教酒链霉菌NRRL 3882中钙霉素生物合成后修饰基因calD的功能分析

【目的】钙霉素是二价阳离子载体,是一类含有吡咯环的聚醚类抗生素,广泛用于细胞二价阳离子的生物学功能研究。本文以钙霉素生物合成基因簇中cal D基因为研究对象,通过蛋白质同源序列比对、基因敲除、回补验证及HPLC/MS分析,对cal D基因的功能进行表征。【方法】对cal D基因功能进行生物信息学预测。选用钙霉素产生菌Streptomyces chartreusis NRRL 3882,通过PCR-targeting的方法对cal D基因进行敲除获得突变株,再将cal D基因克隆到链霉菌整合质粒上,通过接合转移技术将cal D回补到缺失株中。使用HPLC/MS技术对菌株发酵产物进行分析。【结果】生物信息学预测Cal D蛋白酶属于氧化还原酶。获得cal D基因敲除突变株Δcal D及基因回补菌株Δcal D:cal D。HPLC/MS检测到cal D基因的缺失菌株大幅降低钙霉素产生能力,与野生型菌株相比,突变株中积累更多的3-Hydroxylcezomycin和更少的氮-去甲基钙霉素。【结论】cal D参与钙霉素的生物合成。cal D的缺失导致3-Hydroxylcezomycin的积累,推测cal D负责钙霉素生物合成途径中苯并噁唑环3位上羟基转化成酮基的氧化反应。初步阐明了cal D基因在钙霉素生物合成途径中的功能机制。     

A natural plasmid uniquely encodes two biosynthetic pathways creating a potent anti-MRSA antibiotic

BackgroundUnderstanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin.Conclusions/SignificancePlasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.     

Toward steadfast growth of antibiotic research in China: From natural products to engineered biosynthesis

Antibiotics are widely used for clinical treatment and preventing or curing diseases in agriculture. Cloning and studies of their biosynthetic gene clusters are vital for yield enhancement and engineering new derivatives with new and prominent activities. In recent years, research in this aspect is impressively active in China. This article reviews biosynthetic progress on 28 antibiotics, including polyketides, nonribosomal peptides, hybrid polyketide-nonribosomal peptides, peptidyl nucleoside, nucleoside, and others. Their biosynthetic mechanisms were disclosed, and their derivatives with new structures/activities were obtained by gene inactivation, mutasynthesis and combinatorial biosynthesis.     

Chemical synthesis and in silico molecular modeling of novel pyrrolyl benzohydrazide derivatives: Their biological evaluation against enoyl ACP reductase (InhA) and Mycobacterium tuberculosis

In efforts to develop new antitubercular agents, we report here the synthesis of a series of novel pyrrole hydrazine derivatives. The molecules were evaluated against inhibitors of InhA, which is one of the key enzymes involved in type II fatty acid biosynthetic pathway of the mycobacterial cell wall as well as inhibitors of Mycobacterium tuberculosis H37Rv. The binding mode of compounds at the active site of enoyl-ACP reductase was explored using the surflex-docking method. The model suggests one or two H-bonding interactions between the compounds and the InhA enzyme. Some compounds exhibited good activities against InhA in addition to promising activities against M. tuberculosis.     

Structural characterization of pyrrolic cross-links in collagen using a biotinylated Ehrlich's reagent

The structures of pyrrolic forms of cross-links in collagen have been confirmed by reacting collagen peptides with a biotinylated Ehrlich's reagent. This reagent was synthesized by converting the cyano group of N-methyl-N-cyanoethyl-4-aminobenzaldehyde to a carboxylic acid, followed by conjugation with biotin pentyl-amine. Derivatization of peptides from bone collagen both stabilized the pyrroles and facilitated selective isolation of the pyrrole-containing peptides using a monomeric avidin column. Reactivity of the biotinylated reagent with collagen peptides was similar to that of the standard Ehrlich reagent, but heat denaturation of the tissue before enzyme digestion resulted in the loss of about 50% of the pyrrole cross-links. Identification of a series of peptides by mass spectrometry confirmed the presence of derivatized pyrrole structures combined with between 1 and 16 amino acid residues. Almost all of the pyrrole-containing peptides appeared to be derived from N-terminal telopeptide sequences, and the nonhydroxylated (lysine-derived) form predominated over pyrrole cross-links derived from helical hydroxylysine.     

Mutational biosynthesis of tacrolimus analogues by fkbO deletion mutant of Streptomyces sp. KCTC 11604BP

Tacrolimus (FK506) is an important macrocyclic polyketide showing antifungal and immunosuppressive activities, as well as neuroregenerative properties. Tacrolimus biosynthetic machinery should incorporate the shikimate-derived 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) as a biosynthetic starter unit into the biosynthetic line of tacrolimus. fkbO is a homologue of rapK encoding chorismatase related to the biosynthesis of starter unit DHCHC from chorismate in the rapamycin biosynthetic gene cluster. FkbO and RapK are good targets for mutational biosynthesis to produce novel analogues of tacrolimus, ascomycin, and rapamycin, which could be important drugs for clinical application in the treatment of cancer and immune and neurodegenerative diseases. To make novel tacrolimus analogues, we prepared an fkbO in-frame deletion mutant, Streptomyces sp. GT110507, from a tacrolimus high producer. We scrutinized the cyclic carboxylic acids that were possibly incorporated instead of DHCHC by precursor-directed mutasynthesis using Streptomyces sp. GT110507 to lead tacrolimus analogues. Among them, trans-4-hydroxycyclohexanecarboxylic acid and 3-hydroxybenzoic acid were successfully incorporated into the tacrolimus backbone, which led to the production of 31-desmethoxytacrolimus and TC-225, respectively. Especially, adding of trans-4-hydroxycyclohexanecarboxylic acid produced a high amount (55 mg/L) of 31-desmethoxytacrolimus. Interestingly, in the rapK mutant, it has been reported that the incorporation of cyclohexanecarboxylic acid (CHC) led to 39-desmethoxy rapamycin. However, in Streptomyces sp. GT110507, CHC is not successfully incorporated. This discrepancy should reflect the differences in the DHCHC biosynthesis mechanism and/or substrate specificity of starter unit loading machineries (FkbP and RapP) of tacrolimus and rapamycin.     

Two new pyrrole derivatives isolated from Salacia amplifolia Merr

Two new pyrrole derivatives Salaciamole (1), Salaciaglycoside A (2), long with one known compound 1H-pyrrole-3-carboxylic acid (3) were isolated from the roots of Salacia amplifolia Merr (Hippocrateaceae). The structures of the new compounds were deduced from their comprehensive spectroscopic analysis including IR, HR-EI-MS, ¹H NMR, ¹³C NMR, DEPT, COSY, HMBC and HMQC. And the structure of the known compound was identified by comparison of their spectral data with those reported in the literature.     

The complete targeted profile of the organic acid intermediates of the citric acid cycle using a single stable isotope dilution analysis, sodium borodeuteride reduction and selected ion monitoring GC/MS

The quantitative profiling of the organic acid intermediates of the citric acid cycle (CAC) presents a challenge due to the lack of commercially available internal standards for all of the organic acid intermediates. We developed an analytical method that enables the quantitation of all the organic acids in the CAC in a single stable isotope dilution GC/MS analysis with deuterium-labeled analogs used as internal standards. The unstable α-keto acids are rapidly reduced with sodium borodeuteride to the corresponding stable α-deutero-α-hydroxy acids and these, along with their unlabeled analogs and other CAC organic acid intermediates, are converted to their tert-butyldimethylsilyl derivatives. Selected ion monitoring is employed with electron ionization. We validated this method by treating an untransformed mouse mammary epithelial cell line with well-known mitochondrial toxins affecting the electron transport chain and ATP synthase, which resulted in profound perturbations of the concentration of CAC intermediates.     

Synthesis and structure-activity relationships of a series of pyrrole cannabinoid receptor agonists

We designed and synthesized a series of pyrrole derivatives with the aim of investigating the structure-activity relationship (SAR) for the binding of non-classical agonists to CB(1) and CB(2) cannabinoid receptors. Superposition of two pyrrole-containing cannabinoid agonists, JWH-007 and JWH-161, allowed us to identify positions 1, 3 and 4 of the pyrrole nucleus as amenable to additional investigation. We prepared the 1-alkyl-2,5-dimethyl-3,4-substituted pyrroles 10a-e, 11a-d, 17, 21, 25 and the tetrahydroindole 15, and evaluated their ability to bind to and activate cannabinoid receptors. Noteworthy in this set of compounds are the 4-bromopyrrole 11a, which has an affinity for CB(1) and CB(2) receptors comparable to that of well-characterized heterocyclic cannabimimetics such as Win-55,212-2; the amide 25, which, although possessing a moderate affinity for cannabinoid receptors, demonstrates that the 3-naphthoyl group, commonly present in indole and pyrrole cannabimimetics, can be substituted by alternative moieties; and compounds 10d, 11d, showing CB(1) partial agonist properties.     

A novel triterpenoid 16-hydroxy betulinic acid isolated from Mikania cordata attributes multi-faced pharmacological activities

The aerial parts of extensively used ethnomedicinal plant Mikania cordata (Burm. f.) Robinson growing wild in Bangladesh were investigated to isolate and characterize compounds responsible for the bioactivities of the plant. In the present study, a new derivatives of betulinic acid, 16-hydroxy betulinic acid [3β,16-dihydroxy-lup-20(29)-en-28-oic] was isolated and the structure of the compound was determined by NMR spectroscopic means and comparing with available literature data. The isolated compound was then investigated for different pharmacological activities including antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic potential employing different methods. The compound showed potent antibacterial activity with inhibition zone of diameter ranging from 12.0 to 17.5?mm and antifungal activity with mycelial growth inhibition ranging from 37.6 to 54.5%. The MIC values for antibacterial and antifungal activities ranged from 31.5–125 and 250–1000?μg/mL respectively. The compound (50 and 100?mg/kg body weight) showed potent peripheral and central analgesic activity with 55.19% and 41% of writhing inhibition at 90?min after administration of the compound and the highest 55.98%, 79.18% elongation of reaction time, respectively. In anti-inflammatory activity screening, the compound (100?mg/kg b.w.) revealed the highest 77.08% edema inhibition at 4?h after administration of carrageenan. In antipyretic assay, 16-hydroxy betulinic acid displayed a strong antipyretic effect in yeast-induced rats. From the present study it is apparent that 16-hydroxy betulinic acid might play vital role to establish M. cordata as ethnomedicinal plant to treat wound, cuts and fever.     

Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii

Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.     

Cloning and Expression of Genes Required for Coronamic Acid (2-Ethyl-1-Aminocyclopropane 1-Carboxylic Acid), an Intermediate in the Biosynthesis of the Phytotoxin Coronatine

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.     

Characterization of the N-methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of K_m 276 μM, k_cat 1.26 min⁻¹ and k_cat/K_m 76.2 M⁻¹ s⁻¹. These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.     

Anticancer and antimicrobial glycosides from ipomoea bahiensis

Four new antimicrobial glycosides have been isolated from Ipomoea bahiensis. Spectroscopic properties and basic and acidic hydrolysis characterized them as derivatives of 11 -hydroxy hexadecanoic and 11-hydroxy tetradecanoic acid, glycosidically linked in the 11-position to a trisaccharide unit composed of glucose, rhamnose and fucose which is esterified by tiglic and 3-hydroxy-2-methylbutyric acid. One of these compounds revealed significant activity against Sarcoma 180 in mice.     

Kolavane and kaurane diterpenes from the stem bark of Xylopia aethiopica

The stem bark of Xylopia aethiopica has yielded four diterpenes, two of them novel. Three of the diterpenes were identified as (?)-kaur-16-en-19-oic acid and its 7-oxo and 7β-hydroxy derivatives. The fourth was the novel kolavane derivative 2-oxo-kolav-3,13-dien-15-oic acid, a type of compound not previously recorded in the Annonaceae.     

Biotransformation of oleic acid by alcaligenes sp. 5–18, a bacterium tolerant to high concentrations of oleic acid

We isolated from soil 160 bacterial strains which are tolerant to high concentrations of oleic acid, and examined their ability to transform oleic acid to new fatty acid derivatives. One of the isolated strains, Alcaligenes sp. 5–18, produced several compounds from oleic acid: 3-hydroxyoleic acid, 3-hydroxyhexadecenoic acid, octadecadienoic acid, hexadecadienoic acid, and tetradecadienoic acid. These compounds are intermediates in the β-oxidation pathway of oleic acid, and their accumulation is probably due to defective β-oxidation of oleic acid in the microorganism. Neither hydroxy nor enoic derivatives of fatty acids with a carbon chain length shorter than 14 were produced.     

Calcimycin mediates mycobacterial killing by inducing intracellular calcium-regulated autophagy in a P2RX7 dependent manner

Phenotypic screening led to the identification of calcimycin as a potent inhibitor of Mycobacterium bovis BCG (M. bovis BCG) growth in vitro and in THP-1 cells. In the present study, we aim to decipher the mechanism of antimycobacterial activity of calcimycin. We noticed that treatment with calcimycin led to up-regulation of different autophagy markers like Beclin-1, autophagy-related gene (Atg) 7, Atg 3 and enhanced microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) to LC3-II conversion in macrophages. This calcimycin-mediated killing of intracellular M. smegmatis and M. bovis BCG was abrogated in the presence of 3-methyladenine (3-MA). We also demonstrate that calcimycin binding with purinergic receptor P2X7 (P2RX7) led to increase in intracellular calcium level that regulates the extracellular release of ATP. ATP was able to regulate calcimycin-induced autophagy through P2RX7 in an autocrine fashion. Blocking of either P2RX7 expression by 1-[N,O-bis(5-Isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) or reducing intracellular calcium levels by 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxy-methyl) ester (BAPTA-AM) abrogated the antimycobacterial activity of calcimycin. Taken together, these results showed that calcimycin exerts its antimycobacterial effect by regulating intracellular calcium-dependent ATP release that induces autophagy in a P2RX7 dependent manner.     

Biotransformation of ent-pimaradienoic acid by cell cultures of Aspergillus niger

Microbial transformation stands out among the many possible semi-synthetic strategies employed to increase the variety of chemical structures that can be applied in the search for novel bioactive compounds. In this paper we obtained ent-pimaradienoic acid (1, PA, ent-pimara-8(14),15-dien-19-oic acid) derivatives by fungal biotransformation using Aspergillus niger strains. To assess the ability of such compounds to inhibit vascular smooth muscle contraction, we also investigated their spasmolytic effect, along with another five PA derivatives previously obtained in our laboratory, on aortic rings isolated from male Wistar rats. The microbial transformation experiments were conducted at 30 °C using submerged shaken liquid culture (120 rpm) for 10 days. One known compound, 7α-hydroxy ent-pimara-8(14),15-dien-19-oic acid (2), and three new derivatives, 1β-hydroxy ent-pimara-6,8(14),15-trien-19-oic acid (3), 1α,6β,14β-trihydroxy ent-pimara-7,15-dien-19-oic acid (4), and 1α,6β,7α,11α-tetrahydroxy ent-pimara-8(14),15-dien-19-oic acid (5), were isolated and identified on the basis of spectroscopic analyses and computational studies. The compounds obtained through biotransformation (2–5) did not display a significant antispasmodic activity (values ranging from 0% to 16.8% of inhibition); however the previously obtained diterpene, methyl 7α-hydroxy ent-pimara-8(14),15-dien-19-oate (8), showed to be very effective (82.5% of inhibition). In addition, our biological results highlight the importance to study the antispasmodic potential of a large number of novel diterpenes, to conduct further structure–activity relationship investigations.     

Biosynthesis of Auxin by the Gram-Positive Phytopathogen Rhodococcus fascians Is Controlled by Compounds Specific to Infected Plant Tissues

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.     

Synthesis and evaluation of novel chloropyrrole molecules designed by molecular hybridization of common pharmacophores as potential antimicrobial agents

In an attempt to identify new potential lead as antimicrobial agent, 31 novel chloropyrrole derivatives of aroyl hydrazones and chalcones incorporating common pharmacophore of pyoluteorin derivatives were synthesized. Antimicrobial activity of the synthesized compounds was evaluated using broth dilution technique. Based on biological evaluation data it was observed that activity increases as the number of chlorines on pyrrole core increases. Few 1H-pyrrole-2-carbohydrazide derivatives shows activity equivalent to the standard drug ciprofloxacin. Thus, these compounds can act as potential lead for further antibacterial studies.