18.14 Fungi as cell factories

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).

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; 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). 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 such as polysaccharides, triterpenes, polyphenols, proteins, amino acids, and organic germanium ions which, either actually, or by reputation, have medicinal value (and, given the apparent importance of the ‘placebo effect’, it may not be important whether the patient is convinced they have been cured or are chemically cured; providing they are cured).

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).

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). 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; Yan et al., 2015).

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. These synthetic polymers are ubiquitous in the modern world but the global environmental problems they pose are caused by their careless disposal. Poly-(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. Unfortunately, the properties that make PET so useful to us in our daily lives also endow it with an alarming resistance to biodegradation, with the potential of it lasting for centuries in most natural environments. Most applications that employ PET, such as single-use beverage bottles, clothing, packaging, and carpeting employ crystalline PET, which is recalcitrant to catalytic or biological depolymerisation due to the limited accessibility of the ester linkages.

PET can be depolymerised to its constituents if the ester bonds of the polymer can be cleaved. Doing this with available chemical techniques is too costly to be a viable recycling solution. Recently, a newly discovered bacterium isolated from outside a bottle-recycling facility in Japan, Ideonella sakaiensis, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. When grown on PET, this strain produces two enzymes capable of hydrolysing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymatically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol; so, yielding the monomers for further plastics manufacture (Yoshida et al., 2016; Austin et al., 2018). This paragraph suggests the essentials of a plastics pollution remediation technology; devising it is only a matter of time. What is the prospect of generating recombinant fungi that possess this metabolic activity? First, is it a bacterial technology? Probably not, we think, and to whet your mycological appetite we will mention just one more study with plastics remediation potential.

Ahuactzin-Pérez et al. (2018) discovered that Pleurotus ostreatus degrades and uses (as carbon and energy source) high concentrations of di-(2-ethyl hexyl) phthalate (DEHP), and Ferrer-Parra et al. (2018) showed that Fusarium culmorum produced a range of esterase enzymes when challenged with DEHP. Phthallates are plasticisers, primarily used as additives in plastics like polyvinyl chloride (PVC) polymers to make them more flexible. Phthalates are easily released from plastics, through direct release, leaching, and abrasion because they are not chemically bound, and phthalate esters are one of the most frequently detected persistent organic pollutants in the environment (Gao & Wen, 2015). In laboratory animal studies, some phthalates have been associated with developmental and reproductive toxicity and they are generally considered to be toxins that interfere with endocrine systems in mammals (Hauser & Calafat, 2005). If common ascomycete and basidiomycete fungi can produce enzyme systems enabling them to use such pollutants for growth, they provide an opportunity for bioremediation of plastic waste. Pleurotus ostreatus degrades lignin efficiently, grows well in both liquid and solid fermentation systems, and is an ideal candidate for genome engineering into a plastic-eating Oyster mushroom.

In this book we have tried to show you what 21st century mycology has to offer. If you have read the entire book (congratulations!), 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.

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 do you go from here?

Updated July, 2018