Three new polyketides from vasR2 gene over-expressed mutant strain of Verrucosispora sp. NS0172 / 中国天然药物

Over-expression of the pathway specific positive regulator gene is an effective way to activate silent gene cluster. In the curret study, the SARP family regulatory gene, vasR2, was over-expressed in strain Verrucosispora sp. NS0172 and the cryptic gene cluster responsible for the biosynthesis of pentaketide ansamycin was partially activated. Two tetraketides (1 and 2) and a triketide (3) ansamycins, together with five known compounds (4-8), were isolated and elucidated from strain NS0172OEvasR2. Their NMR data were completely assigned by analysis of their HR-ESI-MS and

Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog / 中国天然药物

In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.

Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog / 中国天然药物

In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.

Identification of 3-demethylchuangxinmycin from Actinoplanes tsinanensis CPCC 200056 / 药学学报

Chuangxinmycin (CM) from Actinoplanes tsinanensis was an antibiotic discovered by Chinese scientists about 40 years ago. It contains a new heterocyclic system of indole fused with dihydrothiopyran, whose biosynthetic mechanism remains unclear. CM is used as an oral medicine in the treatment of bacterial infections in China. The simple structure makes CM as an attractive candidate of structure modification for improvement of antibacterial activity. Recently, we analyzed the secondary metabolites of Actinoplanes tsinanensis CPCC 200056, a CM producing strain, as a natural CM analogue. We discovered the first natural CM analogue 3-demethylchuangxinmycin (DCM) as a new natural product. Compared to CM, DCM exhibited a much weaker activity in the inhibition of the bacterial strains tested. The finding provides valuable information for the structure-activity relationship in the biosynthesis of CM.

Comparative analysis of rapamycin biosynthesis clusters between Actinoplanes sp. N902-109 and Streptomyces hygroscopicus ATCC29253 / 中国天然药物

The present study was designed to identify the difference between two rapamycin biosynthetic gene clusters from Streptomyces hygroscopicus ATCC29253 and Actinoplanes sp. N902-109 by comparing the sequence and organization of the gene clusters. The biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus ATCC29253 was reported in 1995. The second rapamycin producer, Actinoplanes sp. N902-109, which was isolated in 1995, could produce more rapamycin than Streptomyces hygroscopicus ATCC29253. The genomic map of Actinoplanes sp. N902-109 has been elucidated in our laboratory. Two gene clusters were compared using the online software anti-SMASH, Glimmer 3.02 and Subsystem Technology (RAST). Comparative analysis revealed that the organization of the multifunctional polyketide synthases (PKS) genes: RapA, RapB, RapC, and NRPS-like RapP were identical in the two clusters. The genes responsible for precursor synthesis and macrolactone modification flanked the PKS core region in N902-109, while the homologs of those genes located downstream of the PKS core region in ATCC29253. Besides, no homolog of the gene encoding a putative type II thioesterase that may serve as a PKS "editing" enzyme accounted for over-production of rapamycin in N902-109, was found in ATCC29253. Furthermore, no homologs of genes rapQ (encoding a methyltransferase) and rapG in N902-109 were found in ATCC29253, however, an extra rapM gene encoding methyltransferase was discovered in ATCC29253. Two rapamycin biosynthetic gene clusters displayed overall high homology as well as some differences in gene organization and functions.

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Entre 13 linhagens de fungos filamentosos isolados a partir de amostras de solo agrícola, tubérculos e de material em compostagem, duas foram selecionadas em função da capacidade de crescer em meio líquido contendo amido como única fonte de carbono, a 45ºC, e produzir consideráveis quantidades de glucoamilase. Essas linhagens, identificadas como Aspergillus flavus A1.1 e Thermomyces lanuginosus A13.37, foram utilizadas para desenvolvimento de experimentos para avaliar os efeitos do tipo de amido (milho e mandioca), do pH inicial do meio de cultura (4,0; 5,0 e 6,0) e da temperatura de incubação (40 e 45ºC), em um modelo fatorial (2x3x2), sobre a produção da glucoamilase. O tipo de amido usado como fonte de carbono para o cultivo dos fungos influenciou significativamente a produção de glucoamilase por A. flavus, sendo obtida uma maior quantidade da enzima em meio contendo amido de mandioca do que em meio com amido de milho. A produção da enzima por T. lanuginosus também foi maior em meio contendo amido de mandioca, porém, a diferença não foi estatisticamente significativa. As atividades enzimáticas sobre amido (0,3 per center), maltose (0,3 per center) ou sobre mistura de 0,3 per center de amido com 0,1 per center de maltose, indicaram que as enzimas de Aspergillus hidrolisaram, preferencialmente, o amido, embora tenham mostrado atividade considerável sobre a maltose. A maior liberação de glicose a partir da mistura de substratos sugeriu que o fungo em questão possa secretar dois tipos diferentes de enzimas. Enzimas produzidas por T. lanuginosus hidrolisaram o amido e a maltose e não liberaram maiores teores de glicose quando o substrato constou de mistura de amido e maltose. As enzimas de Aspergillus e Thermomyces apresentaram elevada temperatura ótima de atividade (65 e 70ºC, respectivamente) com boa termoestabilidade na ausência de substrato (manutenção de 50 per ceter da atividade por 5 e 8h respectivamente), além de estabilidade em ampla faixa de pH. Os resultados apresentados indicam uma importante fonte alternativa de glucoamilase para uso no processamento industrial de amido.

Production of keratinase enzyme by thermophilic Streptomyces thermovulgaris

Seventeen thermophilic actinomycete isolates were tested for their keratinolytic activities. Results indicate that five isolates have keratinase activity. Streptomyces thermovulgaris proved to be superior in keratinase formation and it was selected for the subsequent investigation. The optimum conditions for keratinase production and feather solubilizaiton of S. thermovulgaris were studied. Data revealed that S. thermovulgaris grew better with good yield of keratinase activity by growing it on the fementation medium [100 m.500 ml flasks], in which the nitrogen and the carbon sources were replaced with sterile chicken feather pieces [1.5 g%], 1.0 g% starch and 0.1 g% K[2] HPO[4] using tap water with a homogenized spore suspension [containing approximately 8.2 x 10[-3] spores ml[-1]] of 3 days old culture. Inoculated flasks were incubated at 50°C for 4 days under static conditions

Comparison of thermostable phosphatase and proteases from thermophilically disposed transition species and thermophilic actinomycetes.

Thermoactinomycetes vulgaris is a thermophilic actinomycetes growing optimally at 50 degrees C and Streptomyces albus, S. coelicolor, S. fasciculus and S. olivochromogenes are thermophilically disposed transition species of actinomycetes, which have optimum biomass at 40 degrees C. The acid/alkaline phosphatase and acid/alkaline/neutral protease enzyme from Streptomycetes species showed enzyme activity up to 90 degrees C. In comparison to phosphatases and proteases from T. vulgaris it was concluded that these thermophilically disposed transition species showed macromolecular thermostability i.e. thermostable enzymes and protein.

Studies on the keratinolytic enzymes of thermophilic actinomycetes.1-Production of thermostable keratinase enzyme and identification of keratinolytic thermophilic actinomycetes

One hundred and ninety thermophilic actinomycetes were isolated from different sources. Screening was carried out according to growth temperatures. Obligate thermophilic actinomycetes were picked up and examined according to their keratinolytic activity. Results indicated that five isolates have keratinase activity. The two most active keratinolytic actinomycete isolates C52 and G114 were selected and subjected to complete identification. The cultural, morphological and physiological characteristics of both isolates indicated that they belong to Theinoactinomyces vulgaris and Microbispora thermodiastatica. The effect of nutritional and environmental conditions on the growth and keratinase activity of Th. vulgaris C52 were studied. Data revealed that Th. vulgaris C52 grew better with good yield of keratinase activity by growing it on the modified Kosmatchev medium [50 ml medium/500 ml clinical bottle], in which the nitrogen and the carbon sources of the Kosmatchev medium were replaced with 1.6% [W/V] sterile chicken feather pieces and 1% [W/V] starch, using tap water with an initial pH of 7.5, inoculated with 2% [v/v] of homogenized spore suspension [containing approximately 7.5 x 10 4 spores ml-1] of one day old culture and incubated at 50§C for 4 days under static condition

Studies on the lceratinolytic enzymes of thermophilic Actinomycetes II-Partial purification and characterization of thermostable keratinase enzyme from Thermoactinomyces vulgaris C52

The thermostable keratinase enzyme, produced by Thermoacetinomyces vulgaris CS2 in liquid modified. Kosmatchev medium under the optimum condition, was purified 224-fold with an Overall yield of 36.08% of the original activity and specific activity 748.67 units mg [-1] protein by ammonium sulphate precipitation and ion exchange chromatography [DEAF-Cellulose]. Maximal activity of the enzyme was obtained at 55-60 and pH 8.4-85. It was stable at 45-55'in the pH 8.4-8.5. Also the enzyme was slightly activated by CaC12 MgSO4, FeSO4 and CuSO4, but strongly inhibited by KFeCN, KCN, iodine and iodoacedic acid. The partially purified enzyme actively hydrolyzed all keratinaceous waste materials used

Farmer's lung disease in north-western India--a preliminary report.

A study of farmer's lung (FL) disease was carried out in 197 subjects engaged in farming and having respiratory complaints of varying duration. It revealed that 13.2% of the subjects had precipitating antibodies against thermophilic actinomycetes, with Faenia rectivirgula (Micropolyspora faeni) alone accounting for 85% of the positive reactions. Precipitating antibodies against Thermoactinomyces vulgaris and T. thalpophilus were observed only in 1.5% and 0.5% of the subjects, respectively. Two subjects concomitantly demonstrated F. rectivirgula and T. vulgaris-specific serum precipitins. Sixty (30%) of the subjects related their respiratory symptoms to exposure to wheat straw/thresher's dust or other vegetable substrata in the working environment. Based upon a suggestive clinical history, roentgenography, pulmonary function studies and demonstration of serum precipitins against F. rectivirgula, FL was diagnosed in 4 subjects whose salient features are presented and discussed. To the best of our knowledge, this is the first authentic report on FL from India. A comprehensive epidemiological survey is indicated to determine the prevalence of FL in different geo-climatic regions of the country.

Air spora of thermophilic actinomycetes in the atmosphere of Cairo, Egypt

The distribution of thermophilic actinomycetes in the atmosphere of Cairo was studied. The concentrations of these organisms were higher in the crowded districts than in the quieter residential ones. Maximum counts were recorded during December, higher counts during January and November were also obtained. Seven genera of thermophilic actinomycetes were recorded in the atmosphere of Cairo: Streptomyces, thermomonospora [Saccharomonospora], thermoactinomyces, Micropolyspora, Microbispora, Pseudonocardia and Actinobifida. The first two genera were more frequent than other genera. The genus streptomyces included four series [of which the gray series was most frequent] and 30 different species, of these species S. thermovulgaris and S. sp. 15 [12] were the most common. All thermomonospora and thermoactinomyces isolates were identified as Th. viridis [saccharomonospora viridis] and T. vulgaris, respectively. Micropolyspora was represented by M. faeni, M. Caesia and M. thermoviridis. Species of the other genera were microbispora, bispora pseudonocardia thermophila and actinobifida alba. Results indicate that thermophilic actinomycetes known to cause farmer's lung disease and other respiratory troubles were commonly isolated from the atmosphere of the crowded district "Zeitoun" in Cairo