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jueves, 21 de mayo de 2015

Las levaduras pueden producir morfina y opiáceos

Biotechnology is about to make morphine production as simple as brewing beer. A paper published on 18 May in Nature Chemical Biology reports the creation of a yeast strain containing the first half of a biochemical pathway that turns simple sugars into morphine — mimicking the process by which poppies make opiates. Combined with other advances, researchers predict that it will be only a few years — or even months — before a single engineered yeast strain can complete the entire process.

Dueber and colleagues’ work does not reach that goal. But it demonstrates that, given the right genes and biochemical machinery, yeast can convert glucose into the intermediate compound (S)-reticuline — the first half of the poppy’s morphine-production pathway.

Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate (S)-reticuline from glucose in Saccharomyces cerevisiae. To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L-3,4-dihydroxyphenylalanine (L-DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L-DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L-tyrosine to (S)-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae, will enable low-cost production of many high-value BIAs.

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jueves, 7 de mayo de 2015

X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis

Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome1. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin2, 3. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation4, 5, 6, 7, 8, 9, 10, 11, 12. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain13, 14, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450–X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro—in particular the installation of the second crosslink in GPA biosynthesis—occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.


http://www.nature.com/nature/journal/v521/n7550/images_article/nature14141-f1.jpg

Domain labels for NRPS proteins (Tcp9–12): A, adenylation (selected amino acids indicated above the module: Hpg, 4-hydroxyphenylglycine; Dpg, 3,5-dihydroxyphenylglycine); C, condensation; E, epimerization; T, thiolation/peptidyl carrier protein (PCP); TE, thioesterase; X, domain of unknown function. Essential P450-catalysed aglycone rigidification takes place through crosslinking of aromatic side chains (OxyA–C, OxyE). Each crosslinking reaction is performed by a specific Oxy protein, with the products of each Oxy protein indicated schematically; standard ring nomenclature is indicated on the teicoplanin aglycone in red lettering.

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Complex archaea that bridge the gap between prokaryotes and eukaryotes

The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but the identity and nature of the putative archaeal ancestor remain a subject of debate. Here we describe the discovery of ‘Lokiarchaeota’, a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in phylogenomic analyses, and whose genomes encode an expanded repertoire of eukaryotic signature proteins that are suggestive of sophisticated membrane remodelling capabilities. 

Our results provide strong support for hypotheses in which the eukaryotic host evolved from a bona fide archaeon, and demonstrate that many components that underpin eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic ‘starter-kit’ to support the increase in the cellular and genomic complexity that is characteristic of eukaryotes.

Metagenomic reconstruction and phylogenetic analysis of Lokiarchaeum.
a, Schematic overview of the metagenomics approach. BI, Bayesian inference; ML, maximum likelihood. b, Bayesian phylogeny of concatenated alignments comprising 36 conserved phylogenetic marker proteins using sophisticated models of protein evolution (Methods), showing eukaryotes branching within Lokiarchaeota. Numbers above and below branches refer to Bayesian posterior probability and maximum-likelihood bootstrap support values, respectively. Posterior probability values above 0.7 and bootstrap support values above 70 are shown. Scale indicates the number of substitutions per site. c, Phylogenetic breakdown of the Lokiarchaeum proteome, in comparison with proteomes of Korarchaeota, Aigarchaeota (Caldiarchaeum) and Miscellaneous Crenarchaeota Group (MCG) archaea. Category ‘Other’ contains proteins assigned to the root of cellular organisms, to viruses and to unclassified proteins.

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jueves, 30 de abril de 2015

ParMRC es la maquinaria mitótica conocida mas simple

Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles

Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments1, 2, 3, 4, 5, 6. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments3, 7, 8, 9. Growing ParMRC spindles push sister plasmids to the cell poles9, 10. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.
ParM doublets in E. coli cells, imaged by cryo[hyphen]ET.

Fig. a, A mutant of ParM that hydrolyses ATP more slowly (D170A) was overexpressed in E. coli cells. Tomographic slices show large bundles of ParM blocking cell division. This experiment was performed two times. b, The ParMRC operon driven from highcopynumber plasmid pDD19. Tomographic slice showing an example of observed doublets. c, Tomographic slice for a mediumcopynumber plasmid (pKG321). d, Tomographic slice for a lowcopynumber plasmid, emulating the native lowcopynumber R1 plasmids (pKG491, ‘miniR1’ replicon) in E. coli (see Supplementary Videos 5 and 6 to view entire tomograms). Each experiment with different copynumber plasmids was performed once. e, Schematic depicting proposed asynchronous plasmid DNA segregation. Bipolar ParM spindles are seeded when replication has produced two parC centromeric regions, still in close proximity. Each seeds one unipolar ParM filament, which then come together in an antiparallel fashion to form the segregating bipolar spindle. Nonproductive unipolar filaments or spindles that lack plasmid attachment will be destroyed through the dynamic instability of ParM. This is in contrast to earlier ideas in which all sister plasmids would be segregated through one bundle of filaments, containing double the number of unipolar filaments as the copy number of the plasmid in the cell19.

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Bacterias que se comportan como cristales vivos


Fast-Moving Bacteria Self-Organize into Active Two-Dimensional Crystals of Rotating Cells


We investigate a new form of collective dynamics displayed by Thiovulum majus, one of the fastest-swimming bacteria known. Cells spontaneously organize on a surface into a visually striking two-dimensional hexagonal lattice of rotating cells. As each constituent cell rotates its flagella, it creates a tornadolike flow that pulls neighboring cells towards and around it. As cells rotate against their neighbors, they exert forces on one another, causing the crystal to rotate and cells to reorganize. We show how these dynamics arise from hydrodynamic and steric interactions between cells. We derive the equations of motion for a crystal, show that this model explains several aspects of the observed dynamics, and discuss the stability of these active crystals.

Figure 2
Fig. 1. A large bacterial crystal in dark-field illumination. The bright glow of individual cells results from light scattering off intercellular sulfur globules. The illumination of cells differs because the concentration of sulfur globules varies between cells. The scale bar is 10  μm.

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jueves, 9 de abril de 2015

The future of the postdoc

There is a growing number of postdocs and few places in academia for them to go. But change could be on the way.



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martes, 24 de marzo de 2015

An Escherichia coli Mutant That Makes Exceptionally Long Cells

Although Escherichia coli is a very small (1- to 2-μm) rod-shaped cell, here we describe an E. coli mutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 μm. These extremely elongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division.

IMPORTANCE Escherichia coli is usually a very small bacterium, 1 to 2 μm long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usual E. coli cell. E. coli filaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects of E. coli physiology that are difficult to investigate with small cells.      

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