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A novel actinomycete, designated strain NEAU-SH16(T) was isolated from a soil sample collected from the riverbank of Wusong river in Shanghai and characterized using a polyphasic approach. Morphological and chemotaxonomic properties of strain NEAU-SH16(T) were consistent with the description of the genus Catellatospora. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain NEAU-SH16(T) formed a distinct branch with the closest relatives Catellatospora coxensis DSM 44901(T) (99.2 %) and Catellatospora citrea DSM 44097(T) (99.0 %), an association that was supported by a bootstrap value of 78 % in the neighbour-joining tree and also recovered with the maximum-likelihood algorithm. However, the low level of DNA-DNA relatedness and some different physiological characteristics allowed the strain to be distinguished from its closest relatives. Thus, it is proposed that strain NEAU-SH16(T) represents a novel Catellatospora species. Catellatospora aurea sp. nov. The type strain of Catellatospora aurea is NEAU-SH16(T) (=CGMCC 4.7147(T) = DSM 46719(T)).

A novel actinobacterium, designated strain NEAU-ML12(T), was isolated from a millipede (Kronopolites svenhedind Verhoeff), which was collected from Fenghuang Mountain in Wuchang, Heilongjiang Province, north China. The strain was characterized using a polyphasic approach. Strain NEAU-ML12(T) was found to have morphological and chemotaxonomic characteristics typical of the members of the genus Rhodococcus. 16S rRNA gene sequence similarity analysis showed that the strain NEAU-ML12(T) belongs to the genus Rhodococcus, and was most closely related to Rhodococcus tukisamuensis Mb8(T) (98.9 %) and Rhodococcus koreensis DNP505(T) (97.7 %). Phylogenetic analysis based on 16S rRNA gene sequences also demonstrated that strain NEAU-ML12(T) should be classified in the genus Rhodococcus, forming a distinct clade with R. tukisamuensis Mb8(T) supported by a 99 % bootstrap value. However, the DNA-DNA relatedness between strain NEAU-ML12(T) and R. tukisamuensis Mb8(T) was found to be 41.9 ± 0.7 %. Furthermore, strain NEAU-ML12(T) could also be differentiated from R. tukisamuensis Mb8(T) and other closely related strains (R. koreensis DNP505(T) and Rhodococcus maanshanensis M712(T)) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-ML12(T) represents a novel species of the genus Rhodococcus, for which the name Rhodococcus kronopolitis sp. nov. is proposed. The type strain is NEAU-ML12(T) (=CGMCC 4.7145(T) = DSM 46702(T)).

A Gram-stain positive, filamentous bacterial strain, designated strain NEAU-TWSJ13(T), was isolated from the rhizosphere of a marigold (Tagetes erecta L.) plant collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. The strain was observed to form abundant aerial hyphae differentiated into spherical sporangia. 16S rRNA gene sequence similarity studies showed that strain NEAU-TWSJ13(T) belongs to the genus Streptosporangium, being most closely related to Streptosporangium fragile DSM 43847(T) (98.6 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that it formed a phyletic line with S. fragile DSM 43847(T), Streptosporangium jomthongense NBRC 110047(T) (98.4 % 16S rRNA gene similarity) and Streptosporangium violaceochromogenes DSM 43849(T) (97.6 % 16S rRNA gene similarity). A combination of DNA-DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-TWSJ13(T) can be distinguished from S. fragile DSM 43847(T) and S. jomthongense NBRC 110047(T). Moreover, strain NEAU-TWSJ13(T) can also be differentiated from S. violaceochromogenes DSM 43849(T) and other Streptosporangium species showing high 16S rRNA gene sequence similarity (98.0 %) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-TWSJ13(T) represents a novel species of the genus Streptosporangium, for which the name Streptosporangium subfuscum sp. nov. is proposed. The type strain is NEAU-TWSJ13(T) ( = CGMCC 4.7146(T) = DSM = 46724(T)).

Three new cyclopentenone derivatives (1-3) were isolated from the rare actinomycete Actinoalloteichus nanshanensis NEAU 119. Their structures were elucidated by extensive spectroscopic analysis. Compound 1 showed moderate cytotoxic activity against human lung adenocarcinoma cell line A549, human leukemia cell line K562, and human renal carcinoma cell line ACHN with an IC50 of 14.67, 11.87, and 23.36 μg ml(-1), respectively.

Cycloheximide (1) and actiphenol (2) have been isolated from numerous Streptomyces species. Cloning, sequencing, and characterization of a gene cluster from Streptomyces sp. YIM65141 now establish that 1 and 2 production is governed by single biosynthetic machinery. Biosynthesis of 1 features an acyltransferase-less type I polyketide synthase to construct its carbon backbone but may proceed via 2 as a key intermediate, invoking a provocative reduction of a phenol to a cyclohexanone moiety in natural product biosynthesis.

A novel actinomycete, designated strain NEAU-zh8(T), was isolated from a root of Viola philippica Car collected in China and characterized using a polyphasic approach. 16S rRNA gene sequence similarity studies showed that strain NEAU-zh8(T) belongs to the genus Micromonospora, being most closely related to Micromonospora chokoriensis 2-9(6)(T) (99.9 %), Micromonospora saelicesensis Lupac 09(T) (99.3 %) and Micromonospora lupini Lupac 14N(T) (99.0 %). gyrB gene analysis also indicated that strain NEAU-zh8(T) should be assigned to the genus Micromonospora. The cell-wall peptidoglycan consisted of meso-diaminopimelic acid and glycine. The major menaquinones were MK-10(H4), MK-10(H2) and MK-10(H6). The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The major fatty acids were iso-C15:0, C16:0 and C17:0 10-methyl. A combination of DNA-DNA hybridization results and some physiological and biochemical properties indicated that strain NEAU-zh8(T) could be readily distinguished from the closest phylogenetic relatives. Therefore, it is proposed that strain NEAU-zh8(T) represents a novel Micromonospora species, for which the name Micromonospora violae sp. nov. is proposed. The type strain is NEAU-zh8(T) (=CGMCC 4.7102(T)=DSM 45888(T)).

A novel actinomycete, designated strain NEAU-GRX6T, was isolated from mucky soil collected from a stream of Jinlong Mountain in Harbin, Heilongjiang Province, north China, and characterized using a polyphasic approach. The isolate formed irregular sporangia containing motile sporangiospores on the substrate mycelium. The whole-cell sugars were xylose, glucose and galactose. The predominant menaquinones were MK-9(H6), MK-10(H4) and MK-9(H4). The major fatty acids were C16 : 0, C15 : 0, C18 : 1ω9c, C17 : 1ω7c and C18 : 0. The phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol. The DNA G+C content was 67 mol%. 16S rRNA gene sequence similarity studies showed that strain NEAU-GRX6T belonged to the genus Actinoplanes , being most closely related to Actinoplanes palleronii IFO 14916T (97.80 % similarity) and Actinoplanes missouriensis NBRC 102363T (97.76 %). However, the low observed levels of DNA–DNA relatedness allowed the isolate to be differentiated from the above-mentioned species of the genus Actinoplanes . Moreover, strain NEAU-GRX6T could also be distinguished from A. palleronii IFO 14916T and A. missouriensis NBRC 102363T by phenotypic characteristics. Therefore, it is proposed that strain NEAU-GRX6T represents a novel species of the genus Actinoplanes , for which the name Actinoplanes lutulentus sp. nov. is proposed. The type strain is strain NEAU-GRX6T ( = CGMCC 4.7090T = DSM 45883T).

Ubiquitin and ubiquitin-like proteins (Ubls), such as SUMO, are covalently conjugated to their targets by related, but distinct enzymatic conjugation reactions that involve the dynamic E1–E2–E3 enzyme cascade. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, with the help of ATP, to form a covalent thioester bond. Subsequently, Ubls are transferred to E2 to generate a thioester-linked product. In previous studies, we showed the dynamic processes and thioester intermediates of SUMO with its E1 and E2 conjugating enzymes. Studies of the enzyme specificity of the Ubl conjugation cascade are normally carried out by tedious biochemical processes, and the reaction intermediates are often difficult to capture because they are unstable and have short half-lives. Here, using our recently developed robust quantitative FRET-based technology, we describe systematic investigations of enzymatic specificity and thioester intermediate determination of ubiquitin with its E1–E2 ligases in conjugation with SUMO and its ligases. Our technology easily determined the strong specificity of enzyme–substrate interactions and thioester intermediates in ubiquitination and SUMOylation cascades. The traditional FRET pair ECFP/EYFP lacked adequate signals for these assays. However, in contrast, the highly sensitive FRET pair CyPet/YPet was easily harnessed to define the reaction specificities and intermediates. In addition, the thioester intermediates can be readily monitored by a newly defined FRET index parameter. These results provide an example of a systems biology approach to determine Ubl conjugation specificity and demonstrate that a robust FRET technology can be used to identify enzymes and substrates in other Ubl pathways.

A new prenylated indole derivative 3-acetonylidene-7-prenylindolin-2-one (1) was isolated from the endophytic actinobacterium Streptomyces sp. neau-D50, together with four known hybrid isoprenoids, 7-isoprenylindole-3-carboxylic acid (2), 3-cyanomethyl-6-prenylindole (3), 6-isoprenylindole-3-carboxylic acid (4) and 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone (5). The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with data from the literature. Compounds 1 and 2 demonstrated strong cytotoxic activities against human lung adenocarcinoma cell line A549 with an IC50 of 3.3 and 5.1 μg mL− 1, respectively, which are comparable to that of the positive control doxorubicin (4.2 μg mL− 1). Furthermore, compounds 1–4 exhibited potent antifungal activity against phytopathogenic fungi Colletotrichum orbiculare, Phytophthora capsici, Corynespora cassiicola and Fusarium oxysporum.

Streptomyces cyaneogriseus ssp. noncyanogenus is thermotolerant bacterium producing commercially important nemadectin, a kind of macrolide antibiotics, which has been widely used as a biopesticide. Here, we report the complete genome sequence of S. cyaneogriseus ssp. noncyanogenus , which consists of one chromosome (7,762,396 bp) with no plasmid. Genome sequence information contributes to understanding the biosynthesis of nemadectin better and provides the basis for analysis of its thermotolerance and biosynthetical potential.

Streptomyces bingchenggensis is a soil bacterium that produces milbemycins, a family of macrolide antibiotics that are commercially important in crop protection and veterinary medicine. In addition, S. bingchenggensis produces many other natural products including the polyether nanchangmycin and novel cyclic pentapeptides. To identify the gene clusters involved in the biosynthesis of these compounds, and better clarify the biochemical pathways of these gene clusters, the whole genome of S. bingchenggensis was sequenced, and the transcriptome profile was subsequently investigated by microarray. In comparison with other sequenced genomes in Streptomyces, S. bingchenggensis has the largest linear chromosome consisting of 11 936 683 base pairs (bp), with an average GC content of 70.8%. The 10 023 predicted protein-coding sequences include at least 47 gene clusters correlated with the biosynthesis of known or predicted secondary metabolites. Transcriptional analysis demonstrated an extremely high expression level of the milbemycin gene cluster during the entire growth period and a moderately high expression level of the nanchangmycin gene cluster during the initial hours that subsequently decreased. However, other gene clusters appear to be silent. The genome-wide analysis of the secondary metabolite gene clusters in S. bingchenggensis, coupled with transcriptional analysis, will facilitate the rational development of high milbemycins-producing strains as well as the discovery of new natural products.

A novel actinomycete, designated strain NEAU-st4(T), was isolated from a soil sample collected from Shaanxi province, Northwest China and characterized using a polyphasic approach. 16S rRNA gene sequence analysis revealed that strain NEAU-st4(T) has the highest sequence similarities with Actinomycetospora rishiriensis RI109-Li102(T) (99.4 %), Actinomycetospora corticicola 014-5(T) (99.1 %), Actinomycetospora chiangmaiensis YIM 0006(T) (98.8 %) and Actinomycetospora iriomotensis IR73-Li102(T) (98.2 %). However, the low level of DNA-DNA relatedness differentiated strain NEAU-st4(T) from its closest phylogenetic neighbours. The main chemotaxonomic properties of strain NEAU-st4(T), such as the diamino acid of the peptidoglycan, the whole-cell hydrolysates, the predominant menaquinones and the phospholipid profile, supported its classification within the genus Actinomycetospora. The distinctive morphology of this strain compared with that of other members in the genus Actinomycetospora is the formation of sporangia directly on the substrate hyphae. Phenotypic and genotypic differences also allowed the distinction of the strain from closely related species. Consequently, strain NEAU-st4(T) represents a new species of the genus Actinomycetospora, for which the name Actinomycetospora atypica sp. nov. is proposed. The type strain is NEAU-st4(T) (=CGMCC 4.7093(T) = DSM 45873(T)).

A novel actinomycete, designated strain NEAU-ycm2T, was isolated from edible Chinese black ants (Polyrhachis vicina Roger) and characterized using a polyphasic approach. The organism was found to have morphological and chemotaxonomic characteristics typical of the genus Micromonospora . The 16S rRNA gene sequence of strain NEAU-ycm2T showed highest similarity to those of Micromonospora sonneratiae 274745T (99.12 %), Micromonospora pattaloongensis TJ2-2T (98.85 %), Micromonospora pisi GUI 15T (98.76 %), Polymorphospora rubra TT 97-42T (98.42 %) and Micromonospora eburnea LK2-10T (98.21 %). Phylogenetic analysis based on the 16S rRNA gene and gyrB gene demonstrated that strain NEAU-ycm2T is a member of the genus Micromonospora and supported the close phylogenetic relationship to M. sonneratiae 274745T, M. pattaloongensis JCM 12833T and M. pisi GUI 15T. Furthermore, a combination of DNA–DNA hybridization and some physiological and biochemical properties indicated that the novel strain could be readily distinguished from its closest phylogenetic relatives. Therefore, it is proposed that NEAU-ycm2T represents a novel species of the genus of Micromonospora , for which the name Micromonospora polyrhachis sp. nov. is proposed. The type strain is NEAU-ycm2T ( = CGMCC 4.7100T = DSM 45886T).

A novel actinomycete, designated strain NEAU-GH7(T), was isolated from a lake sediment and characterized using a polyphasic approach. Strain NEAU-GH7(T) was Gram-stain positive, aerobic, non-spore-forming and produced spherical sporangia. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain NEAU-GH7(T) formed a monophyletic clade with the closest relative Streptosporangium longisporum DSM 43180(T) (99.0 %), an association that was supported by a bootstrap value of 74 % in the neighbour-joining tree and also recovered with the maximum-likelihood algorithm. However, the low level of DNA-DNA relatedness allowed the strain to be differentiated from its closest relative. Moreover, strain NEAU-GH7(T) could also be differentiated from S. longisporum DSM 43180(T) and other Streptosporangium species showing high 16S rRNA gene sequence similarity (98.0 %) by morphological and physiological characteristics. On the basis of phylogenetic analysis, DNA-DNA hybridization and phenotypic characteristics, strain NEAU-GH7(T) should be classified as a new species of the genus Streptosporangium, for which the name Streptosporangium shengliense [corrected] sp. nov. is proposed. The type strain is NEAU-GH7(T) (=CGMCC 4.7105(T)=DSM 45881(T)).

A novel actinomycete, strain NEAU-st1(T), was isolated from a soil sample collected in Shaanxi province, Northwest China and characterized using a polyphasic approach. 16S rRNA gene sequence similarity studies showed that strain NEAU-st1(T) belongs to the genus Nonomuraea, being most closely related to Nonomuraea rosea GW12687(T) (98.91 %), Nonomuraea solani NEAU-Z6(T) (98.44 %), Nonomuraea rhizophila YIM67092(T)(98.24 %) and Nonomuraea monospora PT708(T) (98.02 %); similarities to sequences of other type strains of the genus Nonomuraea were lower than 98 %. Both tree-making algorithms used also supported the position that strain NEAU-st1(T) formed a distinct clade with its most closely related species. Morphological and physiological characteristics confirmed that the strain belongs to the genus Nonomuraea and distinguished it from its most closely related species. DNA-DNA hybridization further differentiated strain NEAU-st1(T) from its nearest phylogenetic neighbours. These results suggested that strain NEAU-st1(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea shaanxiensis sp. nov. is proposed. The type strain is NEAU-st1(T) (=CGMCC 4.7096(T) = DSM 45877(T)).

Two new pentanorlanostane metabolites from a soil fungus Curvularia borreriae strain HS-FG-237

A novel ikarugamycin-producing actinomycete, designated strain NEAU-Da3T, was isolated from soybean root [Glycine max (L.) Merr.] and characterized using a polyphasic approach. 16S rRNA gene sequence similarity studies showed that strain NEAU-Da3T belonged to the genus Streptomyces , and was most closely related to Streptomyces carpaticus NRRL B-16359T (99.5 %), Streptomyces cheonanensis VC-A46T (99.3 %) and Streptomyces xiamenensis MCCC 1A01550T (97.2 %); similarities to other type strains of species of the genus Streptomyces were lower than 97.1 %. The maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences showed that the isolate formed a distinct phyletic line with S. carpaticus NRRL B-16359T, S. cheonanensis VC-A46T and S. xiamenensis MCCC 1A01550T. This branching pattern was also supported by the tree reconstructed with the neighbour-joining method. A comparative study between strain NEAU-Da3T and the type strains of the closest related species of the genus Streptomyces revealed that it differed from them in morphological, physiological and biochemical characteristics. Therefore, it is proposed that strain NEAU-Da3T represents a novel species of the genus Streptomyces , for which the name Streptomyces harbinensis sp. nov. is proposed. The type strain is NEAU-Da3T ( = CGMCC 4.7047T = DSM 42076T).

Forster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research. This powerful tool can elucidate protein interactions in either a dynamic or steady state. We recently developed a series of FRET-based technologies to determine protein interaction dissociation constant and for use in high-throughput screening assays of SUMOylation. SUMO (small ubiquitin-like modifier) is conjugated to substrates through an enzymatic cascade. This important posttranslational protein modification is critical for multiple biological processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate SUMO from its substrate. Here, we describe a novel quantitative FRET-based protease assay for determining the kinetics of SENP1. Our strategy is based on the quantitative analysis and differentiation of fluorescent emission signals at the FRET acceptor emission wavelengths. Those fluorescent emission signals consist of three components: the FRET signal and the fluorescent emissions of donor (CyPet) and acceptor (YPet). Unlike our previous method in which donor and acceptor direct emissions were excluded by standard curves, the three fluorescent emissions were determined quantitatively during the SENP digestion process from onesample. New mathematical algorithms were developed to determine digested substrate concentrations directly from the FRET signal and donor/acceptor direct emissions. The kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of SENP1 catalytic domain for pre-SUMO1/2/3 were derived. Importantly, the general principles of this new quantitative methodology of FRET-based protease kinetic determinations can be applied to other proteases in a robust and systems biology approach.

The widespread application of conventional activated sludge treatment process has been employed to deal with a variety of municipal and industrial sewage. While the generation of waste activated sludge (WAS) was considerably huge, the management and disposal expenses were substantially costly. A promising process aimed for WAS reduction during the operation process is urgently needed. Thus, increasing attentions emphasizing on the improved or novel sludge reduction processes should be intensively recommended in the future. This review presents the current and emerging technologies for excess sludge minimization within the process of sewage treatment. The ultimate purpose of this paper is to guide or inspire researchers who are seeking feasible and promising technologies (or processes) to tackle the severe WAS problem.

Two new piperidine alkaloids, streptazones E (1) and F (2), were isolated from Streptomyces sp. NEAU-Z4. Their structures were elucidated on the basis of MS and NMR analysis.

Genome shuffling is an efficient approach for the rapid engineering of microbial strains with desirable industrial phenotypes. In this study, a strategy of incorporating streptomycin resistance screening into genome shuffling (GS-SR) was applied for rapid improvement of doramectin production by Streptomyces avermitilis NEAU1069. The starting mutant population was generated through treatment of the spores with N-methyl-N’-nitro-N-nitrosoguanidine and ultraviolet (UV) irradiation, respectively, and five mutants with higher productivity of doramectin were selected as starting strains for GS-SR. Finally, a genetically stable strain F4-137 was obtained and characterized to be able to yield 992 ± 4.4 mg/l doramectin in a shake flask, which was 7.3-fold and 11.2-fold higher than that of the starting strain UV-45 and initial strain NEAU1069, respectively. The doramectin yield by F4-137 in a 50-l fermentor reached 930.3 ± 3.8 mg/l. Furthermore, the factors associated with the improved doramectin yield were investigated and the results suggested that mutations in ribosomal protein S12 and the enhanced production of cyclohexanecarboxylic coenzyme A may contribute to the improved performance of the shuffled strains. The random amplified polymorphic DNA analysis showed a genetic diversity among the shuffled strains, which confirmed the occurrence of genome shuffling. In conclusion, our results demonstrated that GS-SR is a powerful method for enhancing the production of secondary metabolites in Streptomyces.

A convenient and precise chiral method was developed and validated for measuring indoxacarb enantiomers in cucumber and tomato using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a reversed-phase Chiralpak AD-RH column. The target analytes were extracted by acetonitrile and then purified by solid phase extraction (SPE) using NH2 /Carb combined-cartridge. Parameters including the matrix effect, linearity, precision, accuracy, and stability were used. Then the proposed method was successfully applied to investigate the possible enantioselective degradation of rac-indoxacarb in cucumber and tomato under open conditions. The results indicated that the degradation of indoxacarb enantiomers followed first-order kinetics in cucumber and tomato. The half-lives of (+)-S-indoxacarb in cucumber and tomato were 3.0 and 5.9 days, respectively; while the (-)-R-indoxacarb were 7.3 and 12.2 days, respectively. The data of the half-lives showed that (+)-S-indoxacarb was preferentially degraded in cucumber and tomato. Moreover, indoxacarb degraded faster in cucumber than in tomato.

A novel endophytic actinomycete, designated strain NEAU-J5T was isolated from roots of snap bean (Phaseolus vulgaris L.). Comparative analysis of the 16S rRNA gene sequence indicated that NEAU-J5T is phylogenetically related to members of the family Micromonosporaceae . The whole-cell sugars were galactose, mannose and glucose. The predominant menaquinones were MK-9(H4) and MK-9(H6). The major fatty acids were C16 : 0, C18 : 0, C17 : 1ω7c, iso-C15 : 0 and C17 : 0. The phospholipids were phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and phosphatidylinositol mannoside. The DNA G+C content was 72.2 mol%. On the basis of the morphological and chemotaxonomic characteristics, phylogenetic analysis and characteristic patterns of 16S rRNA gene signature nucleotides, strain NEAU-J5T represents a novel species of a new genus within the family Micromonosporaceae , for which the name Xiangella phaseoli gen. nov., sp. nov. is proposed. The type strain of Xiangella phaseoli is strain NEAU-J5T ( = CGMCC 4.7038T = DSM 45730T).

Borrelidin has high and specific antifungal activity against Phytophthora sojae . To explore the antifungal mechanism of borrelidin against P. sojae , the relationship between the antifungal activity of borrelidin and the concentration of threonine was evaluated. The results demonstrated that the growth-inhibitory effect of borrelidin on the growth of P. sojae was antagonized by threonine in a dose-dependent manner, suggesting that threonyl-tRNA synthetase (ThrRS) may be the potential target of borrelidin. Subsequently, the inhibition of the enzymatic activity of ThrRS by borrelidin in vitro was confirmed. Furthermore, the detailed interaction between ThrRS and borrelidin was investigated using fluorescence spectroscopy and circular dichroism (CD), implying a tight binding of borrelidin to ThrRS. Taken together, these results suggest that the antifungal activity of borrelidin against P. sojae was mediated by inhibition of ThrRS via the formation of the ThrRS-borrelidin complex.

A novel actinomycete, designated strain NEAU-M9(T), was isolated from soybean root (Glycine max (L.) Merr) and characterized using a polyphasic approach. 16S rRNA gene sequence similarity studies showed that strain NEAU-M9(T) belonged to the genus Actinoplanes, being most closely related to Actinoplanes campanulatus DSM 43148(T) (98.85 %), Actinoplanes capillaceus DSM 44859(T) (98.70 %), Actinoplanes lobatus DSM 43150(T) (98.30 %), Actinoplanes auranticolor DSM 43031(T) (98.23 %) and Actinoplanes sichuanensis 03-723(T) (98.06 %); similarity to other type strains of the genus Actinoplanes ranged from 95.87 to 97.56 %. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that the isolate formed a distinct phyletic line with A. campanulatus DSM 43148(T) and A. capillaceus DSM 44859(T). This branching pattern was also supported by the tree constructed with the maximum-likelihood method. However, the low level of DNA-DNA relatedness allowed the isolate to be differentiated from the above-mentioned two Actinoplanes species. Moreover, strain NEAU-M9(T) could also be distinguished from the most closely related species by morphological, physiological and characteristics. Therefore, it is proposed that strain NEAU-M9(T) represents a novel Actinoplanes species, Actinoplanes hulinensis sp. nov. The type strain of Actinoplanes hulinensis is NEAU-M9(T) (= CGMCC 4.7036(T) = DSM 45728(T)).

A borrelidin-producing actinomycete, designated strain NEAU-W2T, was isolated from the root surface of soybean [Glycine max (L.) Merr] and characterized using a polyphasic approach. The organism was found to have morphological and chemotaxonomic characteristics typical of streptomycetes. The G+C content of the DNA was 66.12 mol%. Analysis of the 16S rRNA gene sequence of strain NEAU-W2T revealed that the strain formed a distinct clade within the 16S rRNA gene sequence phylogenetic tree and showed highest similarity (99.61 %) to Streptomyces neyagawaensis ATCC 27449T. However, the DNA–DNA relatedness between strain NEAU-W2T and S. neyagawaensis ATCC 27449T was 58.51 %. Strain NEAU-W2T could also be differentiated from S. neyagawaensis ATCC 27449T and other Streptomyces species showing high 16S rRNA gene sequence similarity (98–99 %), as well as other borrelidin-producing strains, based on morphological and physiological characteristics. On the basis of its physiological and molecular properties, it is proposed that strain NEAU-W2T represents a novel Streptomyces species, Streptomyces heilongjiangensis sp. nov. The type strain is NEAU-W2T ( = CGMCC 4.7004T = ATCC BAA-2424T = DSM 42073T).

A serum-free medium (CHO-SFM) together with a fed-batch process was developed for the cultivation of a recombinant GS-CHO cell line producing TNFR-Fc. According to the metabolic characteristics of GS-CHO cell, a basal medium was prepared by supplementing DMEM:F12:RPMI1640 (2:1:1) with amino acids, insulin, transferrin, Pluronic F68 and some other ingredients. Statistical optimization approaches based on Plackett–Burman and central composite designs were then adopted to identify additional positive determinants and determine their optimal concentrations, which resulted in the final CHO-SFM medium formulations. The maximum antibody titer reached was 90.95 mg/l in the developed CHO-SFM, which was a 18 % and 10 fold higher than that observed in the commercial EX-CELL™ 302 medium (76.95 mg/l) and basal medium (8.28 mg/l), respectively. Subsequently, a reliable, reproducible and robust fed-batch strategy was designed according to the offline measurement of glucose, giving a final antibody yield of 378 mg/l, which was a threefold improvement over that in conventional batch culture (122 mg/l) using CHO-SFM. In conclusion, the use of design of experiment (DoE) method facilitated the development of CHO-SFM medium and fed-batch process for the production of recombinant antibody using GS-CHO cells.

(R)-Ethyl-3-hydroxyglutarate, (R)-3, is an intermediate in the synthesis of the statin side chain. Here, a new two-step, indirect biotransformation pathway involving the formation of ethyl (R)-4-carbamoyl-3-hydroxybutanoate, (R)-2, as an intermediate for (R)-3 production was developed using Rhodococcus boritolerans with ethyl (R)-4-cyano-3-hydroxybutyate, (R)-1, as substrate. Maximum conversion was with 10 g (R)-1/l, 7 g cells/l (dry wt), pH 7.5 and 25°C. A yield of 98 ± 0.5% (w/w) was attained within 8 h.

Chlorination can significantly enhance the antioxidant and antitumor activity of genistein. In this paper, genistein, 8-chlorogenistein, and 3',8-dichlorogenistein were selected to investigate the binding to bovine serum albumin (BSA) using fluorescence spectroscopy and circular dichroism (CD). The results showed that chlorination, especially at position 3', had significant effects on the binding constant value of chlorinated genistein derivatives to BSA; however, the binding site and the binding number were slightly affected. The thermodynamic parameters indicated that hydrophobic and electrostatic forces played important roles in the binding process and the enhanced binding affinity mainly associated with the increase of the hydrophobicity caused by the chlorine atom substitution. Furthermore, the CD data demonstrated that the conformation of BSA was slightly altered in the presence of genistein, 8-chlorogenistein, and 3',8-dichlorogenistein, with different reduced α-helix contents. The results obtained herein will be of biological significance in toxicology investigation and genistein derivative drug design.

The objective of this study was to determine mutagenicity of milbemycin oxime by Ames, micronucleus and sperm malformation tests. In the Ames assay, four genotypic variants of the Salmonella strains (TA97, TA98, TA100 and TA102) carrying mutations in several genes were used. Milbemycin oxime was incubated with them in five different doses both in the presence and absence of metabolic activation (S9) and the result was assessed based on the number of revertant colonies. Concurrently, appropriate positive controls were used so as to validate the test. The average number of revertant colonies per plate treated with milbemycin oxime was less than double as compared to that of the negative control. In the micronucleus assay, Kunming mice were orally treated with 1/5, 1/10, 1/20 of the mice oral LD50, twice (24 h interval), respectively. At 6 h after the second time, all mice were killed and taken out of femur bones, then washed out the bone marrow to make smears on clean slides. 1000 PCE were scored by oil immersion lens. The numbers of micro-nucleated PCE were counted, the micronuclear rates were calculated. The obtained results indicated that milbemycin oxime could not induce the increase of micronuclear rates of PCE in bone marrow. In sperm malformation test, the effect of milbemycin oxime on sperm parameters was investigated following oral doses of 1/5, 1/10, 1/20 LD50 every 5 days for 35 days to male kunming mice. The treated groups showed no significant changes both in sperm abnormality and in a dose-dependent manner.

Milbemycin oxime, isolated from the fermentation of Streptomyces hygroscopicus var. aureolacrimosus, is effective to parasites such as heartworm larvae, adult hookworm and mites. Kunming mice were selected for the acute toxicity study. The animals exposed to milbemycin oxime by single intragastric administration. The pretest showed that gender had effect on the LD50 value. Therefore, 70 male mice were divided into 7 groups at random, each group was given different concentration of milbemycin oxime, so did for the female mice. Each animal was observed for signs of toxicity and behavioral changes at 1 and 3h after test article administration and once daily thereafter for 14 days. Mortality/moribundity checks were performed daily. The LD50 values for milbemycin oxime in male and female mice were 1 832 and 727 mg/kg, respectively, and the 95% confidence interval were 1 637.57～2 022.08 mg/kg and 603.95～868.96 mg/kg, respectively.

In the continuing study of the chemical compositions of the strain Streptomyces bingchenggensis, three new milbemycin derivatives, milbemycin alpha(31) (1), secomilbemycins C (2), and D (3), were isolated. Their structures were established on the basis of extensive spectroscopic analysis.

A bioactivity-guided approach was employed to isolate and determine the chemical identity of bioactive constituents with antifungal activity from Actinoplanes sp. HBDN08. The structure of the antifungal metabolite was elucidated as 5-hydroxyl-5-methyl-2-hexenoic acid on the basis of spectral analysis. This compound showed strong in vitro antifungal activity against Botrytis cinerea, Cladosporium cucumerinum and Corynespora cassiicola, with an IC50 of 32.45, 27.17, and 30.66 mg/L, respectively; however, it only moderately inhibited hyphal growth of Rhizoctonia solani with an IC50 of 61.64 mg/L. The in vivo antifungal activity under greenhouse conditions demonstrated that 5-hydroxyl-5-methyl-2-hexenoic acid could effectively control the diseases caused by B. cinerea, C. cucumerinum and C. cassiicola with 71.42%, 78.63% and 65.13% control values at 350 mg/L, respectively. This strong antifungal activity suggests that 5-hydroxyl-5-methyl-2-hexenoic acid might be a promising candidate for new antifungal agents.

A strain Actinoplanes sp. HBDN08 was screened by PCR-guided method using primers derived from conserved regions of halogenase genes. A new chlorinated isoflavone, 3',8-dichlorogenistein (1), along with 8-chlorogenistein (2) were isolated from the fermentation broth of Actinoplanes sp. HBDN08. Their structures were elucidated on the basis of extensive 1D and 2D NMR as well as HRESI-MS, ESI-MS, UV, and IR spectroscopic analyses. The origin of the two compounds was also investigated by high-performance liquid chromatography (HPLC) analysis. The results demonstrated that they were not biosynthesized but derived from the biotransformation of genistein by Actinoplanes sp. HBDN08. The antioxidant activities of the isolated compounds 1 and 2 were evaluated by using the lipid peroxidation assay. Their antitumor activities were calculated according to the inhibitory rate of cell proliferation against the human breast cancer cell line MDA-MB-231. The results indicated that compounds 1 (IC(50) = 5.2 microM) and 2 (IC(50) = 7.5 microM) showed stronger antioxidant activities than genistein (IC(50) = 13.6 microM). In comparison with the antitumor activities of genistein, those of compounds 1 and 2 increased 7.7- and 2.6-fold, respectively. These results suggest that the PCR-guided screening strategy is a rapid method for obtaining halometabolite-producing strains. Moreover, these results reveal that chlorination has significant effects on the bioactivities of genistein. This could be important information for studying the structure-activity relationships of genistein.

A screening method for asparagine synthetase B (AS-B) inhibitors by reversed-phase high performance liquid chromatography (RP-HPLC) has been established. The contents of asparagines produced in the reaction system can be analyzed by HPLC after the derivatization with 1-fluoro-2,4-dinitrobenzene (DNFB) and used to calculate the total activity of AS-B. The sample was separated on an Agilent C18 column (250 mm x 4.6 mm, 5 microm) at the temperature of 30 degrees C with the elution of 50 mmol/L sodium acetate buffer (pH 6.2)-acetonitrile (15:85, v/v) as mobile phase at a flow rate of 1.0 mL/min. The detection wavelength was set at 365 nm. The enzyme reaction system consisted of 100 mmol/L Tris (tris(hydroxymethyl)aminomehane)-HCl buffer (pH 8.0), 100 mmol/L NaCl, 10 mmol/L MgCl2, 5 mmol/L adenosine triphosphate (ATP), 10 mmol/L L-aspartate, 10 mmol/L L-glutamine and 2 microg recombinant soybean AS-B (1 mL of the total volume), then mixed for 1 min and incubated for 15 min at 37 degrees C. After quenching with ethanol and centrifugation, the supernatant was derivatized by DNFB and then separated by HPLC. The amino acids in the reaction system were baseline separated within 6 min. The quantitative analysis of AS-B inhibition was performed by determining its dynamic parameters. The inhibitor L-glutamic acid gamma-methyl ester was used in the enzyme reaction system to test this method and its inhibition constant obtained was close to the literature value. The established method is fast, accurate, sensitive and suitable for high throughput screening AS-B inhibitors.

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To investigate the function of nsdA in Streptomyces bingchengensis, it was cloned and sequenced, which presented an 89.89% identity with that of S. coelicolor. The lambdaRED-mediated PCR-targeting technique was used to create nsdA replacement in the S. bingchengensis_226541 chromosome. The nsdA disruption mutant, BC29, was obtained, which produced more pigment and spores than did the ancestral strain. HPLC analysis revealed that the disruption of nsdA efficiently increased milbemycin A(4) production and nanchangmycin production by 1.5-fold and 9-fold, respectively. Complementation of the nsdA mutation restored the phenotype and antibiotic production. These results showed that nsdA negatively affected sporulation and antibiotic production in S. bingchengensis.

A novel macrolide compound from Streptomyces bingchenggensis : fermentation, isolation, structure elucidation and biological properties

Two new compounds, 3-O-(13-hydroxy-9Z,11E,15E-octadecatrienoyl) cycloeucalenol (1) and 24′-hydroxy-tetracosyl ferulate (2), together with one known compound, tetracosyl ferulate (3), were isolated from the dried stems of Gnetum pendulum. The two new compounds were structurally elucidated by spectroscopic and chemical methods.

Two new seco-milbemycins have been isolated and characterized from the Streptomyces bingchenggensis. On the basis of detailed spectroscopic analysis and comparison with the reported data of milbemycin beta(14), their structures were established to be the seco-milbemycins of milbemycin beta(14). However, milbemycin beta(14) and the two new seco-milbemycins were not converted to each other during purification by silica gel column or storage in the MeOH extract at room temperature. The two new seco-milbemycins are the first discovered from milbemycin-producing microorganisms. They may play an important role in understanding and perfecting the proposed biosynthesis pathways of milbemycins.

Three new alpha-class milbemycins from Streptomyces bingchenggensis, named milbemycins alpha28, alpha29 and alpha30, have been isolated and characterized. On the basis of detailed spectroscopic analysis and comparison with reported data, their structures were determined to be the 22alpha-hydroxy, 23beta-O-(2-methylbutanoyl) milbemycin alpha9, 22alpha-hydroxy, 23beta-O-(2,4-dimethylpentanoyl) milbemycin alpha9 and 22alpha-hydroxy, 23beta-O-(2-methylbutanoyl) milbemycin A4, respectively. Milbemycins alpha28 and alpha29 are the first reported milbemycins that possess both C-26 and C-23 substituents. These three new milbemycins possess potent acaricidal and nematocidal activity.

Subsequent to the publication of the above paper it has been brought to our attention that the species epithet proposed for the taxon represented by strain NEAUGH7 is not grammatically correct as ‘‘Streptosporangium shengliensis’’ should have been proposed as Streptosporangium shengliense. We here propose the corrected name for the taxon represented by strain NEAU-GH7. Description of Streptosporangium shengliense sp. nov.