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.
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.
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.
2. Agrawal, D. C., Tsay, H., Shyur, L., Wu, Y. & Wang, S. (2017) Medicinal Plants and Fungi: Recent Advances in Research and Development. 4, (Springer).
3. Sharma, M. & Sharma, R. (2016) Drugs and drug intermediates from fungi: Striving for greener processes. Critical Reviews in Microbiology 42 (2). doi: 10.3109/1040841X.2014.947240
4. Cuomo, C. A. (Jun 2017) Harnessing Whole Genome Sequencing in Medical Mycology. Current Fungal Infection Reports 11, 52–59. doi: 10.1007/s12281-017-0276-7
5. Raja, H. A., Miller, A. N., Pearce, C. J. & Oberlies, N. H. (Mar 2017) Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community. Journal of Natural Products 80, 756–770. doi: 10.1021/acs.jnatprod.6b01085
6. Vasundhara, M., Kumar, A. & Reddy, M. S. (Nov 2016) Molecular Approaches to Screen Bioactive Compounds from Endophytic Fungi. Frontiers in Microbiology 7. doi: 10.3389/fmicb.2016.01774
7. Silber, J., Kramer, A., Labes, A. & Tasdemir, D. (Jul 2016) From Discovery to Production: Biotechnology of Marine Fungi for the Production of New Antibiotics. Marine Drugs 14, 137. doi: 10.3390/md14070137
8. Tasdemir, D. (Aug 2017). Marine fungi in the spotlight: opportunities and challenges for marine fungal natural product discovery and biotechnology. Fungal Biology and Biotechnology 4, 5. doi: 10.1186/s40694-017-0034-1
9. Kamthan, A., Kamthan, M. & Datta, A. (Aug 2017) Biotechnology for drug discovery and crop improvement. The Nucleus 60 (2), 237–242. doi: 10.1007/s13237-016-0192-1
10. Sarasan, M., Puthumana, J., Job, N., Han, J., Lee, J., & Philip, R. (Jun 2017). Marine algicolous endophytic fungi – A promising drug resource of the era. Journal of Microbiology and Biotechnology 27 (6), 1039-1052. doi: 10.4014/jmb.1701.01036. Retrieved November 28, 2017 from https://www.researchgate.net/profile/Jae-Seong_Lee/publication/315736189_Marine_algicolous_endophytic_fungi_-_A_promising_drug_resource_of_the_era/links/58e4433f0f7e9bbe9c94d353/Marine-algicolous-endophytic-fungi-A-promising-drug-resource-of-the-era.pdf
11. Chambergo, F. S., & Valencia, E. Y. (mar 2016). Fungal biodiversity to biotechnology. Applied Microbiology and Biotechnology, 100 (6), 2567-2577. doi: 10.1007/s00253-016-7305-2. Retrieved November 28, 2017.
12. Chen, J. et al. (Aug 2017) Genomic and transcriptomic analyses reveal differential regulation of diverse terpenoid and polyketides secondary metabolites in Hericium erinaceus. Scientific Reports 7. doi: 10.1038/s41598-017-10376-0
13. Rao, H. C. Y., Rakshith, D., Harini, B. P., Gurudatt, D. M. & Satish, S. (Feb 2017) Chemogenomics driven discovery of endogenous polyketide anti-infective compounds from endosymbiotic Emericella variecolor CLB38 and their RNA secondary structure analysis. Plos One 12. doi: 10.1371/journal.pone.0172848
14. Lee, T. A. V. D. & Medema, M. H. (Apr 2016) Computational strategies for genome-based natural product discovery and engineering in fungi. Fungal Genetics and Biology 89, 29–36. doi: 10.1016/j.fgb.2016.01.006
15. Meyer, V. et al. (Aug 2016) Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper. Fungal Biology and Biotechnology 3 (6). doi: 10.1186/s40694-016-0024-8
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.
References
1. Bennett, J. (Dec 1998) Mycotechnology: the role of fungi in biotechnology. Journal of Biotechnology 66 (2-3), 101–107. doi: 10.1016/S0168-1656(98)00133-32. Agrawal, D. C., Tsay, H., Shyur, L., Wu, Y. & Wang, S. (2017) Medicinal Plants and Fungi: Recent Advances in Research and Development. 4, (Springer).
3. Sharma, M. & Sharma, R. (2016) Drugs and drug intermediates from fungi: Striving for greener processes. Critical Reviews in Microbiology 42 (2). doi: 10.3109/1040841X.2014.947240
4. Cuomo, C. A. (Jun 2017) Harnessing Whole Genome Sequencing in Medical Mycology. Current Fungal Infection Reports 11, 52–59. doi: 10.1007/s12281-017-0276-7
5. Raja, H. A., Miller, A. N., Pearce, C. J. & Oberlies, N. H. (Mar 2017) Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community. Journal of Natural Products 80, 756–770. doi: 10.1021/acs.jnatprod.6b01085
6. Vasundhara, M., Kumar, A. & Reddy, M. S. (Nov 2016) Molecular Approaches to Screen Bioactive Compounds from Endophytic Fungi. Frontiers in Microbiology 7. doi: 10.3389/fmicb.2016.01774
7. Silber, J., Kramer, A., Labes, A. & Tasdemir, D. (Jul 2016) From Discovery to Production: Biotechnology of Marine Fungi for the Production of New Antibiotics. Marine Drugs 14, 137. doi: 10.3390/md14070137
8. Tasdemir, D. (Aug 2017). Marine fungi in the spotlight: opportunities and challenges for marine fungal natural product discovery and biotechnology. Fungal Biology and Biotechnology 4, 5. doi: 10.1186/s40694-017-0034-1
9. Kamthan, A., Kamthan, M. & Datta, A. (Aug 2017) Biotechnology for drug discovery and crop improvement. The Nucleus 60 (2), 237–242. doi: 10.1007/s13237-016-0192-1
10. Sarasan, M., Puthumana, J., Job, N., Han, J., Lee, J., & Philip, R. (Jun 2017). Marine algicolous endophytic fungi – A promising drug resource of the era. Journal of Microbiology and Biotechnology 27 (6), 1039-1052. doi: 10.4014/jmb.1701.01036. Retrieved November 28, 2017 from https://www.researchgate.net/profile/Jae-Seong_Lee/publication/315736189_Marine_algicolous_endophytic_fungi_-_A_promising_drug_resource_of_the_era/links/58e4433f0f7e9bbe9c94d353/Marine-algicolous-endophytic-fungi-A-promising-drug-resource-of-the-era.pdf
11. Chambergo, F. S., & Valencia, E. Y. (mar 2016). Fungal biodiversity to biotechnology. Applied Microbiology and Biotechnology, 100 (6), 2567-2577. doi: 10.1007/s00253-016-7305-2. Retrieved November 28, 2017.
12. Chen, J. et al. (Aug 2017) Genomic and transcriptomic analyses reveal differential regulation of diverse terpenoid and polyketides secondary metabolites in Hericium erinaceus. Scientific Reports 7. doi: 10.1038/s41598-017-10376-0
13. Rao, H. C. Y., Rakshith, D., Harini, B. P., Gurudatt, D. M. & Satish, S. (Feb 2017) Chemogenomics driven discovery of endogenous polyketide anti-infective compounds from endosymbiotic Emericella variecolor CLB38 and their RNA secondary structure analysis. Plos One 12. doi: 10.1371/journal.pone.0172848
14. Lee, T. A. V. D. & Medema, M. H. (Apr 2016) Computational strategies for genome-based natural product discovery and engineering in fungi. Fungal Genetics and Biology 89, 29–36. doi: 10.1016/j.fgb.2016.01.006
15. Meyer, V. et al. (Aug 2016) Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper. Fungal Biology and Biotechnology 3 (6). doi: 10.1186/s40694-016-0024-8
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