Masumi Izawa

Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis

ABSTRACT Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans.

MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea

In many eukaryotic organisms, the non-homologous end-joining (NHEJ) system is a major pathway for the repair of DNA double-strand breaks (DSBs). DNA ligase IV is a component of the NHEJ system and is strictly required for the NHEJ system in Saccharomyces cerevisiae and in Neurospora crassa. To investigate the functions of DNA Ligase IV in Magnaporthe grisea, we generated deletion mutants of MGLIG4, which encodes a homolog of N. crassa DNA Ligase IV. Mutants (mglig4) showed no defects in asexual or sexual growth, and were fully pathogenic. Compared to the wild-type, mglig4 exhibited weak sensitivity to a DNA-damaging agent, camptothecin. In addition, the frequency of targeted-gene replacement was relatively elevated in mglig4, although this varied in a gene-dependent manner. Surprisingly, non-homologous integration of DNA was frequently observed in mglig4 transformants. Our results demonstrate that MgLig4 is involved in, but not essential for, the NHEJ system in M. grisea.

Mycolic acid-containing bacteria activate heterologous secondary metabolite expression in Streptomyces lividans

Mycolic acid-containing bacteria activate heterologous secondary metabolite expression in Streptomyces lividans

4-O-Acetylation and 3-O-Acetylation of Trichothecenes by Trichothecene 15-O-Acetyltransferase Encoded by Fusarium Tri3

In the biosynthesis of Fusarium trichothecenes, the C-3 hydroxyl group of isotrichodermol must be acetylated by TRI101 for subsequent pathway genes to function. Despite the importance of this 3-O-acetylation step in biosynthesis, Tri101 is both physically and evolutionarily unrelated to other Tri genes in the trichothecene gene cluster. To gain insight into the evolutionary history of the cluster, we purified recombinant TRI3 (rTRI3), one of the two cluster gene-encoded trichothecene O-acetyltransferases, and examined to determine whether this 15-O-acetyltransferase can add an acetyl to the C-3 hydroxyl group of isotrichodermol. When a high concentration of rTRI3 was used in the assay (final concentration, 50 μM), we observed 3-O-acetylation activity against isotrichodermol that was more than 105 times less efficient than the known 15-O-acetylation activity against 15-deacetylcalonectrin. The rTRI3 protein also exhibited 4-O-acetylation activity when nivalenol was used as a substrate; in addition to 15-acetylnivalenol, di-acetylated derivatives, 4,15-diacetylnivalenol, and, to a lesser extent, 3,15-diacetylnivalenol, were also detected at high enzyme concentrations. The significance of the trace trichothecene 3-O-acetyltransferase activity detected in rTRI3 is discussed in relation to the evolution of the trichothecene gene cluster.

Indanostatin, a new neuroprotective compound from Streptomyces sp.

Cerebral ischemia is a leading cause of death and long-term disabilities worldwide. In brain ischemia, blood flow disruptions limit the supply of oxygen and glucose to neurons, initiating excitotoxic events. These include activation of glutamate receptors and release of excess glutamate. They induce neuron depolarization and significant increase of intracellular calcium, which activates multiple intracellular death pathways.1 Accumulation of extracellular glutamate also inhibits cystine-glutamate exchanger, resulting in depletion of the intracellular antioxidant glutathione.2,3 In such conditions, reactive oxygen species are generated and implicated in neuronal cell death.4 C6 glioma cells were originally derived from rat neural tumors induced by N-nitrosomethylurea.5 The cells still maintain the characteristics of glial cells and undergo cell death when exposed to glutamate. Thus, they provide a good model for evaluating neuroprotective activity against glutamate toxicity. Several chemicals including antioxidants have been reported to inhibit glutamateinduced cytotoxicity in C6 cells.6–8 In this study, microbial metabolites were screened to find neuroprotective compounds against glutamate toxicity. As a result, a new active compound designated indanostatin (1) was isolated from the cultured broth of Streptomyces sp. RAI20. The molecular formula of indanostatin (1) was determined to be C13H12O6 by high-resolution ESI-MS. 13C and 1H NMR data for 1 are summarized in Table 1. All one-bond 1H–13C connectivities were confirmed by the HMQC9 spectrum. An HMBC10 experiment revealed 1H–13C long-range correlations from two phenolic hydroxy protons (4-OH and 7-OH) to a couple of three aromatic carbons (C-3a, C-4, C-5; C-6, C-7, C-7a), from a singlet methyl (H3-11) to C-4, C-5 and C-6, and from an aromatic proton (H-6) to C-4, C-7 and C-7a, which constructed a 2-methylhydroquinone ring (Figure 1). Long-range couplings from 2-OH to C-1, C-2, C-3 and C-8 connected a hydroxylated quaternary carbon (C-2) to C-1, C-3 and C-8. The existence of a 2-oxopropyl group on C-2 was required by HMBCs from H2-8 and H3-10 to C-9. A four-bond correlation between H-6 and C-1 joined the two partial structures to identify the structure of 1 as 4,7-dihydroxy-5-methyl-2-(2-oxopropyl)indan-1,3dione. The indan skeleton is rarely found in natural products. As 1,3-indanone-containing metabolites, peltatone A11 and caribbazoins A and B12 have been isolated from an insectivorous plant (Drosera peltata var. lunata) and a sponge (Cliona caribboea), respectively. To our knowledge, this is the first reported 1,3-indanone from bacteria. The neuroprotective activity of indanostatin (1) was examined by the MTT method using C6 rat glioma cells. When C6 cells were treated with 100 mM glutamate for 24 h, about 80% of the cells underwent cell death. The compound partially protected C6 cells against glutamate toxicity with an EC50 of 130 nM as shown in Figure 2. In this condition, a-tocopherol inhibited the glutamateinduced cytotoxicity with an EC50 of 47 nM. Indanostatin, however, exhibited no protective activity on N18-RE-105 rat primary retinamouse neuroblastoma hybrid cells13,14 at less than 40mM. Further biological studies are now under way.

Identification of a prodigiosin cyclization gene in the roseophilin producer and production of a new cyclized prodigiosin in a heterologous host.

The prodigiosins are reddish-colored tripyrrole antibiotics biosynthesized by several microorganisms such as Serratia,1 Pseudomonas2 and Streptomyces.3 Streptomyces griseoviridis 2464-S5 produces prodigiosin R14 and roseophilin,5 a unique prodigiosin-related compound containing two pyrrole and one furan moieties (Figure 1), and carries the rph gene cluster involved in their biosynthesis.6 Prodigiosin R1 and roseophilin possess an alkyl chain with the same length and differ in the cyclization pattern (Figure 1). Twenty-one of 25 genes in the rph cluster are homologous to red genes in Streptomyces coelicolor A3(2),7 which produces undecylprodiginine3 and butyl-meta-cycloheptylprodiginine (streptorubin B).8 In the rph cluster, rphG, rphG2, rphG3 and rphG4 showed sufficient homology to redG, a gene involved in cyclization of undecylprodiginine to butyl-meta-cycloheptylprodiginine.9 Thus, we attempted to analyze the functions of these rph genes using a heterologous expression system in S. coelicolor.

Functional analysis of hatomarubigin biosynthesis genes and production of a new hatomarubigin using a heterologous expression system

The function of hatomarubigin biosynthesis genes was analyzed by heterologous expression of the hrb gene cluster. Streptomyces lividans carrying a gene cluster consisting of 25 genes (hrbR1–hrbX) with hrbY was found to produce all the known hatomarubigins including hatomarubigin D, which has a unique dimeric angucycline with a methylene linkage. Gene disruption was used in this heterologous expression system to analyze the function of hrbF, a gene with no homology to any known angucycline biosynthesis genes. A new metabolite was detected in the fermented broth of S. lividans expressing the hrb genes lacking hrbF and was designated hatomarubigin F. This compound was identified as 5-hydroxyhatomarubigin E by NMR spectroscopic analysis, suggesting that HrbF regulates the regiospecificity of oxygenation enzymes.

Curromycin A as a GRP78 downregulator and a new cyclic dipeptide from Streptomyces sp.

Solid cancers are often nutrient-starved because of poor blood circulation and such tumor cells show remarkable tolerance to nutrient deprivation.1 Thus, antitumor substances targeting resistance to nutrient deprivation may show selective activity against solid cancer cells. In the course of a screening for selective cytotoxic compounds under nutrient-deprived conditions, an actinomycete strain RAI364 was found to produce an active substance. Strain RAI364 was identified as a member of the genus Streptomyces based on the 16S rRNA sequence,2 which revealed high identity with those of Streptomyces cuspidosporus NBRC 12378 (99.3%), Streptomyces cuspidosporus NBRC 12379 (99.3%) and Streptomyces ribosidificus NBRC 13796 (99.3%). The producing organism was cultivated in 500-ml Erlenmeyer flasks containing 100ml of a medium consisting of 2.5% glucose, 1.5% soybean meal (Nisshin Oillio Group, Tokyo, Japan), 0.2% dry yeast (Asahi Food & Healthcare, Tokyo, Japan) and 0.4% calcium carbonate (pH 6.2, before autoclave) on a rotary shaker at 27 °C for 4 days. The culture broth (2 l) was centrifuged and the supernatant was extracted with ethyl acetate. The extract was fractionated on a silica gel column using stepwise elution with chloroform-methanol. The active material eluted with chloroform-methanol (20:1) was subjected to preparative silica gel TLC with chloroform-methanol (10:1). The active fraction was concentrated to dryness to give a colorless powder of 1 (45.4 mg). From the 1H and 13C NMR spectra and the specific rotation ([α]D +23.4 (c 0.567, MeOH) and [α]D+39.0 (c 0.115, MeOH) in literature3), 1 was identified as curromycin A3,4 (Figure 1). The chloroform-methanol (100:1) fraction on the silica gel column contained an inactive metabolite 2. A colorless oil of 2 (12.8mg) was obtained by preparative silica gel TLC with chloroform-methanol (10:1). The physicochemical properties of 2 are summarized as follows: [α]D 19− 13.9 (c 0.642, MeOH); high-resolution FAB-MS m/z 199.1082 ([M+H]+, calcd. for C9H15N2O3, 199.1083); UV (MeOH) λmax nm (ε) 219 (3400); IR (ATR) νmax 3207, 1690 cm − 1. The molecular formula of 2 was established as C9H14N2O3 by highresolution FAB-MS. 13C and 1H NMR data for 2 are summarized in Table 1. The HMQC5 spectrum revealed three methyls, including a methoxy and an N-methyl, an olefinic methylene, two methines and three quaternary sp2 carbons. Two amide carbonyl groups were identified based on their chemical shifts (δ 165.8, 159.5) and the molecular formula. A COSY experiment displayed a spin network from H-6 to H-10 (Figure 2). This substructure was expanded to include the amide carbonyl (C-5) due to long-range correlations from 6-H and 9-H to C-5 in the HMBC6 spectrum (Figure 2). 1H–13C longrange couplings from the methoxy to C-9 and from the N-methyl to C-2 and C-6 established their location and an amide bond between C-2 and C-6. The olefinic methylene protons (8-H2) exhibited long-range correlations to C-2, indicating the presence of an acrylamide moiety. Finally, couplings from an amide proton (4-H) to C-2 and C-6 required a cyclic dipeptide structure consisting of dehydroalanine and N,O-dimethylthreonine (or its stereoisomer) residues. A small coupling constant (4.0Hz) between 6-H and 9-H arranged them in gauche conformations. The structure of 2, including the relative stereochemistry, was determined to be cyclo(dehydroalanyl-N, O-dimethylthreonyl) by a NOESY experiment, which revealed NOEs between 11-H3 and 7-H3, between 7-H3 and 10-H3, and between 10-H3 and 6-H, as shown in Figure 2. The cytotoxic activity was evaluated by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) method using MKN45 human gastric cancer cells1 treated with 1 or 2 for 48 h. MTT formazan formed in the cells was dissolved in dimethyl sulfoxide (DMSO) and measured as absorbance at 540 nm. Curromycin A (1) showed potent cytotoxicity against MKN45 cells in a nutrient-deprived medium (Earles salts solution supplemented with 10% fetal bovine serum), with an IC50 of 15 ngml − 1. The growth of MKN45 cells in a normal medium (Dulbeccos modified Eagles medium with 10% fetal bovine serum) was inhibited ~ 50% by 1 at the range of 20–20 000 ngml 1, although no cell death was observed. Anticancer agents such as doxorubicin displayed reverse selectivity in this assay. The IC50 values of doxorubicin were 1.1 μgml 1 in a normal medium and 420 μg ml 1 in a nutrient-deprived medium. No activity was observed on MKN45 cells treated with 2 at o10 μg ml 1.

Actinopyrone D, a new downregulator of the molecular chaperone GRP78 from Streptomyces sp.

A new downregulator of the molecular chaperone GRP78, actinopyrone D, was isolated together with a known related compound, PM050463, from Streptomyces sp. RAG92. The molecular formula of actinopyrone D was established as C25H36O4 by high-resolution FAB-MS. NMR spectroscopic analysis revealed the structure of actinopyrone D, which consists of an α-methoxy-γ-pyrone ring and a C17 side chain containing a cis olefin moiety. Actinopyrone D and PM050463 dose-dependently inhibited 2-deoxyglucose-induced luciferase expression in HT1080 human fibrosarcoma cells transfected with a luciferase reporter plasmid containing the GRP78 promoter. Actinopyrone D inhibited GRP78 protein expression and induced cell death under endoplasmic reticulum stress.

Increased metabolite production by deletion of an HDA1-type histone deacetylase in the phytopathogenic fungi, Magnaporthe oryzae (Pyricularia oryzae) and Fusarium asiaticum

Histone deacetylases (HDACs) play an important role in the regulation of chromatin structure and gene expression. We found that dark pigmentation of Magnaporthe oryzae (anamorph Pyricularia oryzae) ΔMohda1, a mutant strain in which an orthologue of the yeast HDA1 was disrupted by double cross‐over homologous recombination, was significantly stimulated in liquid culture. Analysis of metabolites in a ΔMohda1 mutant culture revealed that the accumulation of shunt products of the 1,8‐dihydroxynaphthalene melanin and ergosterol pathways were significantly enhanced compared to the wild‐type strain. Northern blot analysis of the ΔMohda1 mutant revealed transcriptional activation of three melanin genes that are dispersed throughout the genome of M. oryzae. The effect of deletion of the yeast HDA1 orthologue was also observed in Fusarium asiaticum from the Fusarium graminearum species complex; the HDF2 deletion mutant produced increased levels of nivalenol‐type trichothecenes. These results suggest that histone modification via HDA1‐type HDAC regulates the production of natural products in filamentous fungi.