We think about them in a ton of different ways. Fungi can be a delicacy in food or they can be a deadly poison. They are beneficial mutualists with plants or harmful parasitic pests. But there is one more feature of theirs that has become more and more prominent in recent decades with the emergence of biotechnology and more advanced genetic, proteomic, and metabolomic analysis techniques.
That trait being the use of fungi as a source for biologically active compounds, whether that means as anti-bacterial medications, as nutritional supplements, or as useful industrial compounds. We have learned quite a lot from this lowly, oft-overlooked kingdom of life.
Today we will be looking at a field of study that utilizes the fungus for this very purpose. Mycotechnology is, as the name suggests, a combinatorial field that combines the methods found in biotechnology with materials obtained from the fungi kingdom. But before we go into those processes, let’s talk some background for both.
Discussing The Fungus
Identifying Taxonomy
Let’s first just look at fungi themselves. That alone is difficult to do thanks to the incredible amount of diversity found within the fungi kingdom. While several hundred thousand species are currently known to exist, there is estimated to be several million species of fungi extant in the world today.
Of those currently identified, 3/4ths of the species are found within the phylum of Ascomycota that includes most kinds of filamentous fungi and yeasts. Its sister phylum, Basidiomycota, makes up almost all of the rest of the fungi species, itself hosting what are colloquially termed the “higher fungi”, like your more common mushroom type fungus.
It should be noted that official classifications of fungi are under review and there is presently an attempt to normalize and conglomerate worldwide classifications systems under one combined, agreed upon umbrella. This standardization is sorely required, with many metabolites being reported in papers not even stating the fungi species the molecules were obtained from. The reasons for this are multifold, but let’s go over the main problem.
Originally, the method for identifying fungi was the most obvious, a visual inspection. Classification was done based on morphology alone and that was how species were grouped. For anyone who knows even a little about the fungal life and reproductive cycle, you might begin to see why this isn’t the best idea if one is trying to pin down species from a scientific perspective.
The fungi kingdom and the variety within it also contain a similar variety in stages of life. Some, such as the yeasts, use a budding reproductive system, which doesn’t meaningfully change their overall morphology. But the higher fungi are a different story altogether. Their stringy mats can go through an array of morphological changes, with the commonly known mushroom form actually being the spore reproduction stage, with the familiar shape being the spore producer that is called the “fruiting body”.
The majority of morphological description relied on this sexual body in order to make classifications and this method is even often still used today.While there may be enough distinctiveness in these organs for this sort of sorting, it doesn’t work all the time and it doesn’t allow for proper organization of the lower fungi that do not possess such a form. Also, it was often the case that the growth stage of a fungus would be sorted as a different species than its reproductive stage, leading to a vast amount of confusion that took time and effort to correct.
Therefore, the current International Code of Nomenclature has stated that for fungi, algae, and plants, morphological classification is no longer allowed. It can be used as an additional factor for confirmation, but it cannot be the sole factor.
The (Short) History of Industrial Fungi
The history of fungal usage extends far back into the annals of time. Humans have long focused on the medicinal benefits of fungal compounds, along with their often hallucinogenic properties. But starting in the last century, people began to notice that they had more varied options than just folk remedies across cultures.
They could be grown industrially and have their specific compounds, like penicillin and cyclosporin, mass-produced. This and more quickly grew into a multi-billion dollar industry, with fungi being used to produce even anti-fungal derivatives themselves. The pursuit of pharmaceutical biotechnology truly began with, as just noted, the discovery of penicillin in 1928 by Alexander Fleming.
While there was a bit of a lull in scientific insight in the years directly following Fleming’s revelation, things picked back up in the 1950’s with several innovations coming down the pipeline. Many natural fungal products were isolated in those years, leading to greater production, but the lack of genetic understanding limited how far the field could go at the time. This has been rectified contemporarily due to genetic sequencing and the global market allowing the scientific investigation of more fungal species than ever before.
There has been significant growth in this area over other cultivation opportunities precisely because of how simple land-based filamentous and matted fungi are to grow, unlike many kinds of plants. Bacteria, of course, remain the easiest, but the molecular options they produce are often limited to anti-bacterials (unless we directly modify them to produce desired compounds). Fungi are naturally more diverse in what they can make and we are still learning new things today about the limits of what fungi in the wild have evolved to produce.
A significant amount of modern mycotechnology research has focused on two unique sources of fungal distinctiveness: marine fungi and fungi that have direct, often positive, associations with plants (endophytic fungi). This article thus will be doing the same, as most published studies in the past year have been on one or the other.
The Genes of Fungi
Before we get into those two groups, we should first look at the overall genetic research in fungi and how breaking down the fungal genome works.
Choosing A Barcode
Proper gene papers on fungi first began being published two decades ago. The original paper analyzed the genes for the fungal nuclear ribosome, its subunits, and the entire region in between the two genes referred to as the internal transcribed spacer (ITS). The ITS region is specially important for use in taxonomy and phylogeny due to it being easy to amplify and thus detect using PCR, along with its being highly variable under evolutionary pressure, making it easier to differentiate between species.
This additionally makes it easier to determine how closely related two fungal species are and where to place them in a phylogenetic tree. Different parts of the ITS can be amplified for sequencing depending on which familial relationship one is trying to find, whether as specific as a genus and all the way up to a phylum. Many mycologists, in turn, made the ITS region the official “barcode” for fungi speciation.
In fact, this method of using a particular sequence of DNA in order to determine species across a wide family of organisms is scientifically referred to as DNA barcoding. The Consortium for the Barcode of Life approved the ITS region for fungal identification in 2011, resulting in a combined phylogenetic paper with over 100 authors that set up the tree for fungi phyla, as far as contemporary knowledge allowed.
There has been some argument over the use of the ITS region in particular genera, however, including Penicillium, due to these groups of fungi lacking or having very short barcode spaces in their ITS regions. And it is not helpful that the genera where this is a problem includes many of the species that have produced the most useful compounds obtained from fungi. More complex genetic markings must be used for these species to identify and differentiate them properly, something which is still being done today.
How To Sequence A Genome
Whole genome sequencing and all of the benefits therein is an alternative option that reveals genes needed for every part of the mycology field. Of course, this option is the more expensive choice, but the cost of sequencing entire genomes is going down year by year as technology advances.
The rapid expansion of genome sequencing choices has led to some outdating of past actions taken by mycologists. There are generally two methods for sequencing a whole genome (with random shotgun sequencing being the common option for both). If you’re dealing with a new never before seen fungal species, then the entire genome must be sequenced from scratch.
In the beginning, certain key species, such as those of the Penicillium family, were given priority for sequencing and the kind used at the time is known as Sanger sequencing. This slower and much more costly method sees some use today, but it has largely been supplanted by Next Generation Sequencing technologies.
Building an entire genome from nothing, termed de novo sequencing and assembly, is used to make reference genome assemblies that can have varying levels of detail depending on the technology used. General overviews at a chromosomal level or so are far easier to make than nucleotide by nucleotide sequences, the latter of which can still run into issues when dealing with things like repetitive parts of the genome that cause the sequencing to fail.
After the assemblies are made, they can then be annotated and investigated via modeling and other computer software to determine likely functions of genes based on their sequences. With faster and cheaper sequencing being made available, it opens up options for sequencing multiple members of a species for comparison between each other to better improve the reference assemblies and determine what the variable genome regions are within a species.
Once a reference library is available for a species, that allows for the second type of sequencing to be used, called re-sequencing. This is a way to isolate genetic variants in a particular organism, such as if one is trying to find the gene that a mutated member of a species developed, you can compare its genome to the reference assembly and find out where it varies, as previously mentioned.
Re-sequencing is a technique that has been highly useful for medical professionals when dealing with a disease outbreak (whether bacterial, viral, or fungal), since how the pathogen evolves over time and through its new hosts can be directly mapped with this method. It is also easier to tell if genes connected to drug resistance have been altered and whether certain medicines used against the pathogens will no longer be effective, allowing doctors to switch to an alternative and saving lives in the process.
Creating A Comprehensive Library
Worldwide, there are multiple projects underway to sum up and organize all of the information on fungi that is available, along with their genomes. The Joint Genome Project as a part of the US Department of Energy has been working on sequencing as many species as possible. Their current goal is to have two full species sequences as a representative for every family level grouping in the fungi kingdom of life.
Thus far, they have sequenced over 2100 species and their rate of sequencing is outstripping their capability to actually annotate and understand the genomes being set out, with only just over 800 having been annotated with genes and their likely functions. They are in a unique, though still somewhat undesirable situation, where the technology is outpacing their ability to actually analyze the data they’re receiving.
A major benefit of all of this is that it is rapidly helping scientists re-assess the current phylogenetic trees and rearrange them to match the new knowledge we’re obtaining. The Phylogenetic Species Concept, the model being used now to say what counts as a species or not, requires there to be consistency in the genomic data, so as to avoid classifying an organism as a new species when it really is only a mutational variant.
Even so, while the data is helping to put to rest several debates on where certain species, genuses, and families fit into the fungal tree, there remain many other ongoing debates that even this data is unable to resolve. The complexity and variation among fungi continue to befuddle attempts to have every species piece fit into the puzzle. It may be that certain cases of hybridization and gene uptake mixed together different limbs of the tree, making it that much more difficult for us to untangle them. But efforts are, as always, continuing.
Marine Fungi: The Fungus of the Waters
A common theme in medical discussions today is concern over the growing amount of antibiotic resistant bacteria and the incredibly slow rate of new antibiotics becoming available. Even though more than 350 antimicrobials have been developed to date from fungal sources, the rise in resistance is steadily overwhelming our reserves. This has resulted in infectious diseases becoming one of the primary causes of death globally due to our inability to treat them properly with our existing medications.
The Key Is In The Seas
But there is perhaps some hope for the future, based entirely in the bias of where scientists and researchers have been searching for bioactive compounds in the past. Almost all of the fungi we have investigated thus far have been those found in forested, marshy, or otherwise greenery-filled landscapes. In short, the fungi that exist on land.
The world is far more vast than just the land however and the fungi that live and thrive in the waters of our planet have largely remained completely untapped by science for biologically active compounds for medicine and other fields. With the crisis happening over antibiotic resistance, scientists are finally turning more firmly to this other source of possible escape from the situation we find ourselves in.
Some discoveries have already occurred over the past few years, with one group finding an antimicrobial quinazolin-based compound and another an anthraquinone for industrial uses. The genetic scaffolds being produced from these marine and deep-sea fungi have proven to contain large amounts of novel genetic sequences to be looked into further, any of which may code for an unknown and useful compound.
These secondary metabolites of the fungi are believed to, in some way, help them to survive in their special environments. It is hard to predict what antimicrobial molecules will be found though, since even the microbial diversity of the oceans are themselves poorly understood. Only about 0.1% of the microbial life in the oceans is believed to have been studied thus far and that, at least, has been an active field of study for quite some time, showcasing just how much work there is left to do.
Though there are issues with being able to probe the fungi living in the oceans. The key one being that in order to unearth their genome and study their produced metabolites, it needs to be possible to cultivate the fungi in a lab. And marine fungi (along with marine bacteria) can be extremely finicky to grow without the proper medium and conditions to facilitate that. This is especially so for those fungi that grow around deep sea thermal vents, whom already have the depth of their environment that makes it difficult to even obtain them, let alone grow them elsewhere.
A final complication is that even if we can obtain them and even if we can convince them to grow on media in a lab and even if we can then find useful compounds in their genetics and biology, we still have to take the final step of being able to produce an industrial level of the compound if it is ever going to be possible for it to be distributed worldwide. A novel antibiotic is pointless if we only have grams of it to use.
These are just a few of the headaches that marine mycologists must deal with in their research, but they are still making progress nonetheless.
The History of Marine Antibiotics
Of course, the history of marine fungi and medicine is not entirely so recent. Some compounds were recognized in the past, with one of the first actually being a very early antibiotic in its own right. That would be cephalosporin C that was commercialized in the 1950’s. This was followed up in the 1970’s with gliotoxin, the first compound to be found from a fungi in deep sea sediments. Then indanonaftol A, the first antibiotic from a marine yeast.
Slowly, but steadily, the number of overall compounds from marine fungi has been increasing. 272 of them by 2002, with nearly 200 new compounds being described every other year since. Unfortunately, while many of these compounds are useful indeed, less than 5% turn out to have any antibacterial properties and even fewer that have a strong enough capability to warrant development into a new official medicine.
Arguably one of the most promising in the past two decades has been the molecule pestalone, which features strong antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium. I’m sure you all know the horrors of just the former, so anything that can be designed to fight against it is a major boon to the world. But synthesis of pestalone is complicated. While it was first found in 2001, it took until 2010 before it was fully synthesized and chemically characterized. Efforts to fully utilize it are ongoing.
Conferencing The Future
For present research, look no further than the Second Annual Conference of Marine Fungal Natural Products that met earlier this year in Germany. It was organized and hosted by the GEOMAR Centre for Marine Biotechnology. The purpose of this meeting was to further a systemic approach among marine mycologists and to make sure their individual efforts were being put toward the most efficient methods of finding new fungal compounds.
Fungal interactions with other species were also presented, such as with seaweed, sponges, corals, crustaceans, and more. One of the more intriguing points was the intersection between marine fungi and microalgae, the latter already being itself a major focus of study. There was even a presentation on the topic of mycoviruses in marine ecosystems and the impact they pose to fungal life. The array of biomes, from the tropical to the temperate to the extremely cold Arctic, helped to create a picture of the assortment of fungi available for future research.
The scientists attending the conference were able to all agree that the ability to cultivate the fungi in labs was the major roadblock setting back much of their work. And it wasn’t an issue unique to marine fungi, but all marine microorganisms, leading to them being referred to as a group as the “oceans’ dark matter”.
There have been some technological improvements however that may soon begin to overturn this mystery. The advancement of lab-on-a-chip tech may allow proper environment manipulation and simulation for fungal study. The One-Strain-Many-Compounds (OSMAC) approach for isolating microbial metabolites can also play a role in the future.
But successful cultivation itself runs directly into a secondary problem. Biosynthetic gene clusters (BGCs) are groups of genes that, together, code for biosynthetic pathways. These pathways, in turn, make the very metabolites that scientists are looking to find and discern. When cultivation works though, it causes BGCs to in most cases remain silent and not active as they would be in their native environment. This means that most of the molecular and chemical potential of marine fungi remains locked away behind a genetic barrier preventing us from producing them. It is hoped that OSMAC will be able to help with this as well.
Another way to induce BGC activation that is being tested is co-cultivating marine fungi with marine bacteria, in the hopes that the fungi will begin using their anti-microbial genes in response, un-silencing them. This type of approach was favored by the presenters at the 2017 conference, with nearly a quarter of them including some form of this in their presentations. Epigenetic modification and procedures like heat shock were also shown and they may together find a concrete way to get around this secondary barrier.
That about sums up the efforts being put into marine fungi. The collaboration between researchers in this field is incredibly recent, with the first meeting having just occurred in 2014. So we may have to wait a few years to see a solidification of results, but it is quite clear that marine mycology is a field that may have revolutionary propositions for medicine in the near future.
Endophytic Fungi and Their Healthy Plant Relationships
The second biggest group of fungi receiving a large amount of research are the endophytic fungi, those fungi that are in a symbiotic relationship with plants. There are other kinds of endophytes, obviously, but this niche is almost entirely filled with fungal species thanks to their broad ability to benefit their host plant and protect against pathogens and other invaders.
Pinning Identities To Metabolites
Because of their responsibilities toward their host plant for nourishment and their common attention on fighting off harmful bacteria, this group of fungi have produced a vast assortment of antibacterial compounds as their defenses evolved. The plant-endophyte co-evolution hypothesis also suggests that some of the biosynthetic pathways observed in endophytic fungi may have been obtained from their host plant, as the two often share these sorts of genetic pathways.
Interestingly enough, while a single host plant can have dozens of endophytic fungi species working for their mutual survival, very few of the latter will exhibit important metabolites like antibacterials. This makes sorting through the available fungi a lengthy process. Swifter screening methods have helped, but it is still complex to find the few fungi that are of any use for more in-depth sequencing. There are some species that might be missed in the mass of types, so comprehensive screens have been a major focal point for this field.
Once appropriately isolated, however, there are a number of techniques available to identify bioactive compounds. Spectroscopic data is among the most useful and, combined with chromatographic equipment, can precisely pinpoint the chemical structure of these compounds. Proper identification does require an existing library of metabolites to compare with though, with the Human Metabolome database or the Madison Metabolomics Consortium Database filling that necessity.
There are cases where a compound is so uniquely structured that these databases aren’t enough and that’s when the more powerful equipment, like fragmentation machines and subsequent molecular ion mass spectroscopy, are used. Nuclear magnetic resonance is also helpful in this process. While it would be nice to just screen all metabolites through these accurate systems, the time and money it would take is enormous, so the comprehensive machines are reserved for only the most useful of compounds.
When run through genomic libraries, it has been found that filamentous endophytic fungi contain far higher gene clusters for metabolite biosynthesis than was expected, with many of them being terpene and peptide enzymes for cellular processes. Identifying function for endophytic fungi remains just as controversial as with marine fungi, due to silenced genes, though direct gene knockouts in this case have been found to be positive for forcing gene response.
One of the major discoveries from endophytic fungi was the first anti-cancer drug isolated in the 90’s, Paclitaxel (taxol), from the yew tree and then later its fungi counterparts. It proved to not be very easy to synthesize and produce in large quantities, but it revealed the possibility of anti-cancer compounds being hidden away in the organisms of the world. Since then, Paclitaxel has been found to be a common compound in a huge amount of endophytic fungi, increasing the availability of its production. Though, at least within fungi, the entire purpose of the compound for an evolutionary advantage has yet to be fully understood.
Algicolous Endophytes, The Best of Both Worlds
Quickly, we find ourselves circling back to a previous topic. This particular group of endophytes are memorable because they not only have relationships with plants, but are also marine fungi. They form symbiotic interactions with macroalgae and it has been known for thousands of years that the latter are a source for therapeutic medicine in folk remedies across international cultures.
The secondary metabolites that fungi in relationships with the algae form therefore correspond generally to the same functions and ecological niche. The constant abiotic stresses that macroalgae are sensitive to seem to be a great selectional pressure for the evolutionary development of metabolite compounds, which is then additionally transferred to the algicolous fungi. Furthermore, the species of these fungi found in tropical and subtropical regions have had little to no scientific inquiries up to now.
Isolation of these fungi have an added complication as compared to their land-based counterparts, the need for there to be surface sterilization to remove any possible pathogenic microorganisms on the algae or the fungi. But scientists also have to be careful that the sterilization methods do not kill the fungi as well, which are themselves microorganisms. Often mechanical separation techniques like vortexing and sonication can be used to split them into groups.
Algal endophytes have thus far produced a slew of compounds, including a handful focused on gram-positive bacteria and some with attention given to S. aureus and E. coli. A mixture of antifungal compounds have also been found to be produced by algicolous fungi, adding to a heavily desired set of fungicidal drugs that are needed as some fungal outbreaks of disease have continued to emerge over the years.
With the development of better isolation media, capturing techniques, and more, we are likely to see more advances with this body of fungi in the near future and it is hard to determine just how much more there is to find out there.
A Mycotechnology Medical Future
In some ways, the antibiotic-resistance crisis has been a boon to scientific knowledge and collection, as it has forced researchers to move beyond the normal areas plumbed for specimens and into the less tread regions of the world. It has forced many fields of science to examine their own preconceived biases on what kind of research should be conducted and where.
In the long term, it may prove to have been beneficial to expanding the medical field, but we still have to deal with the short-term of the here and now where infectious diseases continue to be a main cause of death. Hopefully we’ll be able to keep that short-term as short as possible and with the help of mycotechnology, that’s definitely a possibility.
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Photo CCs: Voile blanchâtre champignon aquatique, bactéries Bois de la Citadelle, Lille, décembre 08 from Wikimedia Commons
