Toshio Tsuchida

Organization of the Biosynthetic Gene Cluster for the Polyketide Antitumor Macrolide, Pladienolide, in Streptomyces platensis Mer-11107

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.

One-pot fermentation of pladienolide D by Streptomyces platensis expressing a heterologous cytochrome P450 gene

Pladienolide D is a 16-hydroxylated derivative of pladienolide B, produced by Streptomyces platensis. To facilitate the production of pladienolide D, the gene encoding a pladienolide B 16-hydroxylase from S. bungoensis was introduced into S. platensis. The recombinant produced pladienolide D at a production level comparable to that of pladienolide B.

Three eremophilane derivatives, MBJ-0011, MBJ-0012 and MBJ-0013, from an endophytic fungus Apiognomonia sp. f24023.

Three eremophilane derivatives, MBJ-0011, MBJ-0012 and MBJ-0013, from an endophytic fungus Apiognomonia sp. f24023

Increase in pladienolide D production rate using a Streptomyces strain overexpressing a cytochrome P450 gene

Pladienolide B and its 16-hydroxylated derivative (pladienolide D) are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107 showing strong in vitro and in vivo antitumor activity. While pladienolide B is mainly produced by this strain, pladienolide D is produced to a lesser extent. To facilitate the production of pladienolide D by biotransformation, we found that Streptomyces bungoensis A-1544 was able to hydroxylate pladienolide B at 16-position. We identified psmA from S. bungoensis A-1544, which encoded a pladienolide B 16-hydroxylase PsmA belonging to the CYP105 family of cytochrome P450. To increase the efficiency of pladienolide D production, we constructed recombinant S. bungoensis A-1544 overexpressing psmA and performed biotransformation of pladienolide B to pladienolide D. This biotransformation achieved a production level 15-fold higher than that using the control strain S. bungoensis A-1544/pIJ702.

New chaetoglobosin derivatives, MBJ-0038, MBJ-0039 and MBJ-0040, isolated from the fungus Chaetomium sp. f24230.

New chaetoglobosin derivatives, MBJ-0038, MBJ-0039 and MBJ-0040, isolated from the fungus Chaetomium sp. f24230

Branchiibius hedensis gen. nov., sp. nov., an actinobacterium isolated from a Japanese codling (Physiculus japonicus).

A novel, Gram-stain-positive bacterial strain, Mer 29717(T), was isolated from the branchia (gills) of a Japanese codling, Physiculus japonicus, collected from bottom waters of Suruga Bay in Shizuoka, Japan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain represents a distinct lineage within the family Dermacoccaceae and was related most closely to members of the genera Demetria and Yimella. It shared highest 16S rRNA gene sequence similarity (95.1 %) with Yimella lutea YIM 45900(T). Strain Mer 29717(T) contained MK-8(H(2)) and MK-8(H(4)) as menaquinones, and iso-C(16 : 0), C(16 : 0), C(17 : 1) cis-9, C(17 : 0), C(18 : 1) cis-9 and C(19 : 1) cis-10 were the major cellular fatty acids. The cell-wall peptidoglycan of strain Mer 29717(T) was composed of l-Lys, d-Ser, l-Ser, Gly, d-Glu and d-Ala. Polar lipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and one unidentified phospholipid. Mycolic acids were not detected. The G+C content of the DNA of strain Mer 29717(T) was 68 mol%. On the basis of differential chemotaxonomic, physiological and biochemical data, strain Mer 29717(T) is considered to represent a novel species of a new genus, for which the name Branchiibius hedensis gen. nov., sp. nov. is proposed. The type strain of Branchiibius hedensis is Mer 29717(T) ( = NBRC 106121(T)  = DSM 22951(T)).

Cytotoxic sesquiterpenoids MBJ-0009 and MBJ-0010 from a saprobic fungus Nectria sp. f26111.

Cytotoxic sesquiterpenoids MBJ-0009 and MBJ-0010 from a saprobic fungus Nectria sp. f26111

MBJ-0086 and MBJ-0087, new bicyclic depsipeptides, from Sphaerisporangium sp. 33226

We have constructed a library of isolated natural products referred to as the ‘CB library’ for efficient implementation of biological screenings.1 To expand and variegate this library, we have conducted chemical and biological screenings using the advanced compoundidentification system based on accumulated HPLC-MS profiling data combined with strain information designated as ‘MBJ’s special selection’. During screening, we have identified several new natural bioactive compounds such as the cytotoxic eremophilane derivatives MBJ-0009, MBJ-0010, MBJ-0011, MBJ-0012 and MBJ-0013 from Nectria sp. f261112 and Apiognomonia sp. f24023,3 the chaetoglobosin derivatives MBJ-0038, MBJ-0039 and MBJ-0040 from Chaetomium sp. f24230,4 a hydroxamate metabolite MBJ-0003 from Micromonospora sp. 298675 and the aziridine containing-peptide MBJ-0035 from Streptosporangium sp. 32552.6 Further screening was used to isolate new bicyclic depsipeptides designated as MBJ-0086 (1) and MBJ-0087 (2) from the culture broth of Sphaerisporangium sp. 33226 (Figure 1). Here, we describe the fermentation, isolation, structure elucidation and antibacterial activity of 1 and 2. Sphaerisporangium sp. 33226 was isolated from a soil sample collected in Kochi Prefecture, Japan. This producing strain was cultivated in 250-ml Erlenmeyer flasks, each containing 25 ml of a seed medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc., Hokkaido, Japan), 2% glucose (Junsei Chemical, Tokyo, Japan), 2% soy bean powder (SoyPro, J-Oil Mills, Tokyo, Japan), 0.5% yeast extract powder (Oriental Yeast, Tokyo, Japan), 0.25% NaCl (Junsei Chemical), 0.32% CaCO3 (Wako Pure Chemical Industries, Osaka, Japan), 0.0005% CuSO4 5H2O (Wako Pure Chemical Industries), 0.0005% ZnSO4 7H2O (Wako Pure Chemical Industries) and 0.0005% MnCl2 4H2O (Junsei Chemical) for 3 days at 28 1C on a rotary shaker at 220 r.p.m. (pH 7.4). The seed culture (0.5 ml) was transferred into 500-ml Erlenmeyer flasks containing 50 ml of the same medium and cultivated for 4 days at 28 1C on a rotary shaker at 220 r.p.m. After cultivation, an equal volume of n-BuOH was added to the culture broth (2 liters). The n-BuOH extract was evaporated in vacuo and partitioned between water (300 ml) and EtOAc (300 ml, 3 times). The EtOAc-soluble material (1.26 g) was subjected to silica gel medium-pressure liquid chromatography (Si-MPLC, Purif-Pack SI-30, Shoko Scientific, Yokohama, Japan) eluted with a gradient system of n-hexane–EtOAc (0–25% EtOAc) followed by a stepwise solvent system of CHCl3–MeOH (0, 2, 5, 10, 20, 30 and 100% MeOH). Fractions were monitored using the UPLC-DAD-ELS-MS system. The 5% MeOH eluate (553.9 mg) was subjected to Sephadex LH-20 column chromatography (CHCl3/MeOH1⁄4 1:1, GE Healthcare BioSciences AB, Uppsala, Sweden). The target fraction (415.1 mg) was then separated by Si-MPLC (Purif-Pack SI-30) using isocratic elution with 4% MeOH in CHCl3 to give fractions A (199.5 mg) and B (129.6 mg). Fraction A was subjected to a second round of chromatography using Si-MPLC (Purif-Pack SI-30, 3% MeOH in CHCl3, isocratically) to obtain crude 2 (59.5 mg). The pure sample of 2 (8.2 mg, retention time: 11.0 min) was obtained by two preparative HPLC runs on an XSelect CSH C18 column (5.0mm, 19 i.d. 150 mm; Waters, Milford, MA, USA) with 55% aq. MeCN containing 0.1% formic acid (flow rate: 10 ml min–1). Fraction B was re-chromatographed using Si-MPLC (Purif-Pack SI-30, isocratic, 3% MeOH in CHCl3) to give semi-purified 1 (51.5 mg). Final purification of 1 was carried out by reversed-phase HPLC (column: CAPCELL PAK C18 MGII column, 5.0mm, 20 i.d. 150 mm, Shiseido, Tokyo, Japan; solvent: 40% aq. MeCN containing 0.1% Figure 1 Structures of MBJ-0086 (1) and MBJ-0087 (2).

Novel aziridine-containing peptides MBJ-0034 and MBJ-0035 from Streptosporangium sp. 32552.

Novel aziridine-containing peptides MBJ-0034 and MBJ-0035 from Streptosporangium sp. 32552

New hydroxamate metabolite, MBJ-0003, from Micromonospora sp. 29867

In the course of our screening program for new bioactive substances from microorganisms using the advanced compound-identification system designated as ‘MBJ’s special selection,1,2 with which we have selected over 50 strain culture samples and consequently have succeeded in discovering new cytotoxic eremophilane derivatives MBJ-0009, MBJ-0010 and MBJ-0011, MBJ-0012 and MBJ-0013 from Nectria sp. f261113 and Apiognomonia sp. f24023,1 respectively, and new chaetoglobosin derivatives MBJ-0038, MBJ-0039 and MBJ-0040 from Chaetomium sp. f24230.2 Further screening resulted in the isolation of a new hydroxamate metabolite, designated as MBJ-0003 (1, Figure 1a), from the culture of Micromonospora sp. 29867. In this paper, we described the fermentation, isolation, structure elucidation and brief biological activities of 1. Micromonospora sp. 29867 was isolated from a shellfish collected in Suruga Bay, Shizuoka Prefecture, Japan. The strain was cultivated in 250 ml Erlenmeyer flasks, each containing 25 ml of a medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Association, Hokkaido, Japan), 2% glucose (Junsei Chemical, Tokyo, Japan), 2% soy bean powder (Honen SoyPro, J-Oil Mills, Tokyo, Japan), 0.5% yeast extract powder (Oriental Yeast, Tokyo, Japan), 0.25% NaCl (Junsei Chemical), 0.32% CaCO3 (Wako Pure Chemical Industries, Osaka, Japan), 0.0005% CuSO4 5H2O (Wako Pure Chemical Industries), 0.0005% ZnSO4.7H2O (Wako Pure Chemical Industries) and 0.0005% MnCl2 4H2O (Junsei Chemical). The flasks were placed on a rotary shaker (220 r.p.m.) at 28 1C and incubated for 3 days. Aliquots (0.5 ml) of the broth were then transferred to 500 ml Erlenmeyer flasks containing 50 ml of the same medium and were cultured on a rotary shaker (220 r.p.m.) at 28 1C for 4 days. The whole culture broth (2 l) was extracted with an equal volume of n-BuOH. The n-BuOH layer was evaporated in vacuo to dryness and the residue was successively suspended in water (350 ml), then washed with EtOAc (350 ml 3), followed by extraction with n-BuOH (300 ml 2). The n-BuOH layer was evaporated and the extract (2.9 g) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-30, size: 60 (29 g), Shoko Scientific, Yokohama, Japan) and developed with a gradient system of n-hexane– EtOAc (0–25% EtOAc over 12 min and was kept at 25% for 3 min, flow rate: 20 ml min–1), followed by the stepwise solvent system of CHCl3–MeOH (100 ml each of 0, 2, 5, 10, 20, 30 and 100% MeOH). The fractions were monitored by using an UPLC-DAD-ELS-MS system. The 100% MeOH-eluted fraction (608 mg) was then chromatographed by reversed-phase medium-pressure liquid chromatography (Purif-Pak ODS-30, size: 60 (30 g), Shoko Scientific) with the developing solvent of the H2O–MeOH–CHCl3 system (100 ml each of 60, 70, 80 and 100% aqueous MeOH and CHCl3/MeOH1⁄4 1:1). The CHCl3/MeOH eluate (214 mg) was subjected to preparative HPLC on a CAPCELL PAK C18 MGII column (5.0mm, 20 i.d. 150 mm; Shiseido, Tokyo, Japan) with 55% aqueous MeCN containing 0.1% formic acid (flow rate: 10 ml min–1) to give semi-purified 1 (7.9 mg; retention time: 9.0 min). Final purification was carried out by preparative HPLC on an XSelect CSH C18 column (20 i.d. 150 mm; Waters, Milford, MA, USA) with 40% aqueous MeCN containing 0.1% formic acid (flow rate: 10 ml min–1) to afford 1 (1.7 mg; retention time: 16.3 min). 1 was found to be an optically inactive compound; a colorless amorphous solid with UV end and IR (ATR) nmax 3300 and 1650 cm 1 (hydroxy and carbonyl). The molecular formula was established as C37H72N6O8 by negative mode high-resolution electrospray ionization mass spectrometry (m/z 727.5353 [M–H]–, calcd for C37H71N6O8 727.5333). The direct connectivity between protons and carbons was established by a heteronuclear single-quantum coherence spectrum; the tabulated 13C and 1H NMR spectroscopic data for 1 are listed in Table 1. The structure determination of 1 was carried out through analysis of DQF-COSY and constant-time heteronuclear multiple bond correlation (CT-HMBC)4 data as described below. DQF-COSY and CT-HMBC spectra revealed the presence of seven fragments (A to G) as follows (Figure 1b). A cadaverine moiety (fragment C) was revealed by the 1H spin couplings between aliphatic