126 research outputs found
Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids
Background De novo production of multi-hydroxylated diterpenoids is
challenging due to the lack of efficient redox systems. Results In this study
a new reductase/ferredoxin system from Streptomyces afghaniensis (AfR·Afx) was
identified, which allowed the Escherichia coli-based production of the
trihydroxylated diterpene cyclooctatin, a potent inhibitor of human
lysophospholipase. This production system provides a 43-fold increase in
cyclooctatin yield (15 mg/L) compared to the native producer. AfR·Afx is
superior in activating the cylcooctatin-specific class I P450s CotB3/CotB4
compared to the conventional Pseudomonas putida derived PdR·Pdx model. To
enhance the activity of the PdR·Pdx system, the molecular basis for these
activity differences, was examined by molecular engineering. Conclusion We
demonstrate that redox system engineering can boost and harmonize the
catalytic efficiency of class I hydroxylase enzyme cascades. Enhancing
CotB3/CotB4 activities also provided for identification of CotB3 substrate
promiscuity and sinularcasbane D production, a functionalized diterpenoid
originally isolated from the soft coral Sinularia sp
A role for AID in chromosome translocations between c-myc and the IgH variable region
Chromosome translocations between oncogenes and the region spanning the immunoglobulin (Ig) heavy chain (IgH) variable (V), diversity (D), and joining (J) gene segments (Ig V-JH region) are found in several mature B cell lymphomas in humans and mice. The breakpoints are frequently adjacent to the recombination signal sequences targeted by recombination activating genes 1 and 2 during antigen receptor assembly in preâB cells, suggesting that these translocations might be the result of aberrant V(D)J recombination. However, in mature B cells undergoing activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM), duplications or deletions that would necessitate a double-strand break make up 6% of all the Ig V-JH regionâassociated somatic mutations. Furthermore, DNA breaks can be detected at this locus in B cells undergoing SHM. To determine whether SHM might induce c-myc to Ig V-JH translocations, we searched for such events in both interleukin (IL) 6 transgenic (IL-6 tg) and AIDâ/â IL-6 tg mice. Here, we report that AID is required for c-myc to Ig V-JH translocations induced by IL-6
Natural and anthropogenic lead in sediments of the Rotorua lakes, New Zealand
Global atmospheric sources of lead have increased more than 100-fold over the past century as a result of deforestation, coal combustion, ore smelting and leaded petroleum. Lead compounds generally accumulate in depositional areas across the globe where, due to low solubility and relative freedom from microbial degradation, the history of their inputs is preserved. In lakes there is rapid deposition and often little bioturbation of lead, resulting in an excellent depositional history of changes in both natural and anthropogenic sources. The objective of this study was to use sediments from a regionally bounded set of lakes to provide an indication of the rates of environmental inputs of lead whilst taking into account differences of trophic state and lead exposure between lakes. Intact sediment gravity cores were collected from 13 Rotorua lakes in North Island of New Zealand between March 2006 and January 2007. Cores penetrated sediments to a depth of 16â30 cm and contained volcanic tephra from the 1886 AD Tarawera eruption. The upper depth of the Tarawera tephra enabled prescription of a date for the associated depth in the core (120 years). Each core showed a sub-surface peak in lead concentration above the Tarawera tephra which was contemporaneous with the peak use of lead alkyl as a petroleum additive in New Zealand. An 8 m piston core was taken in the largest of the lakes, Lake Rotorua, in March 2007. The lake is antipodal to the pre-industrial sources of atmospheric lead but still shows increasing lead concentrations from <2 up to 3.5 ÎŒg gâ1 between the Whakatane eruption (5530 ± 60 cal. yr BP) and the Tarawera eruption. Peaks in lead concentration in Lake Rotorua are associated with volcanic tephras, but are small compared with those arising from recent anthropogenic-derived lead deposition. Our results show that diagenetic processes associated with iron, manganese and sulfate oxidation-reduction, and sulfide precipitation, act to smooth distributions of lead from anthropogenic sources in the lake sediments. The extent of this smoothing can be related to changes in sulfate availability and reduction in sulfide driven by differences in trophic status amongst the lakes. Greatest lead mobilisation occurs in mesotrophic lakes during seasonal anoxia as iron and manganese are released to the porewater, allowing upward migration of lead towards the sedimentâwater interface. This lead mobilisation can only occur if sulfides are not present. The sub-surface peak in lead concentrations in lake sediments ascribed to lead alkyl in petroleum persists despite the diagenetic processes acting to disperse lead within the sediments and into the overlying water
The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila
The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Îsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila
ES&T Guest Comment: Celebrating Bidlemanâs 1988 âAtmospheric Processesâ
Since its 1988 appearance in ES&T, Terry F. Bidlemanâs article, âAtmospheric processes: wet and dry deposition of organic compounds are controlled by their vapor-particle partitioningâ, has had a notable impact on the field of contaminant science. The paper has been cited in over 600 journal articles published by authors from every continent. Far from fading into obscurity, the paperâs influence has been remarkably consistent. Citations over the last year match the annual average attained since publication
Evolution of Vitamin B 2 Biosynthesis. A Novel Class of Riboflavin Synthase in Archaea â
The open reading frame MJ1184 of Methanococcus jannaschii with similarity to riboflavin synthase of Methanothermobacter thermoautotrophicus was cloned into an expression vector but was poorly expressed in an Escherichia coli host strain. However, a synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of a protein with an apparent subunit mass of 17.5 kDa. The protein was purified to apparent homogeneity. Hydrodynamic studies indicated a relative mass of 88 kDa suggesting a homopentamer structure. The enzyme was shown to catalyze the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 24 nmol mg K1 min K1 at 40 8C. Divalent metal ions, preferably manganese or magnesium, are required for maximum activity. In contrast to pentameric archaeal type riboflavin synthases, orthologs from plants, fungi and eubacteria are trimeric proteins characterized by an internal sequence repeat with similar folding patterns. In these organisms the reaction is achieved by binding the two substrate molecules in an antiparallel orientation. With the enzyme of M. jannaschii, 13 C NMR spectroscopy with 13 C-labeled 6,7-dimethyl-8-ribityllumazine samples as substrates showed that the regiochemistry of the dismutation reaction is the same as observed in eubacteria and eukaryotes, however, in a non-pseudo-c 2 symmetric environment. Whereas the riboflavin synthases of M. jannaschii and M. thermoautotrophicus are devoid of similarity with those of eubacteria and eukaryotes, they have significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalyzing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor. Some Archaea have eubacterial type riboflavin synthases which may have been acquired by lateral gene transfer
Lipid transfer from plants to arbuscular mycorrhiza fungi
Arbuscular mycorrhiza (AM) symbioses contribute to global carbon cycles as plant hosts divert up to 20% of photosynthate to the obligate biotrophic fungi. Previous studies suggested carbohydrates as the only form of carbon transferred to the fungi. However, de novo fatty acid (FA) synthesis has not been observed in AM fungi in absence of the plant. In a forward genetic approach, we identified two Lotus japonicus mutants defective in AM-specific paralogs of lipid biosynthesis genes (KASI and GPAT6). These mutants perturb fungal development and accumulation of emblematic fungal 16:1 omega 5 FAs. Using isotopolog profiling we demonstrate that C-13 patterns of fungal FAs recapitulate those of wild-type hosts, indicating cross-kingdom lipid transfer from plants to fungi. This transfer of labelled FAs was not observed for the AM-specific lipid biosynthesis mutants. Thus, growth and development of beneficial AM fungi is not only fueled by sugars but depends on lipid transfer from plant hosts
Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants
Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simianâhuman immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants
Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis
In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis
Enhancing Production of Bio-Isoprene Using Hybrid MVA Pathway and Isoprene Synthase in E. coli
The depleting petroleum reserve, increasingly severe energy crisis, and global climate change are reigniting enthusiasm for seeking sustainable technologies to replace petroleum as a source of fuel and chemicals. In this paper, the efficiency of the MVA pathway on isoprene production has been improved as follows: firstly, in order to increase MVA production, the source of the âupper pathwayâ which contains HMG-CoA synthase, acetyl-CoA acetyltransferase and HMG-CoA reductase to covert acetyl-CoA into MVA has been changed from Saccharomyces cerevisiae to Enterococcus faecalis; secondly, to further enhance the production of MVA and isoprene, a alanine 110 of the mvaS gene has been mutated to a glycine. The final genetic strain YJM25 containing the optimized MVA pathway and isoprene synthase from Populus alba can accumulate isoprene up to 6.3 g/L after 40 h of fed-batch cultivation
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