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We report here the draft genome sequence of Streptomyces sp. strain AM-2504, a microorganism producing a broad range of biotechnologically relevant molecules. The comparative analysis of its 16S rRNA sequence allowed the assignment of this strain to the Streptomyces kasugaensis species, thus fostering functional characterization of the secondary metabolites produced by this microorganism.

To be an effective microbial biocide, Streptomyces kasugaensis should be converted into spore during cultivation process for successful long-term storage. By statistical design methods, culture conditions including medium components and operating parameters were optimized and more than 100 times increase in spore yield was achieved. Addition of spent culture fluid (100 ppm), EDTA (30 ppm), mycophenolic acid (32 ppm) with combination of pH up-shock (5.5 to 8.5) increased total viable cell and spore conversion rate, resulting in 1.6×107 (spore/mL) in 5 days of culture in a fermenter. This result provides a practical method for obtaining high spore number for commercial production of Streptomyces kasugaensis as a microbial pesticide.

Biosynthetic genes of kasugamycin (KSM), an aminoglycoside antibiotic produced by Streptomyces kasugaensis, forms a cluster within the chromosome. A cloned 22.4-kb cluster region contains almost all the enzyme-coding genes required for KSM biosynthesis together with kac338, a gene for KSM acetyltransferase, and kasKLM, a set of genes encoding an ABC transporter, both of which participate in KSM self-resistance as well as kasT encoding a KSM-synthesis-specific transcriptional activator of the biosynthetic genes. Furthermore, rpoZ, encoding a 90-amino acid omega (ω) subunit of RNA polymerase (RNAP), is required for the simultaneous production of KSM and aerial mycelium, and rpoZ-disrupted wild-type S. kasugaensis produces neither KSM nor aerial mycelia. Transcriptional analysis of the biosynthetic genes and forcible expression of kasT in the mutant revealed that the presence of rpoZ, which results in formation of RNAP carrying the ω subunit, facilitates initiation of kasT transcription and is thus crucial for KSM production.

Continuous cultures of immobilized Streptomyces kasugaensis, a kasugamycin producer, were carried out on Celite beads. When using a prototype separator for immobilized-cell separation and recycling, the continuous operation could not be sustained for an extended period as a result of an excessive loss of immobilized cells caused by the poor performance of the separator. Accordingly, the immobilized-cell separator was revised to provide better immobilized-cell settling and thus recycling into the reactor. In a subsequent culture using the revised separator, a stable operation was maintained for over 820 h with a high kasugamycin productivity. The kasugamycin productivity ranged from 9.8 to 16.1 mg/L/h, which was about 14- to 23-fold higher than that in a batch suspended-cell culture. When the original feeding medium concentration was doubled at the end of the continuous culture, the productivity became severely impaired for several reasons, which will be discussed. An excessive formation of free cells and loss of immobilized cells through the separator were also observed.

We isolated a kasugamycin-producing actinomycete, strain MF861-C4, which had spiny spore surface. This strain was different from Streptomyces kasugaensis but rather close to Streptomyces albulus with regard to morphological, cultural, and physiological characteristics, DNA homology and partial base sequences of 16S ribosomal RNA. The strain MF861-C4 was identified as S. albulus.

The kac gene coding for KSM acetyltransferase cloned from kasugamycin (KSM)-producing Streptomyces kasugaensis MB273-C4 (Hirasawa et al., 1993 ; JP. A-05-23187) was investigated for its correlation with KSM-productivity. Genomic Southern hybridization analysis indicated that kac existed in the genome of all the KSM-producing Streptomyces strains tested, i.e., S. kasugaensis MB273-C4 and M338-M1, and S. celluloflavus JCM4126 as well as Streptomyces sp. MF861-C4 that has characteristics taxonomically and phylogenically remote from those of the formers. We also found in experiments using Streptomyces sp. MF861-C4 that kac gene was readily deleted during storage as a lyophile with skim milk at 10°C or as a frozen 20% sucrose suspension at −20°C of its aerial mycelia, accompanying the loss of KSM productivity.

1-Acetyl-β-carboline, isolated as a metabolite of an actinomycete for the first time, was obtained from the culture filtrate of Streptomyces kasugaensis. The separated compound was identified by spectral means. Production of 1-acetyl-β-carboline was substantially rised after addition of equimolar amounts of DL-tryptophan and DL-alanine into the cultivation medium.

Production of 3-methylthioacrylic acid (3-MTAA) by Streptomyces kasugaensis SK 619 was controlled by methionine and by quality and concentration of the carbon source. Maltose (5%) and methionine (5 g l -1 ) added to the production medium positively influenced production of 3-MTAA. 3-Methylthioacrylic acid, a potential herbicide, inhibited growth of Photobacterium phosphoreum at concentrations of 50-200 μg per disc

Streptomyces kasugaensis gave arginine auxotrophic mutants at high frequency, The coupled loss and reappearance of plasmid deoxyribonucleic acid with arginine auxotrophy suggested that the insertion of the plasmid into chromosomal deoxyribonucleic acid caused the arginine auxotrophy.