Continuing fascination of exploration in natural substances from microorganisms^†

^†This work is a translation of an original work in Japanese in the Japan Society for Bioscience, Biotechnology, and Agrochemistry http://doi.10.1271/kagakutoseibutsu.54.10

Abstract

In the search for novel organic compounds, I think it is of paramount importance not to overlook the pursuit of microorganism diversity and the abilities those microorganisms hold as a resource. In commemoration of Professor Satoshi Ōmura’s Nobel Prize in Physiology or Medicine, I will briefly describe the microorganism that produces avermectin and then discuss how innovating isolation methods and pioneering isolation sources have opened the door to numerous new microorganism resources. Furthermore, as exploratory research of substances views the world from many different angles—from biological activity to a compound’s physiochemical properties—it is possible to discover a novel compound from a well-known microorganism. Based on this, I will discuss the future prospects of exploratory research.

Key words:

• Natural compounds
• physicochemical screening

Novel avermectin-producing actinomycete species and the development of microorganism resources

Avermectin-producing microorganism, Streptomyces avermectinius MA-4680^T

This actinomycete was isolated in 1974 from soil collected in Kawana, Ito City, Shizuoka Prefecture. Gray aerial mycelia grew on the agar medium, and oval spores linked to form a long spiral (Fig. 1).

Fig. 1. Colonies (A) and scanning electron micrograph of aerial spore chains (B) of the avermectin-producing strain, Streptomyces avermectinius MA-4680^T, grown on agar medium.

Initially, after it was discovered to produce avermectin, this strain was classified into the Streptomyces genus based on its characteristic morphology and received its trivial name, S. avermitilis, from the substance it produces.¹⁾ The species name was not the official nomenclature until formally proposed 20 years later after studies on its taxonomy, morphology, and chemical composition in addition to the phylogenetic studies based on genetic sequence that developed in recent years. It was classified into the genus Streptomyces based on an important characteristic of the genus (a cell wall containing LL-diaminopimelic acid) and morphology. More detailed phenotypic and phylogenetic studies verified that this was in fact a new species, leading to its official name being proposed in Int. J. Syst. Evol. Microbiol., the authority on bacterial naming.²⁾ At the writing of this manuscript, the authors were notified that while it is accepted as a new species, the common name “S. avermitilis” cannot be used as the official nomenclature; the strain was officially named S. avermectinius. Thus, one strain was given two names, but I will omit those circumstances here as I discussed them in Hosenkin to Ikiru.³⁾ The important message is that S. avermectinius MA-4680^T (the same strain as S. avermitilis MA-4680^T) is a taxonomically novel species, and Prof. Ōmura’s simple response was, “Let’s use S. avermectinius from now on.”

Innovation of isolation methods and development of isolation sources

The Drug Discovery group (commonly, Ōmura Group) centered on the Department of Drug Discovery Sciences at the Kitasato Institute (University), Kitasato Institute for Life Sciences. This group conducted exploratory research on the bioactive substances from microorganisms, concentrating on actinomycetes and filamentous fungi, and discovered about 480 compounds.⁴⁾ We were in charge of isolating, culturing, and classifying the actinomycetes and tested various isolation methods to acquire new microorganism resources. Many innovative isolation methods were tested: use of antibiotic resistance and heat tolerance to isolate rare actinomycetes, use of chemotaxis to isolate motile actinomycetes, isolation after sonication of the soil aggregate, use of gellan gum in the place of agar as a solid agent, and finally—to achieve the greatest diversity in isolated samples—isolation of actinomycetes from plant leaves and desert sand.^5–7)

In the 1990s, Prof. Ōmura offered me this thought: “Humans have only isolated 10% of the microorganism species inhabiting the earth. Couldn’t something be done to isolate those microbes that haven’t been isolated?” He may have worded it differently, but the message is the same. Prof. Ōmura always said, “You mustn’t imitate others.” With these words constantly at the back of my mind, I continued to isolate actinomycetes.

When viewing the agar plates, I always made a conscious effort to isolate many different kinds of actinomycetes. By slightly shifting the angle at which I was approaching the task, I found an overwhelming number of one type of colony appearing on the plates compared to the other strains. I thought these strains might be supporting the survival and growth of the other strains in the soil or on the agar medium. I tentatively called this the dominant microorganisms and isolated seven strains from plates displaying the same appearance. These strains were cultured in Tryptic Soy Broth, and the resulting cell culture supernatant was smeared on agar plates consisting of glucose-peptone-meat extract (GPM) medium (1.0% d-glucose, 0.5% peptone, 0.5% meat extract, 0.3% NaCl, 1.2% agar). The soil diluent was smeared on, and the number and type of colonies were observed after cultivation. Two of the supernatants prepared from each of the seven dominant strains clearly increased the number and type of colonies in comparison with those when they were not added. After investigating many possible factors that could have led to the increases, we identified factors that were highly homologous to bacteria-derived superoxide dismutase (SOD). The increased number of microbial strains was seen even with commercially available SOD derived from bovine erythrocytes; when applied in combination with catalase, the effects were even greater.

To test whether the GPM medium itself, typically used for isolation of bacteria and actinomycetes, was generating reactive oxygen species, we quantified the reactive oxygen species (). Using different numbers (from one to five) of GMP medium agar pieces, we quantified the accumulated amount of reduced cytochrome C by generated from the medium. We found that the amount of reduced cytochrome C accumulated increased in tandem with the length of reaction time and the number of agar pieces, verifying that the medium itself generates ⁸⁾ (Fig. 2). In addition to GPM medium, Nutrient Broth and Tryptic Soy Broth were also found to generate . Furthermore, we analyzed each component in GPM medium to identify those that generate . Meat extract, a natural component, was found to generate large amounts of ; moreover, we detected superoxide anion (), hydrogen peroxide (H₂O₂), and hydroxyl radical (•OH), which is considered the most toxic reactive oxygen species molecule. However, singlet oxygen was not detected. The agar medium was already known to generate and H₂O₂, but it was the first knowledge that •OH had been detected^9,10) (Fig. 3). In further studies, ascorbic acid and other radical scavengers were found to increase the number of colonies; using this method, we have reported one new family, three new genera, and nine new species to date (Table 1). Of those, one strain, Patulibacter minatonensis KV-614^T, was recognized as part of the new family Patulibacteraceae (Fig. 4).¹¹⁾ The phylogenetic tree for the 16S rRNA/DNA sequence of this strain built using the currently registered database revealed interesting results. The 16S rRNA/DNA sequences of colonies that appeared only after incubation for 3 months in 100-fold diluted standard medium or PCR products amplified from the DNA directly extracted from soil samples among those registered to database were closely related to the sequence of KV-614^T on the phylogenetic tree. These results suggested that the culture method using radical scavengers enabled isolation of bacteria that had not been previously isolated due to their sensitivity to reactive oxygen species.¹²⁾ Furthermore, to determine how prevalent these strains are in nature, we built specific primers and tested 43 samples from various soils. The strains were detected in 31 samples (72%) and found to be widely distributed.¹³⁾ The novel species Conexibacter arvalis KV-962^T and KV-963 were isolated using fivefold diluted nutrient agar without radical scavenger after 21 days cultivation (Table 1).

Fig. 2. Detection of superoxide anion () generated from GPM agar medium (A), various nutritional media (B), and GPM medium components (C).

Fig. 3. Detection of reactive oxygen species generated from GPM medium.

Table 1. New taxa isolated by agar media supplemented with a scavenger of reactive oxygen species.

Scavenger		SOD	Catalase	SOD +catalase	Ascorbic acid	Rutin
New taxon
New family
Patulibacter minatonensis KV-614^T		1
New genus
Humibacillus xanthopallidus KV-663^T			1
Humihabitans oryzae KV-657^T			1
Oryzihumus leptocrescens KV-628^T, KV-647, KV-656		1	1	1
New species
Arthrobacter humicola KV-651^T				1
Arthrobacter oryzae KV-653^T			1
Demequina salsinemoris KV-810^T, KV-811, KV-816					2	1
Microbacterium aoyamense KV-492^T				1
Microbacterium deminutum KV-483^T				1
Microbacterium pumilum KV-488^T				1
Microbacterium pygmaeum KV-490^T				1
Microbacterium terricola KV-448^T		1
*Conexibacter arvalis KV-962^T, KV-963

Notes: Soil samples: Field and paddy field in Saitama prefecture, Bank in Tokyo, Amamioshima island.

*Isolated with Nutrient agar (Difco) diluted fivefold, for 21 days cultivation.

Fig. 4. Taxonomical characteristics of Patulibacter minatonensis KV-614^T.

Through our research, for the small portion of microorganisms that were difficult to isolate using conventional methods, we successfully developed new methods. I fear I may have only partially reciprocated the guidance I received from Prof. Ōmura, but I have worked on this for over 15 years.

To expand microorganism resources, it is of paramount importance to also expand isolation sources. Over the past few years, we have been pouring effort into isolating actinomycetes that exist inside of plant roots. Fig. 5 shows the presumed genera and count of the actinomycetes isolated from the internal plant root, the soil surrounding to the plant root (rhizosphere), and non-rhizospheric general soil. After sterilizing the surface of the plant root with hypochlorous acid and ethanol and then grinding it, the sample was cultured for 4–12 weeks at 27 °C before strains were isolated. We attempted to identify the genera and species of the isolated strains by analyzing their 16S rRNA sequences.

Fig. 5. New genera and new species of actinomycetes isolated from plant roots.

In each of the 16 different types of plant samples, the proportion of Streptomyces was low, and numerous, rare actinomycetes other than Streptomyces were isolated. The dominant genus isolated from plant samples varied from sample to sample; however, numerous types were present. In plant sample No. 4, Goodyera procera, a total of 80 strains were isolated with the predominate genera being Micromonospora (28.8%, 23 strains, eight species), Polymorphospora (16.3%, 13 strains, one species), and Sphaerisporangium (11.3%, nine strains, two species). There were only four strains of Streptomyces, the genus most frequently found in soil. Moreover, the strain named and proposed as a new genus, Phytohabitans suffuscus K07-0523^T,¹⁴⁾ was part of these findings, and another nine strains identical to this strain were isolated. Strains of this genus were also isolated from sorrel and fishwort plants and from G. procera collected at another location. After further taxonomic studies, three new species (P. rumisis, P. houttuyneae, and P. flavus) were proposed. The high frequency of Actinoallomurus species isolated became another characteristic of these root samples, leading to the newly proposed Actinoallomurus radicium K08-0128^T, A. liliacearum K10-0485^T, and A. vinaceus K10-0528^T. It was reported that Actinoallomurus strains produce diverse substances, and we discovered a new substance, actinoalloride, from a strain identified as the same genus.¹⁵⁾ As a result, we published two new genera and seven new species collected from the inside of plant roots.¹⁶⁾

In six soil samples taken from the rhizosphere and soil, Streptomyces was the predominant genus, and the frequency of isolating rare actinomycetes was low, indicating that the frequency of colonies differs from the internal root. It is thought that Streptomyces generally accounts for over 90% of actinomycetes isolated from soil samples, and our results also support that statement.¹⁷⁾ I have spent many years isolating actinomycetes from soil samples, and the overwhelming majority of those were Streptomyces strains. To acquire a particular rare actinomycete, it is typically necessary to devise various methods for selective isolation by taking advantage of the strain’s characteristics such as antibiotic resistance, heat tolerance of spores, or motility. However, from these results, it is difficult to call them rare actinomycetes because of the appearance frequency of these actinomycetes inside plant roots. The primary factor leading to the large difference in the types of strains isolated from the rhizosphere and root interior and the role of endophytic microorganisms remain unresolved. Furthermore, it is possible that the actinomycetes living near the surface layer are exterminated by surface sterilization. We cannot debate the flora with only these results, but we can conclude that we isolated many rare actinomycetes using plant roots as sample materials, which led to the expansion of available microorganism resources for the exploration of novel substances. In addition to actinoallolide described above, a novel compound trehangelin was identified from an actinomycete isolated from a root sample using a screening method described in the next section.

Another approach to acquire novel compounds

The Ōmura group routinely combined two approaches in their exploratory research to discover novel substances: searching based on bioactivity and using characteristics of the chemical structures. Prof. Ōmura would always tell us, “Microorganisms don’t make useless things,” and through exploratory research, the big discovery of staurosporine proved just that (Table 2). This compound was discovered by isolating a nitrogenous compound that demonstrated a positive Dragendorff reaction and evaluating its bioactivity. At the time of the discovery, the antifungal activity and the hypotensive action were known, but it was almost 10 years later that the protein kinase inhibition activity was found; since then, staurosporine has been widely used as a biochemical reagent. Moreover, the cyclic polypeptide Dityromycin was found to have translation elongation factor inhibitory activity 37 years after its discovery in 1977, and the producing microorganism is being used once again. These examples emphasize the importance of discovering novel compounds and preserving the microorganism that produce them.

Table 2. Compounds discovered by physicochemical screening and their biological activities.

	Compound	Year discovered	Producing microorganism	Biological activity (Year)
Doragendorff’s reaction	Pyrindicin	1973	Streptomyces griseoflavus NA-15^T	Antimicrobial (1973)
	NA-337A	1974	Streptomyces sp. NA-377	Hypolipidemic (1974)
	TM-64	1975	Thermoactinomyces antibiotics TM-64	Deteriorated reflex of the cornea (1975)
	Quinoline-2-methanol	1976	“Kitasatoa griseophaeus” PO-1227^T	Hypoglycaemic (1976)
	Dityromycin	1977	Streptomyces sp. AM-2504	Antimicrobial (1977), Inhibition of EF-G (2014)
	Staurosporine	1977	Lentzea albida AM-2282	Antifungal and Antitumor (1977), Inhibition of protein kinases (1986)
	1,3-Diphenethylurea	1978	Streptomuces sp. AM-2498	Antidepressant (1978), Adipocyte differentiation promoter (2011)
	Herquline	1979	Penicillium herquei Fg-372	Herquline B; Inhibition of platelet aggregation (1996)
	Neoxaline	1979	Aspergillus japonicus Fg-551	Inhibition of tubulin polymerization (1974)
	Reductinomycin	1981	Streptomyces xanthochromogenus AM-6201	Antitumor, Antibacterial and Antivirus (1981)
	Sespendole	2004	Pseudobotrytis terrestris FKA-25	Inhibition of cholesteryl ester and triacylglycerol, Antimicrobial (2006)
	Spoxazomicin	2011	Streptosporangium oxazolinicum K07-0460^T	Anti-trypanosomal activity (2011)
LC/UV-MS	Actinoallolide	2011	Actinoallomurus fulvus MK10-036	Anti-trypanosomal activity (2011)
	Mangromicin	2011	Lechevalieria aerocolonigenes K10-0216	Anti-trypanosomal activity (2011), Antioxidative activity (2013)
	Trehangelin	2012	Polymorphospora rubra K07-0510	Anti-Lipid peroxidation (2013)
				Enhanced production of collagen (2014)
	Iminimycin	2015	Streptomyces griseus OS-3601	Antimicrobial (2015)
	Sagamilactam	2015	Actinomadura sp. K13-0306	Anti-tripanosonal activity (2015)

Notes: Reference, splendid gift from microorganisms – the achievement of Satoshi Ōmura and colladorators – 5th edition (2015).

Recently, this method was developed further and named “physicochemical screening.” The novelty of a sample can be predicted by analyzing the medium extract with LC/UV-MS followed by isolated purification. Using this method, we have begun exploring newly isolated strains and microorganisms producing novel compounds and analyzing old strains known as substance producers that have been preserved for a long time. Thanks to this method, we have uncovered many novel substances with very interesting structures, including actinoallolide,¹⁵⁾ trehangelin,¹⁸⁾ and mangromicin,¹⁹⁾ from the media of rare actinomycetes. These compounds possess antitrypanosomal, cytoprotective, and antioxidative actions, respectively. Furthermore, very recently, a novel substance containing iminimycin²⁰⁾ in its structure was discovered from the media of the Streptomyces griseus strain conserved 43 years ago for the production of streptomycin (Fig. 6). S. griseus is an actinomycete that is frequently isolated from soil samples, and more than 200 substances have been reported as compounds produced by S. griseus. Our results indicate that novel compounds could be obtained by exploiting new screening methods even from such well-known strains.

Fig. 6. Structure of iminimycin A.

Concluding remarks

Actinomycetes are known to produce many secondary metabolites, and it is thought that over half of the discovered microorganism-derived bioactive substances to date are produced by actinomycetes.^21,22) Furthermore, there is great diversity in both the structures and biological activities of these substances.

It is very likely that we have overlooked an abundance of gifts from rare actinomycetes and all other actinomycetes. In addition, compounds that are marked as of no value to humans now could be immeasurable treasures in the future.

We were fortunate to be able to discern a substance with a new skeletal structure from the metabolite of a rare actinomycete. I believe it was the day-to-day joint efforts of researchers with intimate knowledge of actinomycete properties and those very familiar with chemical compounds that brought this good fortune upon us. A strain of a new taxon will not necessarily produce a new substance, but by always keeping the stance of pioneering for novel microorganism resources, the knowledge, experience, and skills that are accumulated along the way are just as important. We must not neglect to hand down these strains of microorganisms and compounds, intellectual property that cannot be seen with the naked eye.

Prof. Ōmura’s Nobel Prize in Physiology or Medicine, following penicillin (1945, Fleming, Florey Chain) and streptomycin (1952, Waksman), is the third prize awarded for discovery of an antibiotic. This country has valuable experience from 70 years ago, when the industrial sector, government, and academia pooled their knowledge in a joint endeavor to manufacture penicillin; only 9 months after the executive committee was established, they succeeded in producing penicillin in Japan. Avermectin was discovered through the collaboration of industry and academia and its discovery has been recognized as a great contribution to the world. Without a doubt, this was accomplished because of Prof. Ōmura’s outstanding foresight and passionate research spirit. In the wake of this award, natural substance exploratory research in Japan is expected to become more active, and I think that if a framework can be built by comprehensively gathering the strengths from different fields, the research will develop even further. Prof. Ōmura illustrated the importance of microorganism resources in natural product research to those inside and outside of the Ōmura group. He always encouraged, “Even though this is humble research that takes time and effort, it is the foundation.” To all of us who are involved in microorganism research, his support has been immeasurable. I would like to take this opportunity to express my deepest gratitude.

On many occasions, Prof. Ōmura would emphasize, “Learn from microorganisms,” and, “Be grateful for microorganisms.” This is certainly his way of saying, “Abilities unbeknownst to us lie hidden within microorganisms; therefore, we must take good care of microorganism resources and pursue ways to utilize them.”

The details of the research described here, while only a small part of the achievements of Satoshi Ōmura, Distinguished Emeritus Professor, Kitasato University, represent my direct involvement. Moreover, the work was carried out by the many researchers and students under Prof. Ōmura’s tutelage, so everyone is overjoyed that Prof. Ōmura has been honored with the Nobel Prize in Physiology or Medicine. A student from 27 years ago sent me a letter expressing joy: “I was only temporarily part of the Ōmura group, but I am deeply honored to have been taught by Prof. Ōmura.” I am sure everyone feels the same.

I would like to acknowledge the great effort displayed by Dr Yuki Inahashi and Dr Atsuko Matsumoto during the isolation, culturing, and classification of rare actinomycetes and by Dr Takuji Nakashima and Dr Inahashi during the isolated purification of substances by physicochemical screening. In addition, I would like to thank Professor Yuzuru Iwai, Visiting Professor of Kitasato Institute for Life Sciences, for his daily guidance starting from the basics on the path to becoming a full-fledged researcher. During the determination of compound structures and evaluation of their bioactivity, Professor Kazuro Shiomi, Prof. Toshiaki Sunazuka, Dr Masato Iwatsuki, and Dr Kazuhiko Otoguro provided immeasurable assistance.

Finally, I would like to express my gratitude to the Institution for Fermentation, Osaka (IFO), for assisting with development of the isolation method and isolation of endophytic microorganisms from plants and for its support and understanding of the importance of research on microorganism resources development. I would also like to include the Laboratory of Microbiology for Drug Discovery led by Dr Nakashima, for which Prof. Ōmura is acting as a research advisor, with the above-mentioned foundation’s funding; the team diligently works toward discovering new substances under the overarching theme of physicochemical screening.

Disclosure statement

No potential conflict of interest was reported by the author.

Notes

^† This work is a translation of an original work in Japanese in the Japan Society for Bioscience, Biotechnology, and Agrochemistry http://doi.10.1271/kagakutoseibutsu.54.10