Isolation of two triterpenoids from Phlomis purpurea, one of them with anti-oomycete activity against Phytophthora cinnamomi, and insights into its biosynthetic pathway.

Phytophthora cinnamomi is an important plant pathogen responsible for dieback diseases in plant genera including Quercus, Fagus, Castanea, Eucalyptus, and Pinus, among others, all over the world. P. cinnamomi infection exerts tremendous ecological and economic losses. Several strategies have been developed to combat this pathogenic oomycete, including the search for novel anti-oomycete compounds. In this work, a Mediterranean vascular plant, Phlomis purpurea, has been screened for secondary bioactivity against this pathogen. The genus Phlomis includes a group of herbaceous plants and shrubs described as producers of many different bioactive compounds, including several triterpenoids. Triterpenoids are well-known molecules synthesized by plants and microorganisms with potent antioxidant, antitumoral, and antimicrobial activities. We have isolated by HPLC-DAD and characterized by HPLC-MS and NMR two nortriterpenoid compounds (phlomispentaol A and phlomispurtetraolone) from the root extracts of P. purpurea. One of them (phlomispentaol A) is active against the plant pathogenic oomycete P. cinnamomi (based on in vitro inhibition bioassays). Based on their chemical structure and their relationship to other plant triterpenoids, oleanolic acid is proposed to be the common precursor for these molecules. The anti-oomycete activity shown by phlomispentaol A represents a promising alternative to counteract the worldwide-scale damage caused to forest ecosystems by this pathogen.

A diet based on cured acorn-fed ham with oleic acid content promotes anti-inflammatory gut microbiota and prevents ulcerative colitis in an animal model.

BACKGROUND: Diets based on meat products are not recommended in the case of ulcerative colitis (UC). The objective here is to test if some traditional cured meat products, as acorn-fed ham (high levels of oleic acid), may be useful for controlling inflammatory diseases as UC in animal models, which could represent a new dietary complementary intervention in the prevention of this inflammatory disease in humans. METHODS: Two rat cohorts have been used: conventional vegetable rat feed and acorn-fed ham. UC was induced with DSS in drinking water ad libitum for 1 week. Short-chain fatty acids (SCFAs) and 16S rRNA metagenomics from bacterial populations were analyzed in cecum samples. Colon samples were analyzed for histological parameters. RESULTS: Acorn-fed ham diet induced changes in gut microbiota composition, with pronounced enrichments in anti-inflammatory bacterial genera (Alistipes, Blautia, Dorea, Parabacteroides). The animals with this diet showed a strong reduction in most parameters associated to ulcerative colitis: disease activity index, macroscopic score of colitis, epitelium alteration in colon mucosa, inflammatory cell density in colon, myeloperoxidase titers in colon, proinflammatory cytokines (IL-17, IFN-γ). Also, acorn-fed ham diet animals showed increased total antioxidant activity an oleic acid levels in plasma, as well as higher short-chain fatty acid concentrations in cecum (isobutyric, isovaleric and valeric). CONCLUSIONS: In the acorn-fed ham cohort, as a result of the dietary intake of oleic acid and low intake of omega-6 fatty acids, a strong preventive effect against UC symptoms was observed.

[Prebiotics: concept, properties and beneficial effects]. / Prebiotics: concept, properties and beneficial effects.

Prebiotics are non-digestible food ingredients (oligosaccharides) that reach the colon and are used as substrate by microorganisms producing energy, metabolites and micronutrients used for the host; in addition they also stimulate the selective growth of certain beneficial species (mainly bifidobacteria and lactobacilli) in the intestinal microbiota. In this article, a multidisciplinary approach to understand the concept of prebiotic carbohydrates, their properties and beneficial effects in humans has been carried out. Definitions of prebiotics, reported by relevant international organizations and researchers, are described. A comprehensive description of accepted prebiotics having strong scientific evidence of their beneficial properties in humans (inulin-type fructans, FOS, GOS, lactulose and human milk oligosaccharides) is reported. Emerging prebiotics and those which are in the early stages of study have also included in this study. Taken into account that the chemical structure greatly influences carbohydrates prebiotic properties, the analytical techniques used for their analysis and characterization are discussed. In vitro and in vivo models used to evaluate the gastrointestinal digestion, absorption resistance and fermentability in the colon of prebiotics as well as major criteria to design robust intervention trials in humans are described. Finally, a comprehensive summary of the beneficial effects of prebiotics for health at systemic and intestinal levels is reported. The research effort on prebiotics has been intensive in last decades and has demonstrated that a multidisciplinary approach is necessary in order to claim their health benefits.

Los prebióticos son ingredientes alimentarios no digeribles (oligosacáridos) que llegan al colon y sirven de sustrato a los microorganismos, originando energía, metabolitos y micronutrientes utilizados por el hospedador y estimulando el crecimiento selectivo de determinadas especies beneficiosas (principalmente, bifidobacterias y lactobacilos) de la microbiota intestinal. En este artículo se realiza una revisión sobre los carbohidratos prebióticos desde diferentes perspectivas, comenzando por las definiciones de prebióticos formuladas a lo largo de los últimos treinta años por científicos y diferentes organismos internacionales. Se realiza una descripción detallada de los prebióticos aceptados, como tales, que presentan propiedades beneficiosas fundamentadas en estudios llevados a cabo en humanos (fructanos tipo inulina y FOS; GOS, lactulosa y oligosacáridos de leche humana), los que se consideran prebióticos emergentes y aquellos que se encuentran en fases iniciales de estudio. Además y teniendo en cuenta que la estructura química de los carbohidratos influye notablemente en sus propiedades prebióticas, se describen las técnicas más utilizadas para su análisis y caracterización. Asimismo, se detallan los modelos in vitro e in vivo más utilizados para estudiar la resistencia de los prebióticos a la digestión y la absorción gastrointestinal, la fermentación de los prebióticos en el colon así como los criterios a tener en cuenta para llevar a cabo ensayos de intervención en humanos. Por último se realiza una amplia descripción de los efectos beneficiosos de los prebióticos para la salud a nivel intestinal y sistémico. Como conclusión, podría decirse que la investigación existente hasta el momento, sobre prebióticos, es extensa y pone de manifiesto que es necesario considerar un gran número de factores para poder atribuir alegaciones de salud a un prebiótico.

Prebióticos; concepto, propiedades y efectos beneficiosos / Prebiotics: concept, properties and beneficial effects

Los prebióticos son ingredientes alimentarios no digeribles (oligosacáridos) que llegan al colon y sirven de sustrato a los microorganismos, originando energía, metabolitos y micronutrientes utilizados por el hospedador y estimulando el crecimiento selectivo de determinadas especies beneficiosas (principalmente, bifidobacterias y lactobacilos) de la microbiota intestinal. En este artículo se realiza una revisión sobre los carbohidratos prebió- ticos desde diferentes perspectivas, comenzando por las definiciones de prebióticos formuladas a lo largo de los últimos treinta años por científicos y diferentes organismos internacionales. Se realiza una descripción detallada de los prebióticos aceptados, como tales, que presentan propiedades beneficiosas fundamentadas en estudios llevados a cabo en humanos (fructanos tipo inulina y FOS; GOS, lactulosa y oligosacáridos de leche humana), los que se consideran prebióticos emergentes y aquellos que se encuentran en fases iniciales de estudio. Además y teniendo en cuenta que la estructura química de los carbohidratos influye notablemente en sus propiedades prebióticas, se describen las técnicas más utilizadas para su análisis y caracterización. Asimismo, se detallan los modelos in vitro e in vivo más utilizados para estudiar la resistencia de los prebióticos a la digestión y la absorción gastrointestinal, la fermentación de los prebióticos en el colon así como los criterios a tener en cuenta para llevar a cabo ensayos de intervención en humanos. Por último se realiza una amplia descripción de los efectos beneficiosos de los prebióticos para la salud a nivel intestinal y sistémico. Como conclusión, podría decirse que la investigación existente hasta el momento, sobre prebióticos, es extensa y pone de manifiesto que es necesario considerar un gran número de factores para poder atribuir alegaciones de salud a un prebiótico (AU)

Prebiotics are non-digestible food ingredients (oligosaccharides) that reach the colon and are used as substrate by microorganisms producing energy, metabolites and micronutrients used for the host; in addition they also stimulate the selective growth of certain beneficial species (mainly bifidobacteria and lactobacilli) in the intestinal microbiota. In this article, a multidisciplinary approach to understand the concept of prebiotic carbohydrates, their properties and beneficial effects in humans has been carried out. Definitions of prebiotics, reported by relevant international organizations and researchers, are described. A comprehensive description of accepted prebiotics having strong scientific evidence of their beneficial properties in humans (inulin-type fructans, FOS, GOS, lactulose and human milk oligosaccharides) is reported. Emerging prebiotics and those which are in the early stages of study have also included in this study. Taken into account that the chemical structure greatly influences carbohydrates prebiotic properties, the analytical techniques used for their analysis and characterization are discussed. In vitro and in vivo models used to evaluate the gastrointestinal digestion, absorption resistance and fermentability in the colon of prebiotics as well as major criteria to design robust intervention trials in humans are described. Finally, a comprehensive summary of the beneficial effects of prebiotics for health at systemic and intestinal levels is reported. The research effort on prebiotics has been intensive in last decades and has demonstrated that a multidisciplinary approach is necessary in order to claim their health benefits (AU)

Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering.

Polyketides, a large family of bioactive natural products, are synthesized from building blocks derived from alpha-carboxylated Coenzyme A thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA. The productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the availability of these precursors in vivo. We describe a metabolic engineering strategy to enhance both the yield and volumetric productivity of polyketide biosynthesis. The genes matB and matC from Rhizobium trifolii encode a malonyl-CoA synthetase and a putative dicarboxylate transport protein, respectively. These proteins can directly convert exogenous malonate and methylmalonate into their corresponding CoA thioesters with an ATP requirement of 2 mol per mol of acyl-CoA produced. Heterologous expression of matBC in a recombinant strain of Streptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement of macrolactone titers. The unusual efficiency of the bioconversion is illustrated by the fact that approximately one-third of the methylmalonate units added to the fermentation medium are converted into macrolactones. The direct conversion of inexpensive feedstocks such as malonate and methylmalonate into polyketides represents the most carbon- and energy-efficient route to these high value natural products and has implications for cost-effective fermentation of numerous commercial and development-stage small molecules.

The mtmVUC genes of the mithramycin gene cluster in Streptomyces argillaceus are involved in the biosynthesis of the sugar moieties.

Mithramycin is a glycosylated aromatic polyketide produced by Streptomyces argillaceus, and is used as an antitumor drug. Three genes (mtmV, mtmU and mtmC) from the mithramycin gene cluster have been cloned, and characterized by DNA sequencing and by analysis of the products that accumulate in nonproducing mutants, which were generated by insertional inactivation of these genes. The mtm V gene codes for a 2,3-dehydratase that catalyzes early and common steps in the biosynthesis of the three sugars found in mithramycin (D-olivose, D-oliose and D-mycarose); its inactivation caused the accumulation of the nonglycosylated intermediate premithramycinone. The mtmU gene codes for a 4-ketoreductase involved in D-oliose biosynthesis, and its inactivation resulted in the accumulation of premithramycinone and premithramycin A , the first glycosylated intermediate which contains a D-olivose unit. The third gene, mtmC, is involved in D-mycarose biosynthesis and codes for a C-methyltransferase. Two mutants with lesions in the mtmC gene accumulated mithramycin intermediates lacking the D-mycarose moiety but containing D-olivose units attached to C-12a in which the 4-keto group is unreduced. This suggests that mtmC could code for a second enzyme activity, probably a D-olivose 4-ketoreductase, and that the glycosyltransferase responsible for the incorporation of D-olivose (MtmGIV) shows some degree of flexibility with respect to its sugar co-substrate, since the 4-ketoanalog is also transferred. A pathway is proposed for the biosynthesis of the three sugar moieties in mithramycin.

Analysis of two chromosomal regions adjacent to genes for a type II polyketide synthase involved in the biosynthesis of the antitumor polyketide mithramycin in Streptomyces argillaceus.

Mithramycin is an aromatic antitumour polyketide synthesized by Streptomyces argillaceus. Two chromosomal regions located upstream and downstream of the locus for the mithramycin type II polyketide synthase were cloned and sequenced. Analysis of the sequence revealed the presence of eight genes encoding three oxygenases (mtmOI, mtmOII and mtmOIII), three reductases (mtmTI, mtmTII and mtmTIII), a cyclase (mtm Y) and an acyl CoA ligase (mtmL). The three oxygenase genes were each inactivated by gene replacement. Inactivation of one of them (mtmOII) generated a non-producing mutant, while inactivation of the other two (mtmOl and mtmOIII) did not affect the biosynthesis of mithramycin. The mtmOII gene may code for an oxygenase responsible for the introduction of oxygen atoms at early steps in the biosynthesis of mithramycin leading to 4-demethylpremithramycinone. One of the reductases may be responsible for reductive cleavage of an intermediate from an enzyme and another for the reduction of a keto group in the side-chain of the mithramycin aglycon moiety. A hypothetical biosynthetic pathway showing in particular the involvement of oxygenase MtmOII and of various other gene products in mithramycin biosynthesis is proposed.

The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster.

Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.

Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin.

Two genes (mtmD and mtmE) were cloned and sequenced from the mithramycin producer Streptomyces argillaceus. Comparison with proteins in databases and enzymatic assays after expression in Escherichia coli showed that they encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively. The mtmD gene was inactivated by gene replacement, generating a nonproducing mutant that accumulates a tetracyclic compound designated premithramycinone. The identification of premithramycinone reveals new aspects of the mithramycin biosynthetic pathway and suggests that at least some glycosylations occur before breakage of the fourth ring.

An ABC transporter is essential for resistance to the antitumor agent mithramycin in the producer Streptomyces argillaceus.

Mithramycin is an antitumor antibiotic synthesized by Streptomyces argillaceus. This producer strain is highly resistant in vivo to mithramycin (MIC 100 micrograms/ml) but sensitive to the related drugs chromomycin and olivomycin (MIC 10 micrograms/ml). From a genomic library of S. argillaceus DNA two cosmid clones were isolated which confer a high level of resistance to mithramycin on S. albus. The resistance genes were mapped by subcloning to a 3.9-kb PstI-PvuII fragment. DNA sequence analysis of this fragment revealed one incomplete and three complete open reading frames. Subcloning experiments demonstrated that resistance to mithramycin is mediated by the genes mtrA and mtrB. The mtrA gene can potentially encode an ATP-binding protein of the ABC transporter superfamily, containing one nucleotide-binding domain and showing similarity with other ABC transporters involved in resistance to daunorubicin, oleandomycin and tetronasin in their respective producer strains. The mtrB gene codes for an integral membrane protein with six putative transmembrane helices. A mithramycin-sensitive mutant was generated in a gene replacement experiment by disrupting the mtrA gene, thus demonstrating that the system encoded by the mtrAB genes is essential for conferring resistance to mithramycin in S. argillaceus.

Characterization of Streptomyces argillaceus genes encoding a polyketide synthase involved in the biosynthesis of the antitumor mithramycin.

Mithramycin (Mtm) is an aromatic polyketide which shows antibacterial and antitumor activity. From a chromosomal cosmid library of Streptomyces argillaceus, a Mtm producer, a clone (cosAR7) was isolated by homology to the actI/III region of S. coelicolor and the strDEM genes of S. griseus. From this clone, a 5.3-kb DNA region was sequenced and found to encode six open reading frames (designated as mtmQXPKST1), five of them transcribed in the same direction. The deduced products of five of these genes resembled components of type-II polyketide synthases. The mtm genes would code for an aromatase (mtmQ), a polypeptide of unknown function (mtmX), a beta-ketoacylsynthase (mtmP) and a related 'chain length factor' (mtmK), an acyl carrier protein (mtmS) and a beta-ketoreductase (mtmT1). The involvement of this gene cluster in Mtm biosynthesis was demonstrated by the Mtm non-producing phenotype of mutants generated in two independent insertional inactivation experiments.