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Sea buckthorn (Hippophae rhamnoides) is an important medicinal plant of Southeast-Asia, enriched with various antioxidant components like β-carotene, alpha-tocopherol, catechin and folacin. Carotenoids are the bioactive substances abundantly present in sea buckthorn berries providing it an enticing yellowish, or orange red color. We performed the phylogenetic analysis, and conserved domain analysis to observe the evolutionary history and functional cites of carotenoids biosynthesis genes respectively. Further relative expression of two carotenoids biosynthesis genes Lycopene β-cyclase (Hr-Lcyb)and phytoene synthase(Hr-Psy) in was conducted in various tissues of sea buckthorn plants. Our results revealed that both the carotenoid synthesis genes Hr-Lcyband Hr-Psyshowed homology with already reported Arabidopsis thalianagenes (AT3G10230and AT5G17230). Relative expression analysis indicated that expression of Hr-Lcyband Hr-Psygenes in leaves and floral organs of sea buckthorn was one-fold higher than other tissues. This study will open the door for plant biologists for further characterization of carotenoids synthesis genes and their regulatory processes in specific tissues of carotenoid rich fruit plants.

Passalora fulva(or Fulvia fulva) is the causal microorganism of tomato leaf mold, the outbreak of which occurs worldwide in greenhouse especially when humidity is high. However, studies on antifungal agents of P. fulvaare still very limited. In this study, a marine-derived Streptomycesalbidoflavusstrain L131 showing potent inhibitory activities against P. fulvawas identified and characterized. The active antifungal components were obtained, and studies on the antifungal mechanisms of the crude extract showed that the antifungal metabolites of L131 caused damage of hyphae and spore development, as well as plasma membrane of P. fulva. In addition, accumulation of endogenous reactive oxygen species of the leaf pathogen was also observed after treatment by culture extracts of L131. To our knowledge, this is the first report on the studies of the antifungal mechanisms against P. fulva, which benefit further development of biocontrol agent against tomato leaf mold disease.

ABSTRACTDrug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosisviability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosisin vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosisgrowth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosisstrains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.

ABSTRACTDiscovering new antibiotics is a major scientific challenge, made increasingly urgent by the continued development of resistance in bacterial pathogens. A fundamental understanding of the mechanisms of bacterial antibiotic resistance will be vital for the future discovery or design of new, more effective antibiotics. We have exploited our intimate knowledge of the molecular mechanism of glycopeptide antibiotic resistance in the harmless bacterium Streptomyces coelicolorto develop a new two-step cell wall bioactivity screen, which efficiently identified a new actinomycete strain containing a previously uncharacterized glycopeptide biosynthetic gene cluster. The screen first identifies natural product extracts capable of triggering a generalized cell wall stress response and then specifically selects for glycopeptide antibacterials by assaying for the induction of glycopeptide resistance genes. In this study, we established a diverse natural product extract library from actinomycete strains isolated from locations with widely varying climates and ecologies, and we screened them using the novel two-step bioassay system. The bioassay ultimately identified a single strain harboring the previously unidentified biosynthetic gene cluster for the glycopeptide ristocetin, providing a proof of principle for the effectiveness of the screen. This is the first report of the ristocetin biosynthetic gene cluster, which is predicted to include some interesting and previously uncharacterized enzymes. By focusing on screening libraries of microbial extracts, this strategy provides the certainty that identified producer strains are competent for growth and biosynthesis of the detected glycopeptide under laboratory conditions.

Thirty-five thousand actinomycete extracts were screened for anti-Mycobacterium tuberculosis(M. tb) activity, followed by C18cartridge fractionation of 37 prioritized extracts. Based on MICs against replicating and nonreplicating M. tb, and IC50values against Vero cells to generate selectivity indices, seven fractions from seven different strains were selected for further examination. When cultured in G.S.S. media and extracted with ethyl acetate, the Streptomyces hygroscopicusstrain ECUM 14046 yielded an extract with promising anti-M. tbactivity and a well-defined chromatographic profile. Fractionation by preparative HPLC and subsequent structure elucidation of two active fractions using 1D- and 2D-NMR and MS methods revealed the presence of two cyclohexapeptides, hytramycins V and I, each containing three unusual piperazic acid moieties. The use of 1H iterative full spin analysis (HiFSA) on both hytramycins confirmed that quantum mechanics-simulated spectra match the experimental data, and all JH,Hand δHvalues are consistent with the proposed structures. The absolute configuration of each amino acid moiety was determined by Marfey’s method. The MICs against replicating and, more importantly, nonreplicating M. tbfall into the range of some existing second-line anti-TB drugs, such as streptomycin and capreomycin, respectively. The activities were maintained against M. tbstrains that represent the major global clades, as well as H37Rv-isogenic strains that are resistant to individual clinical anti-TB drugs.

The addition of extracellular ATP (exATP) to four Streptomycesstrains had similar effects: low exATP levels stimulated antibiotic production and high levels reduced it. Compared with antibiotic production, the concentrations of intracellular ATP (inATP) in the tested strains were opposite, which suggests a role of inATP in regulating secondary metabolite production. Under inactivation of the polyphosphate kinase gene (ppk) in Streptomyces lividans, we observed the same results: when the inATP level in the mutant strain was lower than in the parent strain, more antibiotic was produced. Combining all the results, a strong inverse relationship between [inATP] and the secondary metabolite production is suggested by this study.

S-Adenosyl-L-methionine (SAM) is one of the major methyl donors in all living organisms. The exogenous treatment with SAM leads to increased actinorhodin production in Streptomyces coelicolorA3(2). In this study, mutants from different stages of the AfsK-AfsR signal transduction cascade were used to test the possible target of SAM. SAM had no significant effect on actinorhodin production in afsK, afsR, afsS, or actII-open reading frame 4 (ORF4) mutant. This confirms that afsKplays a critical role in delivering the signal generated by exogenous SAM. The afsK-pHJL-KNmutant did not respond to SAM, suggesting the involvement of the C-terminal of AfsK in binding with SAM. SAM increased the in vitroautophosphorylation of kinase AfsK in a dose-dependent manner, and also abolished the effect of decreased actinorhodin production by a Ser/Thr kinase inhibitor, K252a. In sum, our results suggest that SAM activates actinorhodin biosynthesis in S. coelicolorM130 by increasing the phosphorylation of protein kinase AfsK.

Liquid fuels from lignocellulosic materials, such as wood, offer an attractive alternative to fossil fuel. Lignocellulosic biomass is composed of a complex mixture of cellulose, hemicellulose, and lignin. As structural polymers, the matrix of hemicellulose and lignin surround the cellulose component of the plant cell wall to protect it against enzymatic attack. The resistance of this complex to enzymatic attack is a major impediment to commercial bioconversion of lignocellulosic biomass. Furthermore, proportional variability within the mixture of the three major components varies, depending on the species of feedstock used, growing site, climate, age, and the part of the plant harvested. This fact presents process design and operating challenges for the fermentation of sugars derived from lignocellulosic feedstocks. The essentially uncontrolled variability of biomass properties also presents major difificulties in understanding the fundamental processes involved in limiting the digestion of cell walls to fermentable sugars. Energy crops must be optimized for ‘low‐input production’ systems and efficiency of pretreatment and hydrolysis to derive sugars for further fermentation to produce ethanol. Recent advances in the molecular biology of wood formation and cell‐wall biosynthesis have provided new tools to tailor the biomass composition. This review summarizes recent advances in the understanding of the molecular mechanisms regulating the biochemical and developmental processes of woody biomass production. Potential areas of future research in optimizing lignocellulosic feedstock for bioenergy production are identified. © 2007 Society of Chemical Industry and John Wiley & Sons, Ltd

Two novel calix4cryptands were synthesized from 1,3alternate calix4bisazacrown. “Mappemonde II” consists of one 1,3calix4bisazacrown wrapped by a benzocrown ether loop. “Mill II” is composed of two 1,3calix4bisazacrowns linked by two benzocrown ether strands.