Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation’s sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.

Small secreted proteins (SSPs) comprise 40–60% of the total fungal secretome and are present in fungi of all phylogenetic groups, representing the entire spectrum of lifestyles. They are characteristically shorter than 300 amino acids in length and have a signal peptide. The majority of SSPs are coded by orphan genes, which lack known domains or similarities to known protein sequences. Effectors are a group of SSPs that have been investigated extensively in fungi that interact with living hosts, either pathogens or mutualistic systems. They are involved in suppressing the host defense response and altering its physiology. Here, we aim to delineate some of the potential roles of SSPs in saprotrophic fungi, that have been bioinformatically predicted as effectors, and termed in this mini-review as “effector-like” proteins. The effector-like Ssp1 from the white-rot fungus Pleurotus ostreatus is presented as a case study, and its potential role in regulating the ligninolytic system, secondary metabolism, development, and fruiting body initiation are discussed. We propose that deciphering the nature of effector-like SSPs will contribute to our understanding of development and communication in saprophytic fungi, as well as help, to elucidate the origin, regulation, and mechanisms of fungal-host, fungal-fungal, and fungal-bacterial interactions.

Current large-scale pretreatment processes for lignocellulosic biomass are generally accompanied by the formation of toxic degradation products, such as 5-hydroxymethylfurfural (HMF), which inhibit cellulolytic enzymes and fermentation by ethanol-producing yeast. Overcoming these toxic effects is a key technical barrier in the biochemical conversion of plant biomass to biofuels. Pleurotus ostreatus, a white-rot fungus, can efficiently degrade lignocellulose. In this study, we analyzed the ability of P. ostreatus to tolerate and metabolize HMF and investigated relevant molecular pathways associated with these processes. P. ostreatus was capable to metabolize and detoxify HMF 30 mM within 48 h, converting it into 2,5-bis-hydroxymethylfuran (HMF alcohol) and 2,5-furandicarboxylic acid (FDCA), which subsequently allowed the normal yeast growth in amended media. We show that two enzymes groups, which belong to the ligninolytic system, aryl-alcohol oxidases and a dehydrogenase, are involved in this process. HMF induced the transcription and production of these enzymes and was accompanied by an increase in activity levels. We also demonstrate that following the induction of these enzymes, HMF could be metabolized in vitro. Aryl-alcohol oxidase and dehydrogenase gene family members are part of the transcriptional and subsequent translational response to HMF exposure in P. ostreatus and are involved in HMF transformation. Based on our data, we propose that these enzymatic capacities of P. ostreatus either be integrated in biomass pretreatment or the genes encoding these enzymes may function to detoxify HMF via heterologous expression in fermentation organisms, such as Saccharomyces cerevisiae.

During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall. Biological pretreatment, in which lignin is decomposed or modified by white-rot fungi, has also been considered. One disadvantage in biological pretreatment, however, is the consumption of the cellulose by the fungus. Thus, fungal species that attack lignin with only minimal cellulose loss are advantageous. The secretomes of white-rot fungi contain carbohydrate-active enzymes (CAZymes) including lignin-modifying enzymes. Thus, modification of secretome composition can alter the ratio of lignin/cellulose degradation. Pleurotus ostreatus PC9 was genetically modified to either overexpress or eliminate (by gene replacement) the transcriptional regulator CRE1, known to act as a repressor in the process of carbon catabolite repression. The cre1-overexpressing transformant demonstrated lower secreted cellulolytic activity and slightly increased selectivity (based on the chemical composition of pretreated wheat straw), whereas the knockout transformant demonstrated increased cellulolytic activity and significantly reduced residual cellulose, thereby displaying lower selectivity. Pretreatment of wheat straw using the wild-type PC9 resulted in 2.8-fold higher yields of soluble sugar compared to untreated wheat straw. The overexpression transformant showed similar yields (2.6-fold), but the knockout transformant exhibited lower yields (1.2-fold) of soluble sugar. Based on proteomic secretome analysis, production of numerous CAZymes was affected by modification of the expression level of cre1. The gene cre1 functions as a regulator for expression of fungal CAZymes active against plant cell wall lignocelluloses, hence altering the substrate preference of the fungi tested. While the cre1 knockout resulted in a less efficient biological pretreatment, i.e., less saccharification of the treated biomass, the converse manipulation of cre1 (overexpression) failed to improve efficiency. Despite the inverse nature of the two genetic alterations, the expected "mirror image" (i.e., opposite regulatory response) was not observed, indicating that the secretion level of CAZymes, was not exclusively dependent on CRE1 activity.