In the course of a search program to acquire rare actinomycete species from natural habitats, a remarkable organism was isolated from a grass blade that formed true synnemata and had motile arthrospores and was named as the new genus Actinosynnema. By a selective isolation method based on the characteristics of Actinosynnema, Actinosynnema-like organisms have been effectively isolated from soil habitats, and subsequently, the new genus Actinokineospora was discovered. Furthermore, a number of “Catenuloplanes”-like organisms have been obtained. In this way, Sporichthya, Actinosynnema, Actinokineospora and “Catenuloplanes” have come to be recognized as rare genera having motile arthrospores. No doubt these rare genera await establishment of their status in the rational actinomycete taxonomy.
The history of culture collections of actinomycetes is briefly surveyed. Some subcollections that should be undertaken by actinomycete culture collections are also proposed. The first catalogue published in 1900 by Král Bacteriological Laboratory, Prague, contained actinomycete strains. This was the first actinomycete culture collection in the world, but microbiologists no longer have this treasure. Actinomycetes are a very important natural resource, yet there is no organisation currently devoted entirely to the collection and maintenance of actinomycete strains. The KCC culture collection was established by the author in 1955 and incorporated into the Japan Collection of Microorganisms (JCM), RIKEN, in 1983 so that KCC cultures could acquire their longevity. As one of the established culture collections, the KCC Culture Collection published their own list of strains and contributed to several other culture collections allowing them to expand.
We have applied knowledges obtained during studies of antibiotic biosynthesis to improve antibiotic producers as follows; (1) The increase in antibiotic productivity and the production of a new component by gene dosage effect: (2) The production of new analogs of antibiotics by the introduction of heterogeneous biosynthetic gene(s): (3) Selective production of useful components by inactivation or activation of genes involving in antibiotic production. Mutants that do not produce antibacterial, antifungal and antimycoplasma antibiotic kalafungin were isolated from kalafungin-producing Streptomyces tanashiensis and classified into seven distinct biosynthesis phenotypes. By colony hybridization with the genes for polyketide synthase (actI and III) as probes, a clone carrying a large DNA fragment was picked up from a genomic library of this strain. In this DNA fragment, the gene cluster for kalafungin biosynthesis containing at least eight genes (kalI to VII, and a regulatory gene) were identified. Although kalafungin is an intermediate of actinorhodin biosynthesis in S. coelicolor A3(2), this gene cluster for kalafungin biosynthesis in S. tanashiensis was not the same as that in S. coelicolor. When the wild type strain and kal mutants of S. tanashiensis were transformed with the cloned DNA fragment, not only the increase in the production of kalafungin and dihydrokalafungin but also production of a new antimicrobial product named tetrahydrokalafungin were caused. The anthelmintic antibiotic avermectin complex produced by Streptomyces avermitilis is a family of eight closely related components, A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b. The component B1a is the most highly effective. However, since it is not easy to separate the “a” and “b” components in the industrial processes, the “B1” components B1a and B1b and their hydrogenated products are used as an important anthelmintic agent. After a mutagenesis, two kinds of mutants, K2021 and K2034, which produced only the specific components, were obtained. Strain K2034 lacked avermectin B2 5-O-methyltransferase activity and produced the “B” components, B1a, B1b, B2a and B2b. In the other mutant, K2021, the incorporation of L-isoleucine and its keto acid was efficient, but very little L-valine and its keto acid were incorporated into avermectins. This mutant produced the “a” components, A1a, A2a, B1a and B2a. The recombinant strains, which have both phenotypes of the above mutants, were designed by in vivo recombination. The strains produced the components B1a and B2a only. Genetic mapping showed that the locus of the mutation affecting the selectivity of the incorporation of branched-chain keto acids into the avermectin skeleton apparently is sufficiently distant from the gene cluster for avermectin biosynthesis.
Bialaphos (BA) is a tripeptide produced by Streptomyces hygroscopicus SF-1293 and is characterized by the presence of a unique C-P-C bond. Through the biosynthetic studies on BA we have revealed the involvement of three enzymes catalyzing different C-P bond formation mechanisms. Two of them, phosphoenolpyruvate phosphomutase (PEP phosphomutase) and carboxyphosphonoenolpyruvate phosphonomutase (CPEP phosphonomutase), have been purified and characterized to catalyze the reactions of a similar type, i.e., intramolecular rearrangements of phosphate esters to form a C-P bond. On the other hand, the remaining one catalyzes P-methylation of phosphinic acid derivatives. In order to reveal these enzymatic properties in more detail, the corresponding genes were identified in the biosynthetic cluster of BA and then expressed in S. lividans. Introduction of PEP phosphomutase or CPEP phosphonomutase gene into S. lividans using pIJ680 resulted in efficient expression of the corresponding enzymes. On the other hand, the P-methylation enzyme was only expressed in S. lividans when the gene was under control of a thiostrepton-inducible strong promoter of pAK114.
A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a microbial hormone that functions as a key switch for secondary metabolite formation and cell differentiation in Streptomyces griseus. Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases its repression. The positive A-factor signal is transferred, probably via some additional unknown regulatory proteins, to the strR gene, a putative regulator for Sm biosynthesis. The StrR product, in turn, activates the other Sm production genes. A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase present in the same organism. Characterization of AfsR by means of site-directed mutagenesis has revealed that phosphorylated AfsR stimulates globally transcription of antibiotic production genes. It is most likely that the AfsR protein and the AfsR-phosphokinase compose a two-component regulatory system.