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61.
Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis. 相似文献
62.
Darren G. Quin Alexander Riek Stuart Green Andrew P. Smith Fritz Geiser 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2010,155(3):336-340
Sugar gliders, Petaurus breviceps (average body mass: 120 g) like other small wild mammals must cope with seasonal changes in food availability and weather and therefore thermoregulatory and energetic challenges. To determine whether free-ranging sugar gliders, an arboreal marsupial, seasonally adjust their energy expenditure and water use, we quantified field metabolic rates (FMR) and water flux at a seasonal cool-temperate site in eastern Australia. Thirty six male and female sugar gliders were labelled with doubly labelled water for this purpose in spring, summer and autumn. The mean FMR was 159 ± 6 kJ d? 1 (spring), 155 ± 8 kJ d? 1 (summer), and 152 ± 20 kJ d? 1 (autumn) and the mean FMR for the three seasons combined was 158 ± 5 kJ d? 1 (equivalent to 1.33 kJ g? 1 d? 1 or 780 kJ kg? 0.75 d? 1). The mean total body water was 83 ± 2 g, equal to 68.5% of body weight. Mean water flux was 29 ± 1 mL day? 1. Season, ambient temperature or sex did not affect any of the measured and estimated physiological variables, but body mass and total body water differed significantly between sexes and among seasons. Our study is the first to provide evidence for a constant FMR in a small mammal in three different seasons and despite different thermal conditions. This suggests that seasonal changes in climate are compensated for by behavioural and physiological adjustments such as huddling and torpor known to be employed extensively by sugar gliders in the wild. 相似文献
63.
João Batista A Oliveira Claudia G Petersen Fabiana C Massaro Ricardo LR Baruffi Ana L Mauri Liliane FI Silva Juliana Ricci José G FrancoJr 《Reproductive biology and endocrinology : RB&E》2010,8(1):56
Background
Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval. 相似文献64.
M Ozkan SG Desai Y Zhang DM Stevenson J Beane EA White ML Guerinot LR Lynd 《Journal of industrial microbiology & biotechnology》2001,27(5):275-280
Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described
strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation
were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but
one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences
from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate
kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate
kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have
several positive implications with respect to future development of a transformation system for cellulolytic thermophiles.
Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280.
Received 12 September 2000/ Accepted in revised form 20 November 2000 相似文献
65.
66.
The meta-cleavage pathway of Pseudomonas putida mt-2 was simulated using a biochemical systems simulation developed by Regan (1996). A non-competitive inhibition term for catechol-2,3-dioxygenase (C23O) by 2-OH-pent-2,4-dienoate (Ki = 150 μM) was incorporated into the model. The simulation predicted steady state accumulation levels in the μM range for metabolites pre-meta-cleavage, and in the mM range for metabolites post-meta-cleavage. The logarithmic gains L[V-i, Xj] and L[X-i, Xj] clearly indicated that the pathway was most sensitive to the concentration of the starting substrate, benzoate, and the first enzyme of the pathway, toluate-1, 2-dioxygenase (TO). The simulation was validated experimentally; it was found that the amplification of TO increased the steady state flux from 0.024 to 0.091 (mmol/g cell dwt)/h. This resulted in an increased accumulation of a number of the pathway metabolites (intra- and extracellularly), especially cis-diol, 4-OH-2-oxovalerate, and 4-oxalocrotonate. Metabolic control analysis indicated that C23O was, in fact, the major controling enzymic step of the pathway with a scaled control coefficient of 0.83. The amplification of TO resulted in a shift of some of the control away from C23O. Catechol-2,3-dioxygenase, however, remained as the major controling element of the pathway. Copyright 1998 John Wiley & Sons, Inc. 相似文献
67.
Higher-level snake phylogeny inferred from mitochondrial DNA sequences of 12S rRNA and 16S rRNA genes 总被引:3,自引:0,他引:3
Portions of two mitochondrial genes (12S and 16S ribosomal RNA) were
sequenced to determine the phylogenetic relationships among the major
clades of snakes. Thirty-six species, representing nearly all extant
families, were examined and compared with sequences of a tuatara and three
families of lizards. Snakes were found to constitute a monophyletic group
(confidence probability [CP] = 96%), with the scolecophidians (blind
snakes) as the most basal lineages (CP = 99%). This finding supports the
hypothesis that snakes underwent a subterranean period early in their
evolution. Caenophidians (advanced snakes), excluding Acrochordus, were
found to be monophyletic (CP = 99%). Among the caenophidians, viperids were
monophyletic (CP = 98%) and formed the sister group to the elapids plus
colubrids (CP = 94%). Within the viperids, two monophyletic groups were
identified: true vipers (CP = 98%) and pit vipers plus Azemiops (CP = 99%).
The elapids plus Atractaspis formed a monophyletic clade (CP = 99%). Within
the paraphyletic Colubridae, the largely Holarctic Colubrinae was found to
be a monophyletic assemblage (CP = 98%), and the Xenodontinae was found to
be polyphyletic (CP = 91%). Monophyly of the henophidians (primitive
snakes) was neither supported nor rejected because of the weak resolution
of relationships among those taxa, except for the clustering of Calabaria
with a uropeltid, Rhinophis (CP = 94%).
相似文献
68.
Summary The uptake of tungsten by the New Zealand silver beech (Nothofagus menziesii) was investigated by pot trials with seedlings of this species in order to obtain information on the mode of their accumulation of this element as a preliminary to further field studies involving biogeochemical prospecting. Tungsten accumulation was most pronounced in the roots. Under controlled glasshouse conditions, the concentrations of this element in the roots, stems and leaves, were all related to levels in the soil. 相似文献
69.
Identification of a biosynthetic gene cluster and the six associated lipopeptides involved in swarming motility of Pseudomonas syringae pv. tomato DC3000 总被引:1,自引:0,他引:1
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Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to "mine" the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites. 相似文献