Screening of Novel Bioactive Compounds from Plant-Associated Actinomycetes

Abstract

INTRODUCTION Although soil-derived microorganisms have been intensively screened as a source of therapeutically important molecules over a half century 1) , the frequency of discovering structurally new compounds is apparently decreasing these years. This trend seems to imply that the easily accessible microorganisms in soil had been exhausted and there is a need to seek unutilized microorganisms from unexplored sources. Since the role of natural products in the drug discovery is still large, new approaches such as the utilization of eDNA 2) , combinatorial biosynthesis 3) and screening of microorganisms from extreme environments 4) are investigated to discover novel chemical structures. It is likely that the diversity of secondary metabolites relies more or less on the isolation source, namely, the habitat of the producers. As for the interrelationship between plants and microorganisms, a couple of evidences have been provided suggesting the exchange or the transfer of biosynthet-ic gene cluster beyond the kingdom. Taxol, an antitumor diterpene, was first isolated from Pacific yew, Taxus brevi-folia. Recently, the production of taxol was identified in the culture broth of a fungus Taxomyces andreannae which is an endophyte of Pacific yew 5). Another example is the isolation of maytansine from a Celastraceae plant, Maytenus ovatus, and ansamitocin from Actinosynnnema sp 6). Maytansine and ansamitocin are comprised of an ansamycin backbone and a fatty acid side chain, and the structure of their ansamycin backbone is identical. These findings suggest that endo-phytes possibly possess metabolic genes different from the microorganisms in other habitat. We have identified several new bioactive compounds from actinomycetes isolated from live plants (Fig. 1). Two new novobiocin analogs 7) were produced by Streptomyces from Aucuba japonica, and cedarmycins 8) by Streptomyces from Cryptomeria japonica as antimicrobial metabolites. Fistupyrone 9) is a metabolite of Streptomyces from Allium fistulosum which inhibits the infection of Alternaria brassi-cicola to Brassica plant. Furthermore, in the culture broth of S. hygroscopicus from Pteridium aquilinum were found pteridic acids that induce the formation of adventitious roots in hypocotyl of kidney beans with the effectiveness equivalent to that of indoleacetic acid, a plant hormone, at 1 nM 10). 6-Prenylindole 11) and clethramycin 12) are antifungal metabo-lites from Streptomyces sp. Anicemycin is a potent antitu-mor antibiotic produced by S. thermoviolaceus isolated from a leaf of Aucuba japonica. These findings indicate that plant is a potential isolation source of strains producing new bio-active molecules. In this article, our recent results on the screening of novel bioactive compounds from plant-associated actinomycetes are described. 1. Isolation of actinomycetes from live plants In 1978, Hasegawa et al first reported the isolation of an actinomycete which was neither symbiotic nor pathogenic but associated host-specifically with plant and the identification of a new genus Actinosynnema 13). Later, Okazaki et al investigated the plants inhabiting in seashores and proved the existence of endophytic actinomycetes in leaves by showing the scanning electron micrographs of aerial hypha and spore chains growing in plants 14). These studies prompted us to investigate thoroughly the distribution of actino-mycetes in herbaceous and arbor plants along with their potency of producing new bioactive compounds 15). Plant samples were collected in Toyama and Miyagi prefectures , Japan. In order to compare the distribution of actin-omycetes in leaves, stems and roots of a whole plant, we chose healthy seedlings with no apparent physical or physiological damages. Samples were surface-sterilized, and in-cubated on an agar plate at 32°C for a month. The numbers of isolates were determined by counting the colonies different from each other by macroscopic observation of morphology on a Bn-2 agar slant. From 24 species of herbaceous and arbor plants, 398 actinomycete strains were isolated. Actinomycetes were isolated from all plants used in this study, regardless of herbaceous or arbor, or wild or agricultural species, suggesting their wide distribution in association with plant in natural environment. These strains were used for the screening of bioactive compounds. 2. Fistupyrone, an inhibitor of spore germination of Alternaria brassicicola Alternaria brassicicola is the cause of black leaf spot, a major disease of cultivated Brassica plants. In the screening of the inhibitor of infection by A. brassicicola to Chinese cabbage, we identified fistupyrone in the fermentation broth 63 Award Lecture