How to get the most out of fungal biotechnology?

Abstract

During the past decades, the importance of fungal biotechnology in advancing a bioeconomy and a circular economy has been emphasized in both scientific literature, project proposals, awarded grants and social media. Filamentous fungi have been proven to provide sustainable solutions for various industrial applications, ranging from bioremediation and medicine to the production of food, feed, materials, chemicals and energy. This is where we are today, but where could tomorrow’s fungal biotechnology take us? How can the seemingly infinite potential of fungal biotechnology for a circular economy become unlocked? In this editorial, we will cover some of the critical aspects that we believe are essential for the success and impact of fungal biotechnology to a future bioeconomy.

Introduction

For more than a hundred years, filamentous fungi have been important production hosts for proteins, enzymes, certain drugs and platform chemicals such as citric acid [1, 2]. Recently, the value of fungi as so-called single cell biomass and/or for production of heterologous proteins for food applications has been recognized. Moreover, several companies work on fungal production of high-value compounds such as pigments, fragrances and bioactive molecules. The use of fungal biomass for material applications is another hot topic. The list could be much longer—mycostories.com is a great platform keeping track of recent developments within the field.

The number of start-ups within the fungal biotechnology space has thus steadily increased during the last years. Large investments in technology development, commercialization and, in a few cases, upscaling production have been celebrated in the media. Nonetheless, while some start-ups move into commercial production, others announce their stories have come to an end. Why haven’t more fungal biotech solutions yet been realized?

To answer this question, it may be helpful to take a broader view of the processes involved in biotechnological innovation. A recent policy paper published by the European Bio-based Industry Consortium (EBIC) emphasizes that ‘Europe must accelerate the path from lab to factory by enabling biomanufacturing at scale along the value chain’ in order to bring innovations to the market [3]. According to EBIC, not one but two ‘valleys of death’ hinder commercialization of bio-based (and thus fungal-based) innovations: The first valley of death refers to translating laboratory innovation into a proof-of-concept prototype, with key challenges relating to technology development, scaling up and financing. The second valley of death refers to transforming initial prototypes into commercially viable products, with key challenges relating to market penetration, regulatory approvals, a sustainable supply of biomass and financing.

In the context of fungal biotechnology (as far as we can both see and judge), we can say that major hurdles within the first valley of death include limited genetic and molecular knowledge of the fungal production host and lengthy strain development as well as challenges in scale-up because of unpredictable fungal macromorphologies. Although today’s public research funding is often channeled to applied fungal research, many fundamental aspects of fungal biology and physiology is still to be elucidated. Undisputably, such basic research is needed to unlock the full potential of fungi as enablers of the bioeconomy.

With respect to the second valley of death, regulatory processes in the field of fungal biotechnology are indeed very complex and time-consuming. Thus, streamlined regulatory frameworks allowing bold innovations to be realized much faster than today are needed.

Genetic understanding and engineering of fungi

The advancements in genome engineering using modern synthetic biology (such as CRISPR with Cas9 or Cas12a) tools and increasing amounts of available (sequence) data have led to more fungal species being explored [4]. More than 23,000 fungal genomes are available in online databases [5]. This has shortened the (still long) time needed for genetic engineering of fungi. Lately, synthetic toolkits for filamentous fungi [6, 7] have been reported and shared with the community. Many synthetic biology tools developed in more easily manipulatable model organisms (often yeast) are today rapidly translated to fungal systems. Cas ribonucleoparticles (RNPs) circumvent the need for expression of Cas and sgRNAs [8], bypassing the need to develop genetic tools for each new species. Still, engineered fungi are typically achieved through protoplastation, but developing protocols for generating protoplasts and achieving efficient transformation of novel species or even strains can be very time consuming. Less successful efforts are typically not documented in scientific literature even though such information could be very valuable. Protoplast formation is followed manually under the microscope and learning what a “good protoplast” looks like for a set strain demands many rounds of trials followed by long incubation times waiting for colonies to appear. Here, cryopreservation of protoplasts can be a great time saver. Other commonly used approaches include Agrobacterium-mediated transformation and electroporation [5, 9], thus there are many options to test when venturing into a new species. The cellular mechanisms involved in transformation of fungi are yet to be elucidated.

The multicellular morphology of filamentous fungi further challenge gene manipulation and they possess only a limited number of plasmids and selectable markers. Nonetheless, the AMA1 plasmid has been shown to be stable in many different fungal species, providing a tool for assessing constructs across strains. CRISPR activation (CRISPRa) has been demonstrated for activation of biosynthetic clusters [10, 11]. Similarly, CRISPR interference (CRISPRi) has been demonstrated for gene silencing in several fungal species [4]. Before the CRISPR era, RNA interference (RNAi) was used as a tool for silencing genes, often conditionally upon induction. Thus, as of today, the synthetic biology toolbox of filamentous fungi is already quite vast—the challenge is the efficiency and reliability (which still often leaves room for improvement) and the transferability of these tools to fungi that have received less study but are potentially better suited for industrial applications.

Translating fungal knowledge to underexplored species

The kingdom of fungi is estimated to constitute of millions of species [12]. Still, the number of fungal species studied and used for production purposes remains modest and the number of groups working with filamentous fungi is, compared to many other fields, still rather small [2]. Often, fungal start-ups prefer not to relieve their species employed. Traditionally, Trichoderma or Aspergillus spp. for protein production, Fusarium venenatum for food (single cell protein) production and Penicillium chrysogenum or Acremonium chrysogenum for antibiotics production have dominated. Citric acid production by Aspergillus niger is after ethanol the largest volumetric biotech product [13]. Solid state fermentation for obtaining mycelium for applications such as leatherlike materials, composites or packing materials is typically conducted with basidiomycetes such as Trametes versicolor, Pleurotus ostreatus, Ganoderma lucidum and Fomes fometarius [14]. Leatherlike materials have also been obtained through submerged bioreactor cultivation of Trichoderma reesei [15]. There are already a few commercial mycelium-material producers but such materials struggle to compete with other (synthetic) materials both in terms of properties and price. Nevertheless, many of the recognized challenges facing fungal materials can be addressed through genetic engineering of the fungal production host, optimizing the production processes, or combining the material with other biodegradable substances.

From proof-of-concept to production at scale

The relatively low number of species employed today may be due to vast domestication efforts having shaped the commercially employed fungi into cell factories amendable for genetic engineering and fit for large scale production in bioreactors. The morphology of fungi, that typically form large clumps in submerged cultures, challenges their exploitation. A more uniform growth pattern has in many cases been obtained by selection and various classical strain improvement technologies, including mutagenesis and adaptive laboratory evolution and combinations thereof. Such efforts, however, rely on selection of mutants with favorable features. While efforts for establishing fungal biofoundry platforms have been initiated [16], screening filamentous fungi in high throughput still remains challenging after two decades of research efforts by the community and cannot be considered yet as a routine method. Microfluidics, automation and artificial intelligence are expected to advance fungal biotechnology, but examples at higher Technology readiness level (TRL) remain scarce [16, 17].

Further, translating (fungal) research results from small to even medium, let alone pilot or industrial scale is notably difficult. Many start-ups are built around a successful proof-of-concept demonstration, and access to pilot or demo facilities for technology validation remains a bottleneck (first valley of death). Notably, several fungal start-ups have built their own pilots that have enabled them to refine their technology, make prototypes of products or even to produce small batches for commercial use. Recent years have also marked several news reports on start-ups securing funding for full-sized commercial factories for production of fungal biomass e.g. for feed and food applications. Thus, in the coming years we hope to see many new mycoprotein-based products hitting the market.

An innovation-friendly regulatory landscape will accelerate fungal biotechnology

The regulatory landscape for novel fungal products is complex and approval processes for new products can be tedious, requiring rigorous safety assessments. The so-called Novel Food authorization by the European Food Safety Authority (EFSA), is required for food that had not been consumed to a significant degree by humans in the EU before 15 May 1997 [18]. In the US, the FDA regulates compounds to be used i.e. for food, feed and drug applications [19]. Navigating these regulatory hurdles can be time-consuming and costly, posing challenges for start-ups and established companies alike as they seek to commercialize novel fungal applications. There is a clear demand for more transparent and rapid approval processes in the EU, with stakeholders such as EuropaBio calling for an improved regulatory approach for use of microorganisms for food and feed, environmental and agricultural applications as well as in consumer applications such as detergents and cosmetics [20]. As the regulations for products to be used as food are more stringent compared to those to be used as feed or materials, many companies choose to start commercialization of less regulated products such as fish feed or materials. Naturally, investors are hesitant to invest in technologies and products where the path to commercialization and timelines for approval are unknown. A recent breakthrough was when EFSA issued a positive scientific opinion for Fermotein^®, a fungal biomass ingredient - the first fermented fungal biomass novel food in Europe [21]. Moreover, Swedish rainbow trout raised on circular feed made of insects, mussels and mycoprotein [22] sold out in no time. Furthermore, it can be expected that the new EU framework, published on Dec 4, 2025, that allows certain plants used for foods to be altered and sold using the so-called ‘New genomic techniques’ [23], will further support market entry of genetically modified fungal food products. These, and globally many more examples, initiatives and frameworks can help unlocking the full potential of fungi as enablers of a future sustainable bioeconomy, not only in the food sector but also in the materials sector and beyond.

Author contributions

YN and VM wrote this manuscript together. All authors read and approved the final manuscript. This article is an opinion paper, does not constitute legal advice, and is not intended to recommend any particular course of action.

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Competing interests

Yvonne Nygård and Vera Meyer are Editors-in-Chief of Fungal Biology and Biotechnology. Yvonne Nygård is the CSO of Cirkulär AB, one of the companies mentioned in this editorial.