Research review paperFrom fungal secretomes to enzymes cocktails: The path forward to bioeconomy
Introduction
Bioeconomy can be defined as an economy that relies on the production and conversion of renewable biological resources to produce food, energy, and goods. Its development is seen as a way to mitigate the carbon footprint of human activities by reducing at least part of the fossil resources-based economy (Eur. Comm., 2021), and it could help to meet at least half of the UN Sustainable Development Goals (El-Chichakli et al., 2016). In this context, the use of biotechnological solutions (micro-organisms and enzymes) is believed to play an increasing role, as they have been tailored by evolution to efficiently degrade and modify biopolymers and synthesize biological molecules.
The global enzyme market was already valued at ~10 billion US $ in 2019 and is believed to keep growing at a rate between 5 and 7% per year (Grand View Res., 2020). Among these commercial enzymes, carbohydrate-active enzymes (CAZymes) have the highest market share, before proteases and others, and their global market is forecasted to generate 5.4 billion US $ revenue in 2024 (Mark. Res. Future, 2021). In industry, most of the enzyme cocktails used in so-called “white” biotechnologies are of fungal origin (McKelvey and Murphy, 2017), with a wide range of applications (textiles, food & beverages, animal feed, pulp & paper, cosmetics, nutraceuticals & pharmaceuticals, household care, bioenergy, etc.).
In nature, filamentous fungi are heterotrophs and exodigesters, feeding on organic matter. Fungal life and growth hence rely on the extracellular secretion of various proteins involved in nutrients acquisition, sensing and signaling, cell walls building and remodeling, and competition with other organisms. The entire set of proteins secreted under given circumstances by an organism is called a “secretome”, the broad sense of this term includes both freely released proteins and proteins attached to the outer cell wall (Tjalsma et al., 2000; Girard et al., 2013). Using multiple regulation and secretion mechanisms, filamentous fungi can modify their secretome and thus their metabolism in response to changes in their environmental conditions, such as a variation of the available carbon and nitrogen sources (McCotter et al., 2016; Brown et al., 2014). Given that fungi are present in almost all existing ecosystems and have evolved to acquire a broad array of nutritional lifestyles, the nature of these nutrient sources can be remarkably diverse. Fungi can indeed live as saprophytes, feeding on various types of dead organic material, and/or engage in symbiotic or parasitic relationships with plants, insects, animals. These different lifestyles and nutritional strategies are associated to a wide variety of enzymes specialized in the degradation of complex biopolymers encountered in the environment (Krijger et al., 2014; Lowe and Howlett, 2012). Some fungal species, although they thrive in similar environments, use different degradation strategies. For instance, fungal wood decayers have been classified empirically according to the different components of the lignocellulosic substrates they target in nature (Blanchette, 2000; Cragg et al., 2015; Spatafora et al., 2017): white-rot basidiomycetes degrade the lignin moiety extensively using laccases and peroxidases before attacking cellulose, while brown-rot basidiomycetes induce limited lignin modifications that allow them to gain access to cellulose and hemicellulose using both enzymatic and non-enzymatic reactions, and soft-rot ascomycetes primarily attack plant fibres using cellulases and hemicellulases while leaving a high lignin content. Several genomic studies allowed to link these different strategies to distinct enzyme sets and associated genes, and some of them provided additional details by showing how evolution shaped fungal enzymatic arsenals in regards to their adaptation to diverse habitats and lifestyles (Floudas et al., 2012; Hage and Rosso, 2021).
Among the various natural molecules that can be degraded or modified by fungal enzymes, plant-based materials are of particular interest since they are an important reservoir of organic carbon. Lignocellulose in particular is envisioned as a renewable feedstock for the production of biofuels and bioproducts, in order to replace most of the molecules obtained from oil refining that are currently used for the manufacture of consumer products in the fields of health, food, feed, materials, chemicals etc. (Cherubini, 2010; De Bhowmick et al., 2018). One of the main obstacles to the use of this raw material in biorefineries is its recalcitrance, due to the organisation of its constitutive polymers, where crystalline cellulose fibers are surrounded by hemicelluloses and pectin chains and cemented by a lignin matrix (De Bhowmick et al., 2018). An effective way to get access to and transform these polymers is thus to use the natural toolbox provided by saprophytic fungi that have evolved to use plant biomass as a carbon and energy source, and secrete an arsenal of glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, oxidoreductases and hydrogen peroxide-producing enzymes in their secretomes (Bouws et al., 2008). By providing a view of the enzymes secreted by fungal strains grown on various substrates, fungal secretomics is thus a powerful tool to better understand biomass degradation mechanisms and design more efficient biomass-degrading enzyme cocktails.
In this review, we highlight recent advances in the study of fungal secretomes using improved computational and experimental secretomics methods, the progress in the understanding of industrially important fungi, and the discovery of new enzymatic mechanisms and interplays involved in the degradation of renewable resources rich in polysaccharides. We review current biotechnological applications focusing on the benefits and challenges of fungal secretomes for industrial applications with some examples of commercial cocktails of fungal origin containing carbohydrate-active enzymes (CAZymes) and we discuss future trends.
Section snippets
Improved computational and experimental secretomics methods
The identification of fungal secreted proteins is mainly based on a comparison with existing genomic data, and thus depend on the availability of sequenced genomes for the corresponding organisms. In the last decade, the access to faster and cheaper sequencing methods has facilitated the emergence of large-scale genomics initiatives aiming at the exploration of the fungal kingdom in the frame of health, energy, and environmental issues, thus increasing the number of available fungal genomes.
Benefits and challenges of fungal secretomes for industrial applications
In addition to being a major source for enzyme discovery, fungal secretomes can be used as commercial products. There are several possible drivers for such developments. The first one is the cost of downstream processing (DSP), which is already low when working with organisms that secrete enzymes, as there is no need to break-up cells. DSP can be further simplified if advanced purification is not needed for the targeted application. This is also enabled by the high secretory capacity of
Discovery of new enzyme sources
Although many research efforts have already been spent on the development of powerful enzymatic cocktails for a large array of applications, the quest for improved industrial performances is still relevant. In addition, new applications for fungal secretomes also emerge, such as delignification and detoxification of biofuel feedstocks by ligninolytic enzyme cocktails (Plácido and Capareda, 2015), control of algal blooms (Du et al., 2015), metal toxicity assessment (Lebrun et al., 2011) or
Conclusion
In recent years, the advances in -omic technologies and the growing interest in fungal enzymes have led to multiple studies to better understand the complex arsenals of extracellular enzymes secreted by fungi under a variety of environmental conditions, from the industrial workhorses to new fungal strains isolated from biodiversity. The ability of these organisms to produce fine-tuned biomass-degrading enzyme cocktails have already led them to be used as providers of biocatalysts for industrial
Funding
Not applicable.
Declaration of competing interest
The authors declare no conflict of interest.
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Present address: Biomass Technology Laboratory, Université de Sherbrooke, Sherbrooke, Canada.