Chong-Xi Liu

Genome Sequence of the Milbemycin-Producing Bacterium Streptomyces bingchenggensis

Streptomyces bingchenggensis is a soil-dwelling bacterium producing the commercially important anthelmintic macrolide milbemycins. Besides milbemycins, the insecticidal polyether antibiotic nanchangmycin and some other antibiotics have also been isolated from this strain. Here we report the complete genome sequence of S. bingchenggensis. The availability of the genome sequence of S. bingchenggensis should enable us to understand the biosynthesis of these structurally intricate antibiotics better and facilitate rational improvement of this strain to increase their titers.

A new quinoline derivative with cytotoxic activity from Streptomyces sp. neau50

A new quinoline derivative, methyl 8-(3-methoxy-3-methylbutyl)-2-methylquinoline-4-carboxylate (1), was isolated from the endophytic strain Streptomyces sp. neau50, and the structure was elucidated by extensive spectroscopic analysis. Compound 1 showed cytotoxicity against human lung adenocarcinoma cell line A549 with an IC(50) value of 29.3 μg mL(-1).

Micromonospora zeae sp. nov., a novel endophytic actinomycete isolated from corn root (Zea mays L.).

A novel actinomycete, designated strain NEAU-gq9T, was isolated from corn root (Zea mays L.) and characterized using a polyphasic approach. The organism was found to have morphological and chemotaxonomic characteristics typical of the genus Micromonospora. On the basis of 16S rRNA gene sequence similarity studies, strain NEAU-gq9T was most closely related to Micromonospora zamorensis CR38T (99.3%), Micromonospora jinlongensis NEAU-GRX11T (99.2%), Micromonospora saelicesensis Lupac 09T (99.2%), Micromonospora chokoriensis 2-19(6)T (98.9%), Micromonospora coxensis 2-30-b(28)T (98.6%) and Micromonospora lupini Lupac 14NT (98.5%). Phylogenetic analysis based on the 16S rRNA gene and gyrB gene demonstrated that strain NEAU-gq9T is a member of the genus Micromonospora and supported the closest phylogenetic relationship to M. zamorensis CR38T, M. jinlongensis NEAU-GRX11T, M. saelicesensis Lupac 09T, M. chokoriensis 2-19(6)T and M. lupini Lupac 14NT. A combination of DNA–DNA hybridization, morphological and physiological characteristics indicated that the novel strain could be readily distinguished from the closest phylogenetic relatives. Therefore, it is proposed that strain NEAU-gq9T represents a novel species of the genus Micromonospora, for which the name Micromonospora zeae sp. nov. is proposed. The type strain is NEAU-gq9T (=CGMCC 4.7092T=DSM 45882T).

A novel macrocyclic lactone with insecticidal bioactivity from Streptomyces microflavus neau3.

A novel macrocyclic lactone (1) was isolated from the fermentation broth of Streptomycesmicroflavus neau3, and the structure was elucidated by extensive spectroscopic analysis. Compound 1 showed high acaricidal activity against adult mites (IC(50)=11.1 μg mL(-1)), and nematocidal activity against Caenorhabditis elegans (IC(50)=17.4 μg mL(-1)), especially the acaricidal activity against mite eggs with an IC(50) of 37.1 μg mL(-1), which was relative higher than that of the commercial acaricide and nematocide milbemycins A(3)/A(4).

Four New Doramectin Congeners with Acaricidal and Insecticidal Activity from Streptomyces avermitilis NEAU1069

Four new doramectin congeners, 1–4, were isolated from Streptomyces avermitilis NEAU1069. The structures of 1–4 were elucidated on the basis of spectroscopic analysis, including 1D‐ and 2D‐NMR as well as HR‐ESI‐MS, ESI‐MS, UV, and IR, and comparison with literature data. All compounds exhibited noticeable acaricidal and insecticidal activities. Especially compound 2 was found to be the most potent pesticide of the compounds evaluated with the IC50 values of 10.2, 65.1 and 124.4u2005μg/ml against adult two‐spotted spider mites (Tetranychus urticae Koch), two‐spotted spider mite eggs, and Mythimna separata, respectively, which are comparable to those of commercial pesticide milbemycin A3/A4 as positive reference.

Diphenyl etheric metabolites from Streptomyces sp. neau50

Natural products are among the most important resources of the clinically used cancer chemotherapeutic agents. Many compounds with anticancer properties have been isolated from natural sources since actinomycin was discovered in 1940’s. More than 60% of the currently known compounds with anti-neoplasic activity are natural products or their derivatives.1–6 As part of the search for new and biologically active secondary metabolites produced by actinomyces from unexplored and underexplored ecological niches, an endophytic actinomycete Streptomyces sp. neau50 was isolated from healthy root of soybean. Initial screening of the crude extract exhibited cytotoxicity against certain cancer cell lines and inhibitory activity against phytopathogenic fungi. Subsequent isolation resulted in borrelidin as the major active component. To further investigate minor amount of active constituents in this strain, the fermentation was scaled up, and detailed fractionation of the crude extract led to the isolation of a novel diphenyl ether compound, methyl 2-hydroxy-4-(2-hydroy-3methoxy-5-methylphenoxy)-6-methylbenzoate (1), together with a known one, methyl 2-hydroxy-4-(3-hydroxy-5-methylphenoxy)-6methylbenzoate (2). In this paper, we describe the isolation, structure elucidation and bioactivities of 1 and 2. The producing strain, Streptomyces sp. neau50, was isolated by moist incubation and desiccation method from healthy soybean root in Harbin, Heilongjiang province, China.7 The 16S rRNA was sequenced for taxonomic classification (GenBank Accession No: GQ 494994). The strain was maintained in the medium containing 10 g glucose, 3 g maltose, 3 g yeast extract, 0.5 g K2HPO4 3H2O, 0.5 g MgSO4 7H2O, 0.5 g NaCl, 1 g KNO3 and 20 g agar in 1.0 l tap water, pH 7.0. Slant culture was incubated for 6–7 days at 281C. The seed medium consisted of 4 g glucose, 10 g maltodextrin, 4 g yeast extract, 2 g CaCO3 in 1.0 l water, pH 7.2–7.4. All the media were sterilized at 1211C for 20 min. Fermentation was carried out in a 50-l first seed fermentor (containing 30 l seed culture) and a 500-l second fermentor (containing 300 l producing medium), successively. The producing medium comprised 1% glucose, 4% soluble amylum, 0.5% yeast extract, 2.5% soybean powder, 0.5% peptone, 0.2% CaCO3, 0.8% MgSO4 7H2O, 0.6% FeSO4 7H2O, 0.2% ZnSO4 7H2O, 0.2% MnSO4 H2O, 0.05% CoCl2 6H2O, 0.2% Na2MoO4 2H2O, pH 7.0. The fermentation was performed at 281C for 7 days stirred at 100 r min 1 with an aeration rate of 30 m3 of air per hour. The final 300 l of fermentation broth was filtered to afford the mycelial cake. After it was washed with water, the mycelia was extracted twice with 100 l of EtOH for about 24 h. The EtOH extract was diluted to about 30% EtOH and subjected to a Diaion HP-20 resin column, which was eluted with 30, 40, 50, 60, 70 and 80% EtOH (each concentration eluted 2 bed volumes). The eluents at 70 and 80% EtOH were pooled and concentrated in vacuo at 501C. Then part of the concentrated material (32 g) was chromatographed on a silica gel column, and successively eluted with a stepwise gradient of petroleum ether–acetone (100:0–50:50, v/v) to obtain five fractions (fractions I–V) based on the TLC profiles. Fraction III was chromatographed on another silica gel column and eluted with petroleum ether–acetone (90:10–70:30, v/v) to give three subfractions. Subfraction II was then subjected to a Sephadex LH-20 column (GE Healthcare, Glies, UK) and eluted with EtOH to obtain fractions A and B. Finally, fraction B was fractionated by semi-preparative HPLC with column Zorbax SBC18 (5mm, 250 9.4 mm) on Agilent 1100 system (Agilent, Palo Alto, CA, USA). It was eluted with CH3OH–H2O (85:15, v/v) at 1.5 ml min 1. Two main compounds 1 (tR 11.8 min, 8 mg) and 2 (tR 12.7 min, 11 mg) (Figure 1) were isolated. Compound 1 was obtained as colorless oil with UV (EtOH) lmax nm (log e): 211 (4.29), 264 (3.92), 299 (3.52). The absorption peaks in the IR spectrum of 1 suggested the presence of hydroxyl

Neaumycin - A New Macrolide from Streptomyces sp. NEAU-x211

Neaumycin, a new 30-membered macrolide featuring an internal diester bridge, a molecular architecture that is unprecedented among known macrolide natural products, was isolated from a soil actinomycete strain Streptomyces sp. NEAU-x211. The structure of neaumycin was elucidated on the basis of comprehensive mass and NMR spectroscopic interpretation, including the relative stereochemistry of four independent coupling systems.

Micromonospora lycii sp. nov., a novel endophytic actinomycete isolated from wolfberry root (Lycium chinense Mill).

A novel actinomycete, designated strain NEAU-gq11T, was isolated from wolfberry root (Lycium chinense Mill) and characterized using a polyphasic approach. Strain NEAU-gq11T was observed to form rough-surfaced spores that borne singly on the substrate hyphae but aerial mycelia were not developed. The organism showed closest 16S rRNA gene sequence similarity to Micromonospora saelicesensis Lupac 09T (99.4%), and phylogenetically clustered with M. chokoriensis 2–19/6T (99.3%), ‘M. zeae’ NEAU-gq9T (99.3%), M. violae NEAU-zh8T (99.1%), ‘M. jinlongensis’ NEAU-GRX11T (99.1%), M. zamorensis CR38T (99.0%), M. taraxaci NEAU-P5T (98.9%) and M. lupini Lupac 14NT (98.6%). Phylogenetic analysis based on the gyrB gene sequences also indicated that the isolate clustered with the above type strains except M. violae NEAU-zh8T. The cell-wall peptidoglycan consisted of meso-diaminopimelic acid and glycine. The major menaquinones were MK-9(H8), MK-10(H6) and MK-10(H2). The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The major fatty acid was iso-C16:0. Furthermore, some physiological and biochemical properties and low DNA–DNA relatedness values enabled the strain to be differentiated from members of closely related species. Therefore, it is proposed that strain NEAU-gq11T represents a novel species of the genus Micromonospora, for which the name Micromonospora lycii sp. nov. is proposed. The type strain is NEAU-gq11T (=CGMCC 4.7149T=DSM 46703T).

New milbemycins from mutant Streptomyces bingchenggensis X-4

Streptomyces bingchenggensis produced milbemycins, including a1 (A3), a3 (A4), b13, b14, a28, a29, a30 and ST 906, four secomilbemycins A, B, C and D, and two cyclic pentapeptides.1–5 Milbemycins belong to a 16-membered macrolide antibiotic with an outstanding activity against various kinds of mites.6 During a screening program for high production of A3 and A4, a mutant S. bingchenggensis X-4 was obtained by UV treated, N-methyl-N¢nitroso-N-nitroso-guanidine mutation and genetic manipulative techniques. Significant differences of phenotype, such as the morphology of aerial mycelia, and the metabolite HPLC profiles were observed between the wild-type S. bingchenggensis and its mutant strain X-4. In the course of investigating the metabolites of this mutant strain, three new interesting compounds milbemycin b15 (1), seco-milbemycins E (2) and F (3) were isolated from the fermentation broth of S. bingchenggensis X-4. The structure of compound 1 was similar with milbemycin D, which is a highly selective and potent nematocide and insecticide.7 So the bioactivity of compound 1 should be further investigated. Compared with the seco-milbemycins isolated previously,2,3 the hydroxy groups at C-5 were absent in compounds 2 and 3. Furthermore, all the milbemycins3,8 and avermectins9,10 obtained from microorganisms contain the hydroxyl at C-5, and the 5-dehydroxyl derivatives of milbemycins and avermectins can only be obtained using the synthetic methods.11 So seco-milbemycins E and F may have an important role in understanding and perfecting the proposed biosynthesis pathways of milbemycins. The producing strain S. bingchenggensis X-4 was maintained on an 1/2YM slant agar consisting of sucrose 0.4%, skim milk 0.1%, yeast extract (OXOID Basingstoke, Hampshire, UK) 0.2%, malt extract (BD Biosciences, San Jose, CA, USA) 0.5%, agar (BD Biosciences) 2.0% at 28 1C for 12 days.12 A seed 15-l fermentor (FUS-15 L (A), Shanghai Guoqiang Bioengineering Equipment, Shanghai, China) containing 10 l of seed medium (sucrose 1.0%, polypepton 0.2%, K2HPO4 0.05%, skim milk 0.05%)12 was inoculated with 0.5 l of broth cultured in flask with seed medium. The flask with seed medium (2–6 107 spores per ml) was cultured for 30 h at 28 1C on a rotary shake at 250 r.p.m. After incubation for 32 h, the seed broth (3 l) in the 15-l fermentor was transferred into the production 50-l fermentor (FUS-50 L (A), Shanghai Guoqiang Bioengineering Equipment) containing 30-l production medium (16.0% sucrose, 2.0% soybean powder, 0.5% yeast extract, 0.5% meat extract, 0.05% K2HPO4, 0.05% MgSO4 7H2O, 0.005% FeSO4 7H2O and 0.3% CaCO3). 12 The culture temperature was 28 1C and the initial pH was 7.40 sterilized by sparging with steam at 121 1C for 30 min. The dissolved oxygen was maintained above 35% by adjusting the agitation speed. The initial aeration and agitation rate in the 15-l reactor was 1 vvm and 180 r.p.m., whereas those in the 50 l were 0.8 vvm and 150 r.p.m., respectively. In the process of fermentation, the pH was not controlled. The fermentation was performed for 10 days at 28 1C. The fermentation broth (30 l) was filtered. The resulting cake was washed with water, and both filtrate and wash were discarded. Methanol (10 l) was used to extract the washed cake. The MeOH extract was evaporated under reduced pressure to B2 l at 45 1C and the resulting concentrate was extracted three times using an equal volume of EtOAc. The combined EtOAc phase was concentrated under reduced pressure to yield 30 g of oily substances. The residual oily substance was chromatographed on silica gel (Qingdao Haiyang Chemical Group, Qingdao, China; 100–200 mesh) and eluted with a petroleum ether–acetone mixture (100:0–50:50, v/v). The fractions eluted with the petroleum ether–acetone mixture (90:10, v/v) were combined and evaporated to obtain fraction I and the fractions eluted with the petroleum ether–acetone mixture (85:15, v/v) were pooled and concentrated to give fraction II. The fraction I was subjected to Sephadex LH-20 (GE Healthcare, Glies, UK) gel column eluting with MeOH to give subfraction I. The semi-preparative HPLC (Agilent 1100, Zorbax SB-C18, 5mm, 250 9.4 mm i.d.; Agilent, Palo Alto, CA, USA) was applied to obtain pure compounds. The eluates were monitored using a photodiode array detector at 220 nm, and the flow rates were 1.5 ml min 1 at room temperature. The subfraction I was further separated by semi-preparative HPLC using a solvent containing a CH3OH–H2O mixture (95:5, v/v) to obtain compounds

New cytotoxic spectinabilin derivative from ant-associated Streptomyces sp. 1H-GS5

Insects are the most abundant and diverse animal class on earth and they are associated with an amazing variety of microorganisms. The ability of insects to live in unique niche habitats is often facilitated by their associated microbe,1 which perform many important host functions, especially against pathogens, parasitoids or predators by producing metabolites2,3 and the enormous insect-associated microbial communities are a potential source of novel antibiotic metabolites.4,5 To find more insect-associated microorganisms-derived bioactive metabolites, the study of the carpenter ants-associated microorganisms was carried out and a strain Streptomyces sp. 1H-GS5 exhibiting good activities against some lines of tumor cell was obtained. In the further investigation of chemical components from the broth of Streptomyces sp. 1H-GS5, a new spectinabilin derivative (1) along with the known compound, spectinabilin (2) (Figure 1), were isolated. Here the report deals with the details of the isolation, structure elucidation and the bioactivity of the new compound. The strain Streptomyces sp. 1H-GS5 was isolated from the head of ant (Camponotus japonicas Mayr) collected from the Northeast Agricultural University, Harbin of Heilongjiang province, China. The strain was identified as the genus Streptomyces because its 16S rRNA sequence (accession no: KP784764 in the GenBank, National Center for Biotechnology Information) exhibited a high-sequence similarity of 99% with that of Streptomyces spectabilis NBRC 13424 (accession no: AB184393). The strain Streptomyces sp. 1H-GS5 has been deposited in the Pharmaceutical Research Culture Collection, Zhejiang Hisun Group Co., Ltd, with accession no: HS-HX-087. Strain was maintained on a YMS medium containing soluble starch (Bei Jing Ao Bo Xing, Beijing, China) 10 g, yeast extract (Bei Jing Ao Bo Xing) 2 g, KNO3 1 g and agar 20 g in 1.0 l tap water, pH 7.0. The seed medium consisted of glucose (Bei Jing Ao Bo Xing) 20 g, soybean flour (Cormwin, Beijing, China) 15 g and yeast autolysate (Bei Jing Ao Bo Xing) 5.0 g in 1.0 l tap water, pH 7.0. Both the media were sterilized at 121 °C for 20min. Slant cultures were incubated for 6–7 days at 28 °C. A total of 10ml of sterile water was added to the slant of the YMS medium. The spores were scraped and transferred onto a sterile tube containing glass beads; the spore suspension was then filtered through six layers of a sterile filter cheesecloth and adjusted to 107–108 c.f.u. ml 1. A 2.0 ml of the spore suspension was inoculated into a 250-ml flask containing 25ml of seed medium and incubated at 28 °C for 24 h, shaken at 250 r.p.m. Then, 8.0 ml of the culture was transferred into 1 liter Erlenmeyer flask containing 100ml of the producing medium consisting of corn starch (Cormwin) 10%, soybean powder (Bei Jing Ao Bo Xing) 1%, cotton flour (Cormwin) 1%, α-amylase (Bei Jing Ao Bo Xing) 0.02%, NaCl 0.1%, K2HPO4 0.2%, MgSO4·7H2O 0.1%, CaCO3 0.7%, cyclohexanecarboxylic acid 0.1%, pH 7.0, before sterilization. Fermentation was carried out at 28 ° C for 7 days on a rotary shaker at 250 r.p.m. The final 30 l of broth from 300 producing fermentations was filtered. The resulting cake was washed with water. Both the supernatant and the wash water were discarded. Methanol (6 l) was used to extract the washed cake and the MeOH extract was evaporated under reduced pressure to 1 liter at 50 °C and the resulting concentrate was extracted three times using an equal volume of EtOAc. The combined organic layer was concentrated to give a yellow oily residue (14 g). The yellow oily residue was subsequently subjected to silica gel column chromatography (Qingdao Haiyang Chemical Group, Qingdao, China; 100–200 mesh) using an n-hexane-EtOAc stepwise system (100:0–50:50, v/v) to obtained four fractions (fractions 1–4) based on the TLC profiles. The fraction 2 was subjected to Sephadex LH-20 (GE Healthcare, Glies, UK) gel column eluting with CHCl3/MeOH (1:1, v/v) to give fraction 2–1. The fraction 2–1 was further separated by semi-preparative HPLC (Agilent 1100, Zorbax SB-C18, 5 μm, 250× 9.4 mm inner diameter; Agilent, Palo Alto, CA, USA) using CH3OH/H2O mixture (9:1, v/v) with the flow rates of 1.5 ml min − 1 at