Engineering Deoxysugar Biosynthetic Pathways from Antibiotic-Producing Microorganisms A Tool to Produce Novel Glycosylated Bioactive Compounds

AbstractA plasmid (pLN2) was generated in which genes involved in the biosynthesis of L-oleandrose in the oleandomycin producer Streptomyces antibioticus ATCC11891 were cloned. pLN2 was used to direct the biosynthesis of different deoxysugars by exchanging and/or adding genes from other antibiotic biosynthetic clusters. Transfer of the synthesized deoxysugars to the tetracenomycin C aglycon, 8-demethyl-tetracenomycin C, through the use of the “sugar flexible” glycosyltransferase ElmGT, validated the system. Several pLN2 derivatives were constructed by replacement of the oleU 4-ketoreductase gene by different 4-ketoreductase genes. Some of them, such as EryBIV and UrdR, reduced the keto group of the 4-keto intermediates, generating L-olivosyl and D-olivosyl derivatives, respectively. The system was also used to generate an L-rhamnosyl derivative (through a two-gene deletion) and an L-rhodinosyl derivative (through a combination of a gene replacement and a gene addition)

Characterization and engineering of the biosynthesis gene cluster for antitumor macrolides PM100117 and PM100118 from a marine actinobacteria: generation of a novel improved derivative

Additional file 1: Figure S1. Sequence alignment of the cluster PKS domains active sites. LD, loading domain; M1-M20, extension modules. Polyketide synthase domains are as follows: KS, ketosynthase; AT, acyltransferase; KR, ketoreductase; DH, dehydratase; ER, enoilreductase; ACP, acyl carrier protein; CAL, CoA-ligase. Figure S2. Genetic complementation of mutant strains. UPLC analysis of PM100117 (1) and PM100118 (2) production in strains GUA-pS, CPgonP8, CPgonM4, CPgonMT, CPgonSL, CPgonS1, CPgonS2, CPgonCP, CPgonMR and CPgonL1. Figure S3. Antibiotic activity test of compound 5. Diffusion disc assay against Saccharomyces cerevisiae and Micrococcus luteus. The length (cm) of the inhibition-growth halo is indicated by numbers and yellow lines. Methods S2. Antibiotic activity assay. Format: PDF

Searching for glycosylated natural products in actinomycetes and identification of novel macrolactams and angucyclines

Many bioactive natural products are glycosylated compounds in which the sugar components usually participate in interaction and molecular recognition of the cellular target. Therefore, the presence of sugar moieties is important, in some cases essential, for bioactivity. Searching for novel glycosylated bioactive compounds is an important aim in the field of the research for natural products from actinomycetes. A great majority of these sugar moieties belong to the 6-deoxyhexoses and share two common biosynthetic steps catalyzed by a NDP-D-glucose synthase (GS) and a NDP-D-glucose 4,6-dehydratase (DH). Based on this fact, seventy one Streptomyces strains isolated from the integument of ants of the Tribe Attini were screened for the presence of biosynthetic gene clusters (BGCs) for glycosylated compounds. Total DNAs were analyzed by PCR amplification using oligo primers for GSs and DHs and also for a NDP-D-glucose-2,3-dehydratases. Amplicons were used in gene disruption experiments to generate non-producing mutants in the corresponding clusters. Eleven mutants were obtained and comparative dereplication analyses between the wild type strains and the corresponding mutants allowed in some cases the identification of the compound coded by the corresponding cluster (lobophorins, vicenistatin, chromomycins and benzanthrins) and that of two novel macrolactams (sipanmycin A and B). Several strains did not show UPLC differential peaks between the wild type strain and mutant profiles. However, after genome sequencing of these strains, the activation of the expression of two clusters was achieved by using nutritional and genetic approaches leading to the identification of compounds of the cervimycins family and two novel members of the warkmycins family. Our work defines a useful strategy for the identification new glycosylated compounds by a combination of genome mining, gene inactivation experiments and the activation of silent biosynthetic clusters in Streptomyces strains

Effects of size and morphology on swimming performance in juvenile brown trout (<i>Salmo trutta</i> L.)

Our study assesses swimming capacity (speed and stamina) and possible morphometric determinants of locomotor performance of juvenile brown trout (Salmo trutta L.). We addressed these issues at the individual level to have an approach of the functional significance of intraspecific variation in morphological design. Both swimming speed and endurance time showed significant positive relationships with fish length. Size-corrected values of speed and endurance time were negatively correlated suggesting a phenotypic trade-off between burst and prolonged swimming. Size was also highly correlated with all the morphological variables measured. Therefore, we used the residuals of the regressions of those variables on fish length to remove the effect of body size. A principal components analysis (PCA) summarised the 12 morphological variables into two factors, which accounted for 44.3% of the variance. PC1 combined several measures of body depth and width, whereas PC2 represented mainly postanal length relative to abdomen length. Relationships between the scores of the two factors and size-corrected values of maximum swimming speed and endurance time were weak. PC2 showed a significant positive relationship with endurance time; that is, individuals with longer caudal regions were able to swim against water flow for longer periods of time. Stoutness (PC1) showed a marginally significant negative correlation with endurance time. The lack of stronger relationships could be because of the low morphometric variability among the test individuals, all proceeding from the same population, reared in a common environment, and measured at the same ontogenetic stage.</p

Thermal dependence of embryonic growth and development in brown trout

Fertilized eggs from a brown trout Salmo trutta population in northern Spain were incubated in the laboratory at 4, 6, 8, 10, 12, 14, 16 and 18degrees C. Developmental stage and embryo size were monitored by taking samples at regular intervals. Survival was maximal at 8 and 10degrees C and decreased at higher and lower temperatures. Despite starting development, no embryo hatched at 16 and 18degrees C, which suggests an upper thermal limit for development between 14 and 16degrees C. Time required to reach a given ontogenetic stage decreased with increasing temperature. Embryos incubated at lower temperatures were larger at 50% hatching, and these differences.. persisted throughout the subsequent embryonic period until the start of exogenous feeding. A comparison with previously published data indicates low interpopulation variability in thermal sensitivity of embryonic development, even in consideration of the great latitudinal range of the studies. (C) 2003 The Fisheries Society of the British Isles.</p

Thermal sensitivity of growth, food intake and activity of juvenile brown trout

Growth rate, food intake and activity of juvenile brown trout (Salmo trutta L.) were studied at 10 constant temperatures (2.6-22.3 degreesC) and using eight full-sib families. We found between-family differences in growth rate but not significant temperature x family interaction. The effects of temperature were well described by polynomial regressions that allowed the calculation of optima, performance breadths and thermal limits. The obtained values were noticeably higher and the performance breadth wider, than previously reported data for the species. (C) 2001 Elsevier Science Ltd. All rights reserved.</p

Interactions of Myxococcus xanthus and Streptomyces sp. induces antibiotic production and changes in Myxococcus development

Resumen del trabajo presentado al Congreso del ANQUE-ICCE-BIOTEC: Science, the key for a better life; celebrado en Madrid del 1 al 4 de julio de 2014.In natural environments, the microbial populations are complex communities composed of several species of microorganisms that interact with each other competing or collaborating in order to survive. In this work we have studied the interaction between different typical soil bacteria as Myxococcus xanthus and several species of Streptomyces in laboratory conditions. M. xanthus, a motile bacteria, exerts its predatory effect on most of Streptomyces species inducing the lysis of the Streptomyces hyphae in the contact zone. These interactions triggers the production of antibiotics either in M. xanthus or in some of the Streptomyces species but not in all. Nevertheless, induction of silent pathways was observed neither in M. xanthus nor in the Streptomyces species under the conditions assayed. Changes in the differentiation pattern was observed in M. xanthus in the interactions with some species of Streptomyces such as S. griseus that induces the grouping of Myxococcus and the formation of ripplings. These results open new possibilities for increasing the production of some antibiotics by conducting co-culture or by adding the inducer molecules produced by other bacteria to cultures of producing microorganisms (Pérez et al. Microb Biotechnol 2011 4:175 -183).Peer Reviewe