layer in the cell-water interface, thereby protecting the emerging fungal hyphae from surrounding water. Even though hydrophobins have similar functions in fungi, they share less sequence similarity. In the current study, we made a comparative study of the hydrophobin produced by the mushroom Pleurotus floridanus (PfH). Mushroom P. floridanus was cultured in PD broth. The hydrophobin was purified by foam fractionation and characterized in terms of molecular weight, solubility and glycosylation. In the RP-HPLC analysis, the hydrophobin eluted at a retention time of 45.56 min. The molecular weight of the PfH was found to be 13.52 kDa by MALDI-TOF MS and the LC-MS/MS showed no similar sequence in MASCOT database. The hydrophobin gene of P. floridanus was amplified using custom-designed primers and the BLAST analysis showed 80% sequence similarity with the Vmh2-1 gene of Pleurotus ostreatus. The sequence was translated into protein using ExPASy, secondary and tertiary structure predictions were carried out using Jpred4 and Phyre2. The tertiary structure showed 91.5% similarity with the HYD1 hydrophobin of Schizophyllum commune. A comparative study of PfH with Vmh2-1 and HYD1 was performed using bioinformatics tools. Hydrophobic cluster analysis revealed that three of these proteins have uniformity in terms of amphiphilic and non-amphiphilic α-helices, whereas PfH has a unique proline clustering. Physicochemical analysis by ProtParam revealed that PfH shares similar properties with HYD1 and Vmh2-1, which can be correlated with its function.

A study of hydrophobins-modified menaquinone-7 on osteoblastic cells differentiation.

Menaquinone-7 is involved in bone metabolism and can be used to prevent and treat osteoporosis. However, as a fat-soluble vitamin, menaquinone-7 has poor water solubility. As a surfactant, hydrophobins can change the affinity/hydrophobicity of the covered interface. In this study, menaquinone-7 was modified by hydrophobins, and the different addition ratios were explored. Moreover, Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and water contact angle (WCA) measurements indicated that hydrophobins effectively bind to menaquinone-7 and greatly increase the hydrophilicity of the surface of menaquinone-7. Studies on the metabolism of MC3T3-E1 cells showed that compared with native menaquinone-7, HGFI-modified menaquinone-7 can significantly promote osteoblast differentiation but inhibit osteoclast differentiation. Besides, the Mito-Tracker Green experiments show that HGFI-modified menaquinone-7 can significantly promote the activity of mitochondria in cells. These findings indicate that hydrophobins can be used as an effective biomaterial to modify menaquinone-7, promote the formation of osteoblasts, and better to bone balance.

Biomacromolecules and Bio-Sourced Products for the Design of Flame Retarded Fabrics: Current State of the Art and Future Perspectives.

The search for possible alternatives to traditional flame retardants (FRs) is pushing the academic and industrial communities towards the design of new products that exhibit low environmental impact and toxicity, notwithstanding high performances, when put in contact with a flame or exposed to an irradiative heat flux. In this context, in the last five to ten years, the suitability and effectiveness of some biomacromolecules and bio-sourced products with a specific chemical structure and composition as effective flame retardants for natural or synthetic textiles has been thoroughly explored at the lab-scale level. In particular, different proteins (such as whey proteins, caseins, and hydrophobins), nucleic acids and extracts from natural sources, even wastes and crops, have been selected and exploited for designing flame retardant finishing treatments for several fibers and fabrics. It was found that these biomacromolecules and bio-sourced products, which usually bear key elements (i.e., nitrogen, phosphorus, and sulphur) can be easily applied to textiles using standard impregnation/exhaustion methods or even the layer-by-layer technique; moreover, these "green" products are mostly responsible for the formation of a stable protective char (i.e., a carbonaceous residue), as a result of the exposure of the textile substrate to a heat flux or a flame. This review is aimed at summarizing the development and the recent progress concerning the utilization of biomacromolecules/bio-sourced products as effective flame retardants for different textile materials. Furthermore, the existing drawbacks and limitations of the proposed finishing approaches as well as some possible further advances will be considered.

Dynamics of the Phanerochaete carnosa transcriptome during growth on aspen and spruce.

BACKGROUND: The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. RESULTS: Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. CONCLUSIONS: The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.

The Hydrophobin HYTLO1 Secreted by the Biocontrol Fungus Trichoderma longibrachiatum Triggers a NAADP-Mediated Calcium Signalling Pathway in Lotus japonicus.

Trichoderma filamentous fungi are increasingly used as biocontrol agents and plant biostimulants. Growing evidence indicates that part of the beneficial effects is mediated by the activity of fungal metabolites on the plant host. We have investigated the mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by Trichoderma longibrachiatum, which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing Lotus japonicus suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca2+ increase that dissipated within 30 min, followed by the activation of the defence-related genes MPK3, WRK33, and CP450. The Ca2+-dependence of these gene expression was demonstrated by using the extracellular Ca2+ chelator EGTA and Ned-19, a potent inhibitor of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor in animal cells, which effectively blocked the HYTLO1-induced Ca2+ elevation. Immunocytochemical analyses showed the localization of the fungal hydrophobin at the plant cell surface, where it forms a protein film covering the plant cell wall. Our data demonstrate the Ca2+-mediated perception by plant cells of a key metabolite secreted by a biocontrol fungus, and provide the first evidence of the involvement of NAADP-gated Ca2+ release in a signalling pathway triggered by a biotic stimulus.

HFB7 - A novel orphan hydrophobin of the Harzianum and Virens clades of Trichoderma, is involved in response to biotic and abiotic stresses.

Hydrophobins are small secreted cysteine-rich proteins exclusively found in fungi. They are able to self-assemble in single molecular layers at hydrophobic-hydrophilic interfaces and can therefore be directly involved in establishment of fungi in their habitat. The genomes of filamentous mycotrophic fungi Trichoderma encode a rich diversity of hydrophobins, which are divided in several groups based on their structure and evolution. Here we describe a new member of class II hydrophobins, HFB7, that has a taxonomically restricted occurrence in Harzianum and Virens clades of Trichoderma. Evolutionary analysis reveals that HFB7 proteins form a separate clade distinct from other Trichoderma class II hydrophobins and that genes encoding them evolve under positive selection pressure. Homology modelling of HFB7 structure in comparison to T. reesei HFB2 reveals that the two large hydrophobic patches on the surface of the protein are remarkably conserved between the two hydrophobins despite significant difference in their primary structures. Expression of hfb7 gene in T. virens increases at interactions with other fungi and a plant and in response to a diversity of abiotic stress conditions, and is also upregulated during formation of aerial mycelium in a standing liquid culture. This upregulation significantly exceeds that of expression of hfb7 under a strong constitutive promoter, and T. virens strains overexpressing hfb7 thus display only changes in traits characterized by low hfb7 expression, i.e. faster growth in submerged liquid culture. The hfb7 gene is not expressed in conidia. Our data allow to conclude that this protein is involved in defence of Trichoderma against a diversity of stress factors related to the oxidative stress. Moreover, HFB7 likely helps in the establishment of the fungus in wetlands or other conditions related to high humidity.

Comparative genomics and expression levels of hydrophobins from eight mycorrhizal genomes.

Hydrophobins are small secreted proteins that are present as several gene copies in most fungal genomes. Their properties are now well understood: they are amphiphilic and assemble at hydrophilic/hydrophobic interfaces. However, their physiological functions remain largely unexplored, especially within mycorrhizal fungi. In this study, we identified hydrophobin genes and analysed their distribution in eight mycorrhizal genomes. We then measured their expression levels in three different biological conditions (mycorrhizal tissue vs. free-living mycelium, organic vs. mineral growth medium and aerial vs. submerged growth). Results confirmed that the size of the hydrophobin repertoire increased in the terminal orders of the fungal evolutionary tree. Reconciliation analysis predicted that in 41% of the cases, hydrophobins evolved from duplication events. Whatever the treatment and the fungal species, the pattern of expression of hydrophobins followed a reciprocal function, with one gene much more expressed than others from the same repertoire. These most-expressed hydrophobin genes were also among the most expressed of the whole genome, which suggests that they play a role as structural proteins. The fine-tuning of the expression of hydrophobin genes in each condition appeared complex because it differed considerably between species, in a way that could not be explained by simple ecological traits. Hydrophobin gene regulation in mycorrhizal tissue as compared with free-living mycelium, however, was significantly associated with a calculated high exposure of hydrophilic residues.

Familiar Stranger: Ecological Genomics of the Model Saprotroph and Industrial Enzyme Producer Trichoderma reesei Breaks the Stereotypes.

The filamentous fungus Trichoderma reesei (Hypocreales, Ascomycota) has properties of an efficient cell factory for protein production that is exploited by the enzyme industry, particularly with respect to cellulase and hemicellulase formation. Under conditions of industrial fermentations it yields more than 100g secreted protein L(-1). Consequently, T. reesei has been intensively studied in the 20th century. Most of these investigations focused on the biochemical characteristics of its cellulases and hemicellulases, on the improvement of their properties by protein engineering, and on enhanced enzyme production by recombinant strategies. However, as the fungus is rare in nature, its ecology remained unknown. The breakthrough in the understanding of the fundamental biology of T. reesei only happened during 2000s-2010s. In this review, we compile the current knowledge on T. reesei ecology, physiology, and genomics to present a holistic view on the natural behavior of the organism. This is not only critical for science-driven further improvement of the biotechnological applications of this fungus, but also renders T. reesei as an attractive model of filamentous fungi with superior saprotrophic abilities.

Peptaibol, secondary-metabolite, and hydrophobin pattern of commercial biocontrol agents formulated with species of the Trichoderma harzianum complex.

The production of bioactive polypeptides (peptaibiotics) in vivo is a sophisticated adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species for colonizing and defending their natural habitats. This feature is of major practical importance, as the detection of peptaibiotics in plant-protective Trichoderma species, which are successfully used against economically relevant bacterial and fungal plant pathogens, certainly contributes to a better understanding of these complex antagonistic interactions. We analyzed five commercial biocontrol agents (BCAs), namely Canna(®) , Trichosan(®) , Vitalin(®) , Promot(®) WP, and TrichoMax(®) , formulated with recently described species of the Trichoderma harzianum complex, viz. T. afroharzianum, T. simmonsii, and T. guizhouense. By using the well-established, HPLC/MS-based peptaibiomics approach, it could unequivocally be demonstrated that all of these formulations contained new and recurrent peptaibols, i.e., peptaibiotics carrying an acetylated N-terminus, the C-terminus of which is reduced to a 1,2-amino alcohol. Their chain lengths, including the amino alcohol, were 11, 14, and 18 residues, respectively. Peptaibols were also to be the dominating secondary metabolites in plate cultures of the four strains obtained from four of the Trichoderma- based BCAs, contributing 95% of the UHPLC-UV/VIS peak areas and 99% of the total ion count MS peak area from solid media. Furthermore, species-specific hydrophobins, as well as non-peptaibiotic secondary metabolites, were detected, the latter being known for their antifungal, siderophore, or plant-growth-promoting activities. Notably, none of the isolates produced low-molecular weight mycotoxins.

Solution structure and interface-driven self-assembly of NC2, a new member of the Class II hydrophobin proteins.

Hydrophobins are fungal proteins that self-assemble spontaneously to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin-based applications.

Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension.

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

Two forms and two faces, multiple states and multiple uses: Properties and applications of the self-assembling fungal hydrophobins.

The fungal hydrophobins are small proteins that are able to spontaneously self-assemble into amphipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic materials. The self-assembling properties and amphipathic nature of hydrophobins make them attractive candidates for biotechnological applications. Recently, there have been significant advances in the understanding of the structure and assembly of these remarkable proteins. This opens up the way for engineering these proteins to encompass novel functions and for the use of hydrophobins in modification of nanomaterials. This review highlights the important structural aspects of the hydrophobins and the mechanisms by which they assemble and describes recent exciting developments in the use of hydrophobins for cell attachment, drug delivery, and protein purification.

In Silico Evaluation, Phylogenetic Analysis, and Structural Modeling of the Class II Hydrophobin Family from Different Fungal Phytopathogens.

The class II hydrophobin group (HFBII) is an extracellular group of proteins that contain the HFBII domain and eight conserved cysteine residues. These proteins are exclusively secreted by fungi and have multiple functions with a probable role as effectors. In the present study, a total of 45 amino acid sequences of hydrophobin class II proteins from different phytopathogenic fungi were retrieved from the NCBI database. We used the integration of well-designed bioinformatic tools to characterize and predict their physicochemical parameters, novel motifs, 3D structures, multiple sequence alignment (MSA), evolution, and functions as effector proteins through molecular docking. The results revealed new features for these protein members. The ProtParam tool detected the hydrophobicity properties of all proteins except for one hydrophilic protein (KAI3335996.1). Out of 45 proteins, six of them were detected as GPI-anchored proteins by the PredGPI server. Different 3D structure templates with high pTM scores were designed by Multifold v1, AlphaFold2, and trRosetta. Most of the studied proteins were anticipated as apoplastic effectors and matched with the ghyd5 gene of Fusarium graminearum as virulence factors. A protein-protein interaction (PPI) analysis unraveled the molecular function of this group as GTP-binding proteins, while a molecular docking analysis detected a chitin-binding effector role. From the MSA analysis, it was observed that the HFBII sequences shared conserved 2 Pro (P) and 2 Gly (G) amino acids besides the known eight conserved cysteine residues. The evolutionary analysis and phylogenetic tree provided evidence of episodic diversifying selection at the branch level using the aBSREL tool. A detailed in silico analysis of this family and the present findings will provide a better understanding of the HFBII characters and evolutionary relationships, which could be very useful in future studies.

Identification and Characterization of a Predominant Hydrophobin in the Edible Mushroom Grifola frondosa.

Hydrophobins (HFBs) are a group of small, secreted amphipathic proteins of fungi with multiple physiological functions and potential commercial applications. In this study, HFB genes of the edible mushroom, Grifola frondosa, were systematically identified and characterized, and their transcriptional profiles during fungal development were determined. In total, 19 typical class I HFB genes were discovered and bioinformatically analyzed. Gene expression profile examination showed that Gf.hyd9954 was particularly highly upregulated during primordia formation, suggesting its major role as the predominant HFB in the lifecycle of G. frondosa. The wettability alteration profile and the surface modification ability of recombinant rGf.hyd9954 were greater than for the Grifola HFB HGFII-his. rGf.hyd9954 was also demonstrated to form the typical class I HFB characteristic-rodlet bundles. In addition, rGf.hyd9954 was shown to possess nanoparticle characteristics and emulsification activities. This research sheds light on the regulation of fungal development and its association with the expression of HFB genes.

Fungal Cell Factories for Efficient and Sustainable Production of Proteins and Peptides.

Filamentous fungi are a large and diverse taxonomically group of microorganisms found in all habitats worldwide. They grow as a network of cells called hyphae. Since filamentous fungi live in very diverse habitats, they produce different enzymes to degrade material for their living, for example hydrolytic enzymes to degrade various kinds of biomasses. Moreover, they produce defense proteins (antimicrobial peptides) and proteins for attaching surfaces (hydrophobins). Many of them are easy to cultivate in different known setups (submerged fermentation and solid-state fermentation) and their secretion of proteins and enzymes are often much larger than what is seen from yeast and bacteria. Therefore, filamentous fungi are in many industries the preferred production hosts of different proteins and enzymes. Edible fungi have traditionally been used as food, such as mushrooms or in fermented foods. New trends are to use edible fungi to produce myco-protein enriched foods. This review gives an overview of the different kinds of proteins, enzymes, and peptides produced by the most well-known fungi used as cell factories for different purposes and applications. Moreover, we describe some of the challenges that are important to consider when filamentous fungi are optimized as efficient cell factories.

Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins.

Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins) with multiple intramolecular disulphide bonds which are typically difficult to express in a soluble and folded state in the reducing environments found inside a cell. In some cases, the inclusion of a recombinant disulphide isomerase DsbC further enhances the expression levels of correctly folded hydrophobins. Using this protocol, we can achieve milligram levels of protein expression per ml of reaction. While our target proteins are the fungal hydrophobins, it is likely that this protocol with some minor variations can be used to express other proteins with multiple intramolecular disulphide bonds in a natively folded state. Graphic abstract: Figure 1.Workflow for cell-free protein expression and single-step purification using affinity chromatography. A. E. coli S30 lysate prepared as described in Apponyi et al. (2008) can be stored for up to several years at -80°C without any loss of activity in our experience. B. The S30 lysate, plasmid DNA that encodes for the protein of interest along with an affinity tag and components required for transcription and translation are added to the reaction mix. Following a single-step protein purification, the protein of interest can be isolated for further use.