Michizane Hashimoto

Characterization of a gene responsible for the Na+/H+ antiporter system of alkalophilic Bacillus species strain C‐125

An alkali‐sensitive mutant, 38154, of the alkalophilic Bacillus sp. strain C‐125 could not grow at an alkaline pH. The nucleotide sequence of a 3.7 kb parental DNA fragment that recovers the growth of 38154 at alkaline pH has four open reading frames (ORF1–4). By sub‐cloning the fragment, we demonstrated that a 0.25 kb DNA region is responsible for the recovery. Direct sequencing of the mutants corresponding region revealed a G to A substitution. The mutation resulted in an amino acid substitution from Gly‐393 to Arg of the putative 0RF1 product, which was deduced to be an 804‐amino‐acid polypeptide with a molecular weight of 89 070. The N‐terminal part of the putative ORF1 product showed amino acid similarity to those of the chain‐5 products of eukaryotic NADH quinine oxidoreductases. Membrane vesicles prepared from 38154 did not show membrane potential (δψ)‐driven Na+/H+ antiporter activity. Antiporter activity was resumed by introducing a parental DNA fragment which recovered the mutants alkalophily. These results indicate that the mutation in 38154 affects, either directly or indirectly, the electrogenic Na+/H+ antiporter activity. This is the first report which shows that a gene responsible for the Na+/H+ anti‐porter system is important in the alkalophily of alkalo‐philic microorganisms.

FR209602 and Related Compounds, Novel Antifungal Lipopeptides from Coleophoma crateriformis No. 738 : I. Taxonomy, Fermentation, Isolation and Physico-chemical Properties

Novel antifungal lipopeptides, FR209602, FR209603 and FR209604, were isolated from the fermentation broth of a fungal strain No. 738 which was identified as Coleophoma crateriformis from morphological and physiological characteristics. The antibiotics were purified by solvent extraction, HP-20, YMC-ODS and silica gel column chromatography and lyophilization. These compounds were structurally similar to FR901379 previously reported by ourselves which had a sulfate residue in the cyclic peptide portion.

FR220897 and FR220899, Novel Antifungal Lipopeptides from Coleophoma empetri No. 14573

Novel antifungal lipopeptides, FR220897 and FR220899, were isolated from the fermentation broth of a fungal strain No. 14573. This strain was identified as Coleophoma empetri No. 14573 from morphological and physiological characteristics. FR220897 and FR220899 showed antifungal activities against Aspergillus fumigatus and Candida albicans attributed to inhibition of 1,3-β-glucan synthesis. Furthermore, FR220897 was effective in a murine model of systemic candidiasis.

FR227673 and FR190293, Novel Antifungal Lipopeptides from Chalara sp. No. 22210 and Tolypocladium parasiticum No. 16616

Novel antifungal lipopeptides, FR227673 and FR190293, were isolated from the fermentation broths of fungal strains Chalara sp. No. 22210 and Tolypocladium parasiticum No. 16616, respectively. These compounds have the same cyclic peptide nuclear structure as FR901379, with different side chains, and showed antifungal activity against Aspergillus fumigatus and Candida albicans attributed to inhibition of 1,3-β-glucan synthesis.

AS1387392, a novel immunosuppressive cyclic tetrapeptide compound with inhibitory activity against mammalian histone deacetylase

The novel immunosuppressant AS1387392 has been isolated from Acremonium sp. No. 27082. This compound showed a strong inhibitory effect against mammalian histone deacetylase and T-cell proliferation. Further, AS1387392 showed a good oral absorption, and its plasma concentration was higher than that of FR235222, an analog of AS1387392 that inhibited histone deacetylase previously reported. Given these findings, AS1387392 may represent an important new lead in developing immunosuppressant.

FR258900, a novel glycogen phosphorylase inhibitor isolated from Fungus No. 138354. I. Taxonomy, fermentation, isolation and biological activities.

FR258900 is a novel glycogen synthesis activator produced by Fungus No. 138354. This compound was isolated from the culture broth by solvent extraction and reverse-phase column chromatography. FR258900 stimulated glycogen synthesis and glycogen synthase activity in primary rat hepatocytes. FR258900 exhibited a potent inhibitory effect on the activity of liver glycogen phosphorylase, suggesting that this compound may activate hepatic glycogen synthesis via glycogen phosphorylase inhibition. Thus, this glycogen phosphorylase inhibitor may be useful in the treatment of postprandial hyperglycemia in type 2 diabetes.

ASP2397: a novel antifungal agent produced by Acremonium persicinum MF-347833

The novel antifungal agent ASP2397 (Vical’s compound ID VL-2397) is produced by the fungal strain MF-347833 that was isolated from Malaysian leaf litter and is identified here as an Acremonium species based on its morphology, physiological properties and 28S ribosomal DNA sequence. Because of its potential importance for producing novel antifungal agents, we determined the taxonomic and biologic properties of MF-347833. We show here that ASP2397 is a cyclic hexapeptide that chelates aluminum ion and is therefore similar to ferrichrome, a hydroxamate siderophore. However, ASP2397 differs structurally from licensed antifungal agents such as amphotericin B, triazoles and echinocandins. To understand the relationship between chemical structure and biological function, we isolated certain ASP2397 derivatives from the culture broth, and we further chemically converted the metal-free form to other derivatives.

FR207944, an Antifungal Antibiotic from Chaetomium sp. No. 217 I. Taxonomy, Fermentation, and Biological Properties

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-β-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 μg/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED50s of 5.7 and 17 mg/kg respectively.

An expedient synthesis of the amide analog of the potent antifungal lipopeptidolactone FR901469

An expedient synthesis of the lactam analog (2) of the 40-membered lipopeptidolactone antifungal antibiotic, FR901469 (1), is described. The key steps in this synthesis are a novel biotransformation of the natural product to produce the highly versatile linear peptide building block 3, and efficient formation of the 40-membered ring by macrolactamization under high-dilution conditions.

Cloning and heterologous expression of P450Um-1 , a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392

Biotransformation technology involving enzymatic modification of original substrates by organisms such as microbes is a valuable tool in improving pharmacokinetics or physicochemical properties of the base compounds. The fungal metabolite AS1387392 is a histone deacetylase inhibitor with potential as a therapeutic immunosuppressant. However, its paucity of functional groups, essential to synthesizing derivatives, is a drawback. Amycolatopsis azurea JCM-3275 catalyzed hydroxylation of AS1387392 to AS1429716, which may facilitate the synthesis of more derivatives by the additional hydroxyl moiety present in AS1429716. This reaction was inhibited by cytochrome P450 inhibitor metyrapone, indicating that cytochrome P450 may be responsible for the transformation. Degenerate PCR primers were subsequently constructed and used to clone genes encoding cytochrome P450 from the genomic DNA of A. azurea JCM-3275. We cloned an entire novel P450 gene (1209 bp) and named it P450Um-1. Its deduced amino acid sequence was homologous with that of the CYP105 subfamily. Further cloning of the upstream region, which may contain the native promoter site, was followed by insertion of the open reading frame with the upstream area into Streptomycetes high copy vector pIJ702, giving the expression plasmid pNUm-1. P450Um-1 was specifically expressed in Streptomyces lividans TK24, and this recombinant strain converted AS1387392 to AS1429716 without any redox partners. These results show that P450Um-1, a novel bacterial P450, catalyzed hydroxylation of AS1387392 to AS1429716. This resultant recombinant strain is expected to be an efficient biocatalyst with application to more suitable redox systems than those tested here.