18.14 Fungi as cell factories

We have shown throughout this book how fungi relate to their environment and all the other life forms on the planet, and how we have come to depend on the ways we can manipulate fungi for our benefits in agriculture, medicine and industrial biotechnology. Finally, in the previous Section (Section 18.13) we have introduced you to a range of techniques that enable the most detailed manipulation of the fungal genome. Molecular tools have already found application in every area of fungal biology. It’s only a slight exaggeration to say that if you can dream of a genetic manipulation, then there’s a genome editing technology that can now make it happen (McCluskey & Baker, 2017; Kersey et al., 2020).

Hibbett et al. (2013) call the science ‘genome-enabled mycology’ describing it as being characterised:

‘…by the pervasive use of genome-scale data and associated computational tools in all aspects of fungal biology. Genome-enabled mycology is integrative and often requires teams of researchers with diverse skills in organismal mycology, bioinformatics and molecular biology...’

Their paper discusses the technical and social changes that need to be made to enable all fungal biologists to make use of the new data; and it starts a special issue of the journal Mycologia which is devoted to genome-enabled mycology. The next Resources Box directs your attention to several journal special issues and monographic reviews that are worth examining because they show you the incredibly wide range of the interests (and the technologies) of existing mycologists in a way that might inspire you to join the ranks of the practising professionals.

Resources Box 18.3

Learning more about genome-enabled mycology

Several journals in the recent past have published special issues about various aspects of this topic.

CLICK HERE to visit a page providing details of these and a few other sources of information.

‘Fungal cell factories’ is a phrase that is often used in those ‘blue skies’ discussions about what could be done in the future. This is because the metabolic activities of fungi have already been harnessed for so long in applications ranging from food fermentation to pharmaceutical production that they are naturally thought of as indispensable biotechnological tools. The more so because fungal bioprocesses of earlier generations, like those that produce citric acid and penicillin, and those of today’s generation producing lovastatin, have had such positive impacts on human society.

We have discussed how fungi are utilised for industrial production processes throughout this book, most notably in Chapters 11 and 17. The growing amount of information that seems to cascade from the various forms of genomic analysis described in Section 18.12 is demanding application to these industrial processes for our greater good.

The metabolic and enzymatic diversity encoded in the genomes of fungi will continue to be developed for production of new generations of enzymes, pharmaceuticals, chemicals and biofuels. Though there must be many applications which will only emerge with time and further knowledge (Howes et al., 2020; Meyer et al., 2020); there are some which are immediately obvious. Currently, fungal derived enzymes that degrade plant derived biomass are being utilised for the development of bioprocesses for biofuel and renewable chemical production, particularly the growing demand for sustainable production of biochemicals that substitute for chemicals otherwise obtained from fossil fuels.

Filamentous fungi are of great interests as biocatalysts in biorefineries as they naturally produce and secrete a variety of different organic acids that can be used as building blocks in the chemical industry; ideally, in a lignocellulosic biorefineries, the fungus could be considered in a combined approach where it hydrolyses plant biomass wastes and ferments the resulting sugars into different organic acids.

Genomics and metabolomics analyses enable rapid identification of novel secondary metabolites open to industrial exploitation through the design of high yielding fungal cell factories (Karagiosis & Baker, 2012; Khan et al., 2014; Nielsen & Nielsen, 2017; Badalyan & Zambonelli, 2019; Badalyan et al., 2019; Overy et al., 2019; Youssef et al., 2019; Howes et al., 2020). There is no shortage of novel methods to obtain new metabolites by engineering fungal secondary metabolism, but increased yield is the key essential and regulation of secondary metabolite biosynthesis is incompletely understood.

However, the identification of the mcrA gene as a principal regulator of Aspergillus secondary metabolism indicates that further advance in this direction is imminent. The mcrA gene is conserved, and it encodes a transcription factor that regulates transcription of hundreds of genes including at least ten secondary metabolite gene clusters in Aspergillus terreus and Penicillium canescens (Scharf & Brakhage, 2013; Oakley et al., 2016).

Production of recombinant proteins by filamentous fungi was initially focussed on exploiting the extraordinary enzyme synthesis and secretion ability of fungi to produce single recombinant protein products, especially by industrial strains of Aspergillus, Trichoderma, Penicillium and Rhizopus species. Two disadvantages of filamentous fungi as hosts for recombinant protein production became apparent immediately: one is their common ability to produce homologous proteases which could degrade the heterologous protein product and the other is that the protein glycosylation patterns in filamentous fungi and in mammals are quite different.

Specifically, fungi lack the functionally important terminal sialylation of the glycans that occurs in mammalian cells. So, without engineering, filamentous fungi, despite their other advantages, are not the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. Nevertheless, strategies to prevent proteolysis have already met with some success and new scientific information being generated through genomics and proteomics research will extend the biomanufacturing capabilities of recombinant filamentous fungi, enabling them to express genes encoding multiple proteins, making filamentous fungi even better candidates to produce proteins and protein complexes for therapeutic use (Ward, 2012; Fernández & Vega, 2013; Nevalainen & Peterson, 2014).

Most of what we have discussed so far in this Section has either stated or implied submerged (liquid) fermentation of fungi, but it is essential to remember that solid state fermentation is a crucial process for producing enzymes, organic acids, flavour compounds, pharmaceutical agents and food processing (see Chapters 11 and 17; and see review by Ghosh, 2016). Of course, it is also the foundation of the mushroom cultivation industry (Section 11.6; and see Petre, 2015). This last is especially important in relation to potential improvements in the biotechnological procedures for producing mushrooms as healthy and highly nutritive food in their own right, while at the same time using mushroom farming as a bioremediation tool, by using recalcitrant wastes as substrates for the mushroom farming compost before crop production and/or by using spent mushroom compost for soil remediation after cropping (Purnomo et al., 2011; Camacho-Morales & Sánchez, 2015).

Ganoderma is a particularly interesting edible commercial mushroom because it is mainly farmed for use as a traditional Chinese medicine. Fruit bodies of the Ganoderma lucidum species complex contain many bioactive compounds; indeed, well over 400 secondary metabolites have been isolated from various Ganoderma species (Baby et al., 2015; Ahmad et al., 2019, 2021; Zhou et al., 2018). A mixture of Ganoderma lucidum polysaccharides (known as GLP) is the main bioactive component in the water soluble extracts of this mushroom, and there is some evidence that GLP possesses potential anticancer activity (Sohretoglu & Huang, 2018). Interestingly, the triterpenoid squalene, from the culture filtrate of Ganoderma lucidum, has been identified as an effective antagonist of the plant necrosis disease virus in tomato (Sangeetha et al., 2020).

The clinical evidence for antitumor and other medicinal activities of mushroom metabolites comes primarily from some commercialised purified polysaccharides, and polysaccharide preparations can be obtained from medicinal mushrooms cultured in bioreactors. Mushroom polysaccharides do not attack cancer cells directly but produce their antitumor effects by activating various immune responses in the host. Structurally different β-glucans have different affinities toward receptors and thus generate different host responses. Immunomodulating and antitumor activities of these metabolites are related to immune cells such as hematopoietic stem cells, lymphocytes, macrophages, T cells, dendritic cells, and natural killer cells, which are involved in the innate and adaptive immunity, resulting in the therapeutic immune modification (Berovic & Podgornik, 2015; Sudheer et al., 2018; Zmitrovich et al., 2019). On the other hand, the antiviral activity of some compounds from fungi seems to be due mainly to inhibition of enzymes, particularly proteases, needed for the virus to infect humans (Hernández Sánchez et al., 2021).

A wide range of pharmaceutically-interesting metabolites have been found in extracts of Ganoderma, and some have been found to be stimulators of neural stem cell proliferation in vitro, which could be of value in treatment of neurodegenerative diseases (Yan et al., 2015). Other extracts have been assessed for genotoxicity and anti-genotoxicity using comet assays of mouse lymphocytes; no evidence was found for genotoxic chromosomal breakage nor cytotoxic effects by Ganoderma extract in the mouse, nor did it protect against the effects of the mutagen ethyl methanesulfonate. This study found no evidence for the extract having any value in protecting against the test mutagen (Chiu et al. 2000). A more recent study found that although the aqueous extract of Ganoderma lucidum exhibited no genotoxic effect, it did have an antigenotoxic effect. This study used the hen’s egg test for micronucleus induction as a genotoxicity assay (formation of micronuclei during cell division indicates induced chromosome instability and fragmentation) that is different from the assay techniques using mouse lymphocytes mentioned earlier (Çelik & Özparlak, 2019).

Shah (2012) stresses the importance of genotoxicity testing for pharmaceuticals to ensure compliance with the guideline of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH; a unique project that brings together regulatory authorities of Europe, Japan and the United States with pharmaceutical industry representatives).

We mention in Section 13.6 that lack of degradability and growing pollution problems on land, in water courses and in the open seas have led to mounting concern about waste plastics being persistent organic pollutants. These synthetic polymers are ubiquitous in the modern world but the global environmental problems they pose are caused by their careless disposal. We show in Section 13.6 that common ascomycete and basidiomycete fungi can produce enzyme systems enabling them to use such pollutants for growth, so providing several opportunities for genome engineering leading to mycoremediation of plastic waste.

In this book we have tried to show you what 21st century mycology has to offer. If you have read the entire book, well, congratulations on your dedication! But, hopefully, having reached this point you can now appreciate how important fungi are to life on Earth, and particularly, human life on Earth; and that includes your every-day life. You could also take the knowledge to which we guide you and decide to manipulate, control and engineer fungi in ways of which earlier mycologists could only dream in their wildest fantasies. Please also add ‘conserving fungi’ to that to-do list; we can’t imagine life on Earth without fungi and their functions. Indeed, some of our colleagues have claimed that even mycologists are in danger of needing conservation (Minter, 2001; Courtecuisse, 2001), and others have stridently pointed out the importance of fungi (Anonymous Editorial, 2017; Willis, 2018). 

We have shown you what we know about fungi. We will end by pointing out some of the topics about which we remain astonishingly ignorant.

  • We wonder how free cell formation works (Section 3.4). How do some fungi and fungal-like organisms accomplish the three-dimensional positioning of wall- and membrane-forming vesicles to subdivide large volumes of cytoplasm to create motile or non-motile spores? They are the only organisms that do this.
  • We wonder what mechanisms are used to ensure that the nuclear membrane remains intact as the nuclear division progresses (Section 5.6). Another unique characteristic of present day fungi.
  • We wonder how the Spitzenkörper operates, how it is assembled, regulated, and directed (Section 5.10). It is only found in filamentous fungi.
  • We wonder what it is that controls the multinucleated nature of most hyphae and some specific tissues (Sections 5.6 and 5.15). How do fungi (and only fungi) control the synchronicity of mitotic divisions and then the (rapid) migration (Section 4.8) and distribution of daughter nuclei within their hyphae?
  • We wonder how hyphal fusion is managed, and how incompatible fusions trigger the death of hyphae (Section 5.16 and Chapter 7, especially Fig. 7.7, and Chapter 8).
  • We wonder what regulates the placement of septa in hyphae (Section 4.12 and Section 5.17) and how different states of differentiation can be controlled on the two sides of perforated septa.
  • We wonder how the yeast-hyphal dimorphism is controlled (Section 5.18); it’s important in the life-style of so many pathogenic fungi and control may be analogous to multicellular growth regulation in plants (Cogo et al., 2018).
  • We wonder how hyphal branching is controlled in time and space.
  • We wonder how multitudes of independent hyphal apices are orchestrated to create tissue layers (like hymenia; Section 12.7) and how determinate-growth controls are selectively applied to such large hyphal communities.
  • We wonder how the button mushroom (Agaricus bisporus) manages largely to avoid the diseases that might be caused by the populations of viral RNAs that exist in its mycelia.   

You will notice that we have not mentioned the fungal plasma membrane (which is unique in using ergosterol) or the fungal wall (which is also exclusive to fungi); this is because, as well as being established targets for selective toxicity, these structures have been investigated exhaustively. It’s too easy to try a slightly different agent that targets the wall or membrane; another batch of chemicals, another lot of routine tests. We need now to be more imaginative when trying to identify new antifungal drugs and fungicides.

Perhaps we would have more success if we directed our attention to some of the other structures and/or processes that are unique to fungi. Bearing in mind the bulleted list immediately above we would suggest: (a) vesicle transportation and positioning; (b) nuclear membrane dynamics; (c) Spitzenkörper dynamics; (d) nucleus migration and control of nuclear division; (e) hyphal fusion and how programmed cell death is triggered by the non-self-recognition system; (f) septation dynamics and physiological function; (g) control of dimorphism; (h) dynamics of hyphal branching; (i) selective control of determinate growth of hyphal apices.

This is as far as we can go. So, now it’s up to you to decide what will happen in the rest of the 21st century. Where are you taking us from here?

Updated September, 2021