Highlights

The fruiting bodies of many Basidiomycota have been established as food for a long time. Among these are the world’s most popular fungi: the button mushroom (Agaricus bisporus), shiitake (Lentinula edodes), oyster mushrooms (Pleurotus sp.), or the chanterelles (Cantharellus cibarius), as well as non-cultivated genera like Boletus. Others have no significant culinary value, but are known as medicinal mushrooms (e.g., Ganoderma sp.) and have also been used historically. Thus, these fungi and their ingredients are established as toxicologically harmless. In the US, this is marked by the GRAS-status („generally recognized as safe”). The utilization of choice fungal components like enzymes in food is thus significantly easier when compared to the use of compounds from organisms that have no established history as food.

Many Basidiomycota are saprotrophs, which means that they take their nutrients from dead organic substance like tree stems and fallen leaves. For this, they produce a large variety of different, mainly hydrolytic and redox-active enzymes. These are secreted to digest the macronutrients in the immediate vicinity, which are then available for the fungus.

During submerse cultivation of the mycelium, the enzymes are secreted into the medium and can be purified from it. Agro-industrial side-streams can be used to induce the production of specific enzymes by adding the respective macronutrient (see below). The enzymes can then be identified and produced heterologously, e.g., in E. coli or K. phaffii, and used for food applications.

The heterologous production of enzymes was established for the formation of different aroma compounds^1,2 and vegan gels^3,4. In other studies, basidiomycotal enzymes were used for the degradation of the stomach-irritating chlorogenic acid in apple juice and celiac disease-inducing peptides in flour fractions^5,6.

Agro-industrial side-streams like wheat bran, sugar beet fibre, pomaces and spent brewers grains as well as potato peelings are mainly thermally recycled in biogas plants, despite the fact that they still contain valuable nutrients like glucose. These are needed for the cultivation of microorganisms and make up a sizable amount of the respective cultivation costs. Basidiomycota can produce and secrete the enzymes needed to degrade the side-streams (see above) and can thus properly recycle them. This closes bioeconomic cycles, while reducing cultivation costs and increasing the yield of the respective degrading enzyme(s).

A walk through the woods reveals the colourful beauty of fungi to the attentive observer. The underlying natural colourants have been used for the artisan dyeing of mainly textiles for centuries. An industrial application is prevented by the restricted availability of the colourful fruiting bodies. A biotechnological production of the mycelium would be able to remedy this problem, but not all biosynthetic pathways that are responsible for the formation of the colourant in the fruiting body are active in the mycelia. Biological, physical, and chemical inducers like light, temperature, and oxidative stress can help to make these available during submerged fermentation and thus enable the scale up of their production.

So far, the production of the red-orange laetiporic acids from the sulphur shelf, Laetiporus sulphureus, as well as the formation of yellow-orange hispidin from the shaggy bracket, Inonotus hispidus, were established^7-9.

Basidiomycota have a complex life cycle, during which the single hyphae (= cell) contains two nuclei for long periods. For propagation, the nuclei merge inside the fruiting body and meiosis results in genetic recombination. The thus produced spores contain a single nucleus with newly mixed, genetic material and can form a monokaryotic mycelium. Only after a merge with another monokaryot of the correct mating type, a dikaryotic mycelium (with two nuclei per cell) with the ability to form fruiting bodies is formed (compare ¹⁰).

This cycle enables the generation of genetically ‘new’ monokaryots from a parental dikaryon. These can produce enzymes with improved activities and stabilities and thus present an opportunity to produce new enzymatic variants without the use of active genetic manipulation and thus genetic engineering.

This technique was used to gain new variants of a Dye-decolourizing peroxidase from the oyster mushroom Pleurotus sapidus with up to 2,6-times increased enzyme activity, which can be used for the production of different aroma compounds¹¹.

1- Krahe, N. K., Berger, R. G. & Ersoy, F. A DyP-type peroxidase of pleurotus sapidus with alkene cleaving activity. Molecules 25 (2020). https://doi.org/10.3390/molecules25071536

2- Krahe, N. K., Berger, R. G., Kahlert, L. & Ersoy, F. Co-Oxidative Transformation of Piperine to Piperonal and 3,4-Methylenedioxycinnamaldehyde by a Lipoxygenase from Pleurotus sapidus. Chembiochem 22, 2857-2861 (2021). https://doi.org/10.1002/cbic.202100183

3- Khalighi, S., Berger, R. G. & Ersoy, F. Cross-linking of fibrex gel by fungal laccase: Gel rheological and structural characteristics. Processes 8 (2020). https://doi.org/10.3390/pr8010016

4- Khalighi, S., Berger, R. G. & Ersoy, F. Cross-linking of wheat bran arabinoxylan by fungal laccases yields firm gels. Processes 8 (2020). https://doi.org/10.3390/pr8010036

5- Siebert, M., Berger, R. G. & Pfeiffer, F. Hydrolysis of chlorogenic acid in apple juice using a p-coumaryl esterase of Rhizoctonia solani. J. Sci. Food Agric. 99, 6644-6648 (2019). https://doi.org/10.1002/jsfa.9940

6- Ersoy, F. et al. A Prolyl Endopeptidase from Flammulina velutipes Degrades Celiac Disease-Inducing Peptides in Grain Flour Samples. Catalysts 13, 158 (2023). https://doi.org/10.3390/catal13010158

7- Zschätzsch, M. et al. Production of natural colorants by liquid fermentation with Chlorociboria aeruginascens and Laetiporus sulphureus and prospective applications. Engineering in Life Sciences 21, 270-282 (2021). https://doi.org/10.1002/elsc.202000079

8- Bergmann, P. et al. Pilot-Scale Production of the Natural Colorant Laetiporic Acid, Its Stability and Potential Applications. Fermentation 8, 684 (2022).

9- Bergmann, P. et al. Cultivation of Inonotus hispidus in Stirred Tank and Wave Bag Bioreactors to Produce the Natural Colorant Hispidin. Fermentation 8, 541 (2022). https://doi.org/10.3390/fermentation8100541

10- Vreeburg, S., Nygren, K. & Aanen, D. K. Unholy marriages and eternal triangles: how competition in the mushroom life cycle can lead to genomic conflict. Philosophical Transactions of The Royal Society B 371, 20150533 (2016). https://doi.org/10.1098/rstb.2015.0533

11- Krahe, N.-K. et al. Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression. Int. J. Mol. Sci. 22, 1363 (2021). https://doi.org/10.3390/ijms22031363