Two new cyclopentenone metabolites from Streptomyces sp. HU119.

Two previously undescribed cyclopentenone metabolites, (S)-2-(3-acetylamino-2-methyl)propyl-3-butyl-2-cyclopenten-1-one (1) and (S)-2-(3-acetylamino-2-ethyl)propyl-3-butyl-2-cyclopenten-1-one (2), were isolated from the fermentation broth of the strain Streptomyces sp. HU119. The structures of 1 and 2 were determined by the comprehensive spectroscopic analysis, including 1 D, 2 D NMR, MS spectral analysis and the comparison with data from the literature. The absolute configurations were elucidated by experimental and calculated optical rotations (OR). Compounds 1 and 2 displayed weak cytotoxic activity.

Two new compounds from Streptomyces sp. HS-NF-813.

Two new derivatives of cytotoxic substance BE-52211, designed as BE-52211D (1) and BE-52211E (2), were isolated from the fermentation broth of the strain Streptomyces sp. HS-NF-813. Their structures were determined by 1D and 2D NMR techniques, ESI-MS and comparison with data from the literature. The absolute stereochemistry of 1 was elucidated by NMR data of the Mosher ester derivatives. Compounds 1 and 2 showed moderate cytotoxic activity against three human tumor cell lines.

Novel Metabolites from the Endophytic Fungus Chaetomium subaffine L01.

One natural p-terphenyl glycoside, gliocladinin C, and two furano-polyene derivatives, chaetominins A and B, were isolated from potato endophytic fungus Chaetomium subaffine. The absolute configurations of these compounds were elucidated by HR-ESI-MS, NMR, the DP4+ probabilities and electronic circular dichroism (ECD) spectra. Furthermore, gliocladinin C and chaetominin A showed cytotoxic activity against two selected human tumor cell lines (Hep-2 and HepG-2).

A new spectinabilin derivative with cytotoxic activity from ant-derived Streptomyces sp. 1H-GS5.

A new spectinabilin derivative (1) was isolated from the fermentation broth of the ant-derived Streptomyces sp. 1H-GS5, and the structure was elucidated by extensive spectroscopic analysis. Compound 1 showed cytotoxicity against human tumor cell lines A549, HCT-116, and HepG2 with IC50 values of 9.7, 12.8, and 9.1 µg/ml, respectively, which was relative higher than that of spectinabilin.

[Long-term Effect of the Treatment of IgA Nephropathy by Tonifying Shen, Activating Blood Stasis, Dispelling Wind-Dampness Combined with Western Medicine].

Objective To observe the long-term effect of tonifying Shen, activating blood stasis, dispelling wind-dampness (TSABSDWD) combined with Western drugs (WD) for IgA nephropathy. Methods A single center retrospective case-control study was used. The clinical and laboratory examinations, pa- thology of renal biopsy, and treatment programs of IgA nephropathy were obtained from primary IgA ne- phropathy patients (confirmed from renal biopsy at authors' hospital) from Jan 1st, 2008 to Dec 31 , 2008. Patients were assigned to Group A (basic treatment +Chinese herbs) and Group B (basic treatment +Chi- nese herbs + glucocorticoid and/or immune inhibitors). A follow-up visit started from the confirmation of re- nal biopsy to Dec 31, 2008, for at least 12 months. The end point event was defined as entering end stage renal disease (ESRD), estimated glomerular filtration rate (eGFR) decreased by more than 50%, or SCr was doubled. The differences in clinical manifestations, lab indicators and etc. were compared between be- fore treatment and after 1 year of treatment/till the end of follow-ups. The accumulative kidney survival rate was calculated using Kaplan-Meier method. The curve for accumulative kidney survival rate was drawn. Re- sults A total of 219 cases were included, 49 in Group A and 170 in Group B. In Group A, there were 7 pa- tients (14.0%) with Shen deficiency syndrome, 21 cases (43.0%) with Shen deficiency blood stasis syn- drome, 8 (16. 0%) with Shen deficiency wind-dampness syndrome, 13 cases (27. 0%) with Shen deficien- cy blood stasis wind-dampness syndrome. In Group B there were 12 patients (7.1%) with Shen deficiency syndrome, 47 cases (27. 6%) with Shen deficiency blood stasis syndrome, 22 (12.9%) with Shen defi- ciency wind-dampness syndrome, 89 cases (52.4%) with Shen deficiency blood stasis wind-dampness syndrome. No statistical difference in age, sex, or follow-up period between the two groups (P >0.05). Compared with Group A, the disease courser was shorter, 24 h urination increased more, levels of SCr and blood urea nitrogen (BUN) increased higher, plasma albumin decreased lower in Group B (P <0. 05). Compared with before treatment, 24 h urination and counts of urinary red blood cells (RBCs) decreased more in the two groups after 1-year treatment, and decreased further till the end of follow-up (P <0. 05). The total effective rate was 89. 0% (1951219). The total effective rate of Group A was 89. 8% (44/49), with no patient entry into endpoint event. The total effective rate of Group B was 88. 8%(151/170). Totally 5 pa- tients arrived at endpoint event in Group B, 4 in ESRD, 1 with eGFR decreased by more than 50%, or SCr doubled. Compared with Group B, the complete relief rate was higher in Group A (P <0. 01). The accumulative kidney survival rate was 100. 0%, 100. 0%, 98. 0% and 96. 1% in the 219 patients at year 1 , 3, 5, 7, re- spectively using Kaplan-Meier method. Conclusions Programs based on theory of Shen disease wind- dampness in CM and in integrative medicine could be used in treating IgA nephropathy according to differ- ent conditions. Long-term observation showed this program could significantly improve patients' conditions. The 7-year accumulative kidney survival rate was 96. 1%.

Designed biosynthesis of 25-methyl and 25-ethyl ivermectin with enhanced insecticidal activity by domain swap of avermectin polyketide synthase.

BACKGROUND: Avermectin and milbemycin are important 16-membered macrolides that have been widely used as pesticides in agriculture. However, the wide use of these pesticides inevitably causes serious drug resistance, it is therefore imperative to develop new avermectin and milbemycin analogs. The biosynthetic gene clusters of avermectin and milbemycin have been identified and the biosynthetic pathways have been elucidated. Combinatorial biosynthesis by domain swap provides an efficient strategy to generate chemical diversity according to the module polyketide synthase (PKS) assembly line. RESULTS: The substitution of aveDH2-KR2 located in avermectin biosynthetic gene cluster in the industrial avermectin-producing strain Streptomyces avermitilis NA-108 with the DNA regions milDH2-ER2-KR2 located in milbemycin biosynthetic gene cluster in Streptomyces bingchenggensis led to S. avermitilis AVE-T27, which produced ivermectin B1a with high yield of 3450 ± 65 µg/ml. The subsequent replacement of aveLAT-ACP encoding the loading module of avermectin PKS with milLAT-ACP encoding the loading module of milbemycin PKS led to strain S. avermitilis AVE-H39, which produced two new avermectin derivatives 25-ethyl and 25-methyl ivermectin (1 and 2) with yields of 951 ± 46 and 2093 ± 61 µg/ml, respectively. Compared to commercial insecticide ivermectin, the mixture of 25-methyl and 25-ethyl ivermectin (2:1 = 3:7) exhibited 4.6-fold increase in insecticidal activity against Caenorhabditis elegans. Moreover, the insecticidal activity of the mixture of 25-methyl and 25-ethyl ivermectin was 2.5-fold and 5.7-fold higher than that of milbemycin A3/A4 against C. elegans and the second-instar larva of Mythimna separate, respectively. CONCLUSIONS: Two new avermectin derivatives 25-methyl and 25-ethyl ivermectin were generated by the domain swap of avermectin PKS. The enhanced insecticidal activity of 25-methyl and 25-ethyl ivermectin implied the potential use as insecticide in agriculture. Furthermore, the high yield and genetic stability of the engineered strains S. avermitilis AVE-T27 and AVE-H39 suggested the enormous potential in industrial production of the commercial insecticide ivermectin and 25-methyl/25-ethyl ivermectins, respectively.

Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis.

Milbemycin oxime has been commercialized as effective anthelmintics in the fields of animal health, agriculture, and human infections. Currently, milbemycin oxime is synthesized by a two-step chemical reaction, which involves the ketonization of milbemycins A3/A4 to yield the intermediates 5-oxomilbemycins A3/A4 using CrO3 as catalyst. Due to the low efficiency and environmental unfriendliness of the ketonization of milbemycins A3/A4, it is imperative to develop alternative strategies to produce 5-oxomilbemycins A3/A4. In this study, the atmospheric and room temperature plasma (ARTP) mutation system was first employed to treat milbemycin-producing strain Streptomyces bingchenggensis, and a mutant strain BC-120-4 producing milbemycins A3, A4, B2, and B3 as main components was obtained, which favors the construction of genetically engineered strains producing 5-oxomilbemycins. Importantly, the milbemycins A3/A4 yield of BC-120-4 reached 3,890 ± 52 g/l, which was approximately two times higher than that of the initial strain BC-109-6 (1,326 ± 37 g/l). The subsequent interruption of the gene milF encoding a C5-ketoreductase responsible for the ketonization of milbemycins led to strain BCJ60 (∆milF) with the production of 5-oxomilbemycins A3/A4 and the elimination of milbemycins A3, A4, B2, and B3. The high 5-oxomilbemycins A3/A4 yield (3,470 ± 147 g/l) and genetic stability of BCJ60 implied the potential use in industry to prepare 5-oxomilbemycins A3/A4 for the semisynthesis of milbemycins oxime.

Three new cyclopentenone derivatives from Actinoalloteichus nanshanensis NEAU 119.

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.

Characterization and analysis of an industrial strain of Streptomyces bingchenggensis by genome sequencing and gene microarray.

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.

New nemadectin congeners with acaricidal and nematocidal activity from Streptomyces microflavus neau3 Y-3.

Two nemadectin congeners 1 and 2 were isolated from the fermentation broth of a mutant strain (Y-3) of Streptomyces microflavus neau3. Their structures were determined on the basis of extensive spectroscopic analysis and comparison with data from the literature. Compound 2 possessed a 5-membered ring lactone that is unprecedented among known milbemycins and avermectins. Both compounds 1 and 2 exhibited potent acaricidal activity and nematocidal activity. Especially, compound 2 demonstrated impressive acaricidal activity against adult mites with an IC50 of 2.3±0.9 µg/mL and mite eggs with an IC50 of 17.5±2.1 µg/mL and nematocidal activity against Caenorhabditis elegans with an IC50 of 0.7±0.2 µg/mL, which are higher than those of nemadectin and the known commercial acaricide and nematocide milbemycin A3/A4.

Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin.

Milbemycins A3/A4 are important 16-membered macrolides which have been commercialized and widely used as pesticide and veterinary medicine. However, similar to other milbemycin producers, the production of milbemycins A3/A4 in Streptomyces bingchenggensis is usually accompanied with undesired by-products such as C5-O - methylmilbemycins B2/B3 (α-class) and ß1/ß2 (ß-class) together with nanchangmycin. In order to obtain high yield milbemycins A3/A4-producing strains that produce milbemycins A3/A4 as main components, milD, a putative C5-O-methyltransferase gene of S. bingchenggensis , was biofunctionally investigated by heterologous expression in Escherichia coli . Enzymatic analysis indicated that MilD can catalyze both α-class (A3/A4) and ß-class milbemycins (ß11) into C5-O-methylmilbemycins B2/B3 and ß1, respectively, suggesting little effect of furan ring formed between C6 and C8a on the C5-O-methylation catalyzed by MilD. Deletion of milD gene resulted in the elimination of C5-Omethylmilbemycins B2/B3 and ß1/ß2 together with an increased yield of milbemycins A3/A4 in disruption strain BCJ13. Further disruption of the gene nanLD encoding loading module of polyketide synthase responsible for the biosynthesis of nanchangmycin led to strain BCJ36 that abolished the production of nanchangmycin. Importantly, mutant strain BCJ36 (ΔmilDΔnanLD) produced milbemycins A3/A4 as main secondary metabolites with a yield of 2312 ± 47 µg/ml, which was approximately 74 % higher than that of the initial strain S. bingchenggensis BC-109-6 (1326 ± 37 µg/ml).

Two new piperidine alkaloids from Streptomyces sp. NEAU-Z4.

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.

Bioconversion of ethyl (R)-4-cyano-3-hydroxybutyate into (R)-ethyl-3-hydroxyglutarate via an indirect pathway by Rhodococcus boritolerans.

(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.

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).

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 IC(50) values of 10.2, 65.1 and 124.4 µg/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 A(3)/A(4) as positive reference.

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.

Identification and analysis of the germin-like gene family in soybean.

BACKGROUND: Germin and germin-like proteins constitute a ubiquitous family of plant proteins. A role of some family members in defense against pathogen attack had been proposed based on gene regulation studies and transgenic approaches. Soybean (G. max L. Merr.) germin genes had not been characterized at the molecular and functional levels. RESULTS: In the present study, twenty-one germin gene members in soybean cultivar 'Maple Arrow' (partial resistance to Sclerotinia stem rot of soybean) were identified by in silico identification and RACE method (GmGER 1 to GmGER 21). A genome-wide analyses of these germin-like protein genes using a bioinformatics approach showed that the genes located on chromosomes 8, 1, 15, 20, 16, 19, 7, 3 and 10, on which more disease-resistant genes were located on. Sequence comparison revealed that the genes encoded three germin-like domains. The phylogenetic relationships and functional diversity of the germin gene family of soybean were analyzed among diverse genera. The expression of the GmGER genes treated with exogenous IAA suggested that GmGER genes might be regulated by auxin. Transgenic tobacco that expressed the GmGER 15 [corrected] gene exhibited high tolerance to the salt stress. In addition, the GmGER mRNA increased transiently at darkness and peaked at a time that corresponded approximately to the critical night length. The mRNA did not accumulate significantly under the constant light condition, and did not change greatly under the SD and LD treatments. CONCLUSIONS: This study provides a complex overview of the GmGER genes in soybean. Phylogenetic analysis suggested that the germin and germin-like genes of the plant species that had been founded might be evolved by independent gene duplication events. The experiment indicated that germin genes exhibited diverse expression patterns during soybean development. The different time courses of the mRNAs accumulation of GmGER genes in soybean leaves appeared to have a regular photoperiodic reaction in darkness. Also the GmGER genes were proved to response to abiotic stress (such as auxin and salt), suggesting that these paralogous genes were likely involved in complex biological processes in soybean.

5-ketoreductase from Streptomyces bingchengensis: overexpression and preliminary characterization.

To elucidate the biotransformation from 5-oxomilbemycins A(3) and A(4) to milbemycins A(3) and A(4) in Streptomyces bingchengensis, the C5-ketoreductase gene (milF) was cloned using PCR with the specific primer designed from homologous nucleotide sequences. The C5-ketoreductase (MilF) was heterologously expressed in E. coli BL21 (DE3) as a His-tagged fusion protein. The characterization and biotransformation function of purified MilF was verified by in vitro enzyme assay. MilF is an NADPH-dependent reductase. The biotransformation products, analyzed by LC-APCI/MS, were identified as milbemycin A(3) and milbemycin A(4). MilF is thus present in Streptomyces bingchengensis and can transform 5-oxomilbemycins A(3) and A(4) to milbemycins A(3) and A(4). These findings are significant for understanding the biosynthetic pathway of milbemycins in Streptomyces bingchengensis and pave the way to obtain a producer strain of 5-oxomilbemycins directly by targeted milF disruption.

Three new milbemycin derivatives from Streptomyces bingchenggensis.

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.