Brasilicardin A, a New Terpenoid Antibiotic from Pathogenic Nocardia brasiliensis: Fermentation, Isolation and Biological Activity.

Abstract

A novel tricyclic diterpenoid antibiotic, brasilicardin A, was isolated from the culture broth of Nocardia brasiliensis IFM0406. The antibiotic exhibited immunosuppressive activity in a mouse mixed lymphocyte reaction (MLR)assay system and its IC50 value was 0.057jug/ml. Although the inhibitory activity of cyclosporin A (CyA) against IL-2 production was confirmed in the MLRassay system, brasilicardin A did not have the activity. The results of in vitro toxicity testing of brasilicardin A against various human cell lines were comparedwith those of CyA.During our continuing search for bioactive metabolites from pathogenic Nocardia, we isolated and reported new antibiotics, brasiliquinones A~C1>2), brasilidine A3) and nocardicyclins A, B4). Recently we also reported a new 32-membered macrolide antibiotic, brasilinolide A, with immunosuppressive activity from N. brasiliensis IFM 04065>6). Further studies on the active metabolites from the culture broth of strain IFM 0406 led to the isolation of a new terpenoid antibiotic, brasilicardin A.In this paper we report on the fermentation, isolation and biological activity of brasilicardin A. FermentationThe seed culture was prepared by inoculating mycelial fragments of strain IFM 0406 into a 500 ml-Erlenmeyer flask containing 150ml of brain heart infusion medium (BHI, Difco) supplemented with 1% glucose and 1% glycerol. The culture was performed at 32°C for 96 hours on a rotary shaker at 300rpm. The seed culture (150ml) was then inoculated into a 20 liter jar fermenter containing the production medium (15 liters) consisting of2% glycerol, 1%polypeptone and 0.5% meat extract in distilled water. The pH was adjusted to 7.0 with 1 m NaOHbefore sterilization.Adekanol (Asahi Denka Co., Ltd.) was added as the antifoam. Fermentation was carried out at 32°C for 3 days under aeration of 15 liters/minute and agitation at 200 rpm. HPLCAnalysis of Brasilicardin AThe amount of brasilicardin A in the culture broth was estimated by HPLCanalysis using a reverse phase column (LiChrospher 100 RP-18 endcapped, 4mmx125 mm,Merck). The analysis was carried out as follows:

Cloning of the Gene Cluster Responsible for the Biosynthesis of Brasilicardin A, a Unique Diterpenoid

“…Media and growth conditions for IFM 0406 were as described by Komaki et al [17]. Escherichia coli JM110 and plasmids, pUC118 and pUC119, were used for sequence analysis.…”

“…Genomic DNAs were prepared from the transconjugants and subjected to PCR analysis with the following primers: 5Ј-GTGAGCGCGCGCA-CCCGGCGGCACTGGTGGGCGCA-3Ј and 5Ј-AGCGT-GGCCGCGACATCGAGGAGGCGATGTGCTGA-3Ј, which corresponded to the N-terminal and C-terminal coding region of the bra3 and bra5 genes. The productivity of BCA by the disruptant was examined as follows: The disruptants were cultivated by the methods described by Komaki et al [17], except for the addition of thiostrepton (50 m g/ml) to the medium. After full growth cultivation, the culture broth was centrifuged, and compounds were extracted by ethyl acetate from the supernatant.…”

“…Brasilicardin A (BCA), produced by Nocardia brasiliensis IFM 0406, is a representative of this type of compound, which consists of a basic diterpene skeleton with L-rhamnose, Nacetylglucosamine, amino acid, and hydroxybenzoate moieties ( Fig. 1) [17]. BCA was demonstrated to possess potent anti-tumor [18] and immunosuppressive activity [19].…”

Phenalinolactones A–D, terpenoglycoside antibiotics from Streptomyces sp. Tü 6071

“…One example is brasilicardin A, which is produced by Nocardia brasiliensis that exhibits a potent immunosuppressive and cytotoxic activity. [7][8][9] The genetic organization of the phenalinolactone gene cluster in Streptomyces sp. Tü 6071 was published 2006 by the group of Andreas Bechthold.…”

Nocardia amamiensis sp. nov., isolated from a sugar-cane field in Japan

“…Additionally, they are considered to play a significant role as saprophytic organisms in the turnover of naturally occurring organic substances . Nocardia species have industrial uses as well, as they are known to produce bioactive agents such as nocardicin, tubelactomicin A and brasilicardin A (Aoki et al, 1976;Komaki et al, 1999;Igarashi et al, 2000).Strain TT 00-78 T was isolated from soil in a sugar-cane field on Amami Island in Japan, using an SDS/yeast extract pretreatment method (Hayakawa & Nonomura, 1989) and HV agar (Hayakawa & Nonomura, 1987) containing nalidixic acid (20 mg l 21 ). The strain formed thin, flat colonies with sparse, white aerial hyphae on HV agar containing nalidixic acid.…”