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101.
Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica 总被引:1,自引:0,他引:1
Förster A Aurich A Mauersberger S Barth G 《Applied microbiology and biotechnology》2007,75(6):1409-1417
The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess.
Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production
bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l−1 CA with a yield Y
CA of 0.75–0.82 g g−1, whereas at pH 5.0, 87 g l −1 with a yield Y
CA of 0.51 gg−1 were produced. The CA-productivity Q
CA increased from 0.40 g l −1 h−1 at pH 5.0 up to 0.73 g l −1 h−1 at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA
production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression
resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production. 相似文献
102.
During endomitosis, megakaryocytes undergo several rounds of DNA synthesis without division leading to polyploidization. In primary megakaryocytes and in the megakaryocytic cell line CHRF, loss or knock-down of p53 enhances cell cycling and inhibits apoptosis, leading to increased polyploidization. To support the hypothesis that p53 suppresses megakaryocytic polyploidization, we show that stable expression of wild-type p53 in K562 cells (a p53-null cell line) attenuates the cells' ability to undergo polyploidization during megakaryocytic differentiation due to diminished DNA synthesis and greater apoptosis. This suggested that p53's effects during megakaryopoiesis are mediated through cell cycle- and apoptosis-related target genes, possibly by arresting DNA synthesis and promoting apoptosis. To identify candidate genes through which p53 mediates these effects, gene expression was compared between p53 knock-down (p53-KD) and control CHRF cells induced to undergo terminal megakaryocytic differentiation using microarray analysis. Among substantially downregulated p53 targets in p53-KD megakaryocytes were cell cycle regulators CDKN1A (p21) and PLK2, proapoptotic FAS, TNFRSF10B, CASP8, NOTCH1, TP53INP1, TP53I3, DRAM1, ZMAT3 and PHLDA3, DNA-damage-related RRM2B and SESN1, and actin component ACTA2, while antiapoptotic CKS1B, BCL2, GTSE1, and p53 family member TP63 were upregulated in p53-KD cells. Additionally, a number of cell cycle-related, proapoptotic, and cytoskeleton-related genes with known functions in megakaryocytes but not known to carry p53-responsive elements were differentially expressed between p53-KD and control CHRF cells. Our data support a model whereby p53 expression during megakaryopoiesis serves to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. Furthermore, we identify a putative p53 regulon that is proposed to orchestrate these effects. 相似文献
103.
Christian Sturmbauer Corinna Fuchs Georg Harb Elisabeth Damm Nina Duftner Michaela Maderbacher Martin Koch Stephan Koblmüller 《Hydrobiologia》2008,615(1):57-68
Lake Tanganyika, the second-oldest and second-deepest lake in the world, harbors an impressive cichlid fish fauna counting about 250 endemic species that are characterized by a great level of ecological, morphological, and behavioral specialization. This study describes and compares cichlid fish communities at two rocky shores with differential human impact in the south of Lake Tanganyika. Species inventories and depth-dependent abundances were elaborated. About 41 and 46 sympatric cichlid species were recorded in the two study sites, respectively. Variabilichromis moorii was the most abundant species (29–60% of total number of fishes), followed by Aulonocranus dewindti (3–19%), Tropheus moorii (12%), Ophthalmotilapia ventralis (4–10%), Eretmodus cyanostictus (6–11%), and Cyathopharynx furcifer (0.01–9%). All other species had abundances below 5%. It further emerged that large cichlids such as Petrochromis species, Cyathopharynx furcifer, and Lobochilotes labiatus were very rare at one location, with frequencies of 0.55% or less. Territorial sizes of three particularly abundant species, Variabilichromis moorii, Aulonocranus dewindti, and Tropheus moorii, were assessed by behavioral observations. We distinguished between territorial core areas and total defended area, yielding average core areas between 0.4 (V. moorii) and 1.6 m2 (T. moorii), and total defended areas averaging for each species between 1.6 (V. moorii) and 5.0 m2 (A. dewindti) with no significant differences between the two study sites. The data on individual densities are also relevant for evolutionary studies, in that they allow more accurate calculations of effective population sizes. 相似文献
104.
Traditional cultivation-based methods to quantify microbial abundance are not suitable for analyses of microbial communities
in environmental or medical samples, which consist mainly of uncultured microorganisms. Recently, different cultivation-independent
quantification approaches have been developed to overcome this problem. Some of these techniques use specific fluorescence
markers, for example ribosomal ribonucleic acid targeted oligonucleotide probes, to label the respective target organisms.
Subsequently, the detected cells are visualized by fluorescence microscopy and are quantified by direct visual cell counting
or by digital image analysis. This article provides an overview of these methods and some of their applications with emphasis
on (semi-)automated image analysis solutions. 相似文献
105.
Horton R Gibson R Coggill P Miretti M Allcock RJ Almeida J Forbes S Gilbert JG Halls K Harrow JL Hart E Howe K Jackson DK Palmer S Roberts AN Sims S Stewart CA Traherne JA Trevanion S Wilming L Rogers J de Jong PJ Elliott JF Sawcer S Todd JA Trowsdale J Beck S 《Immunogenetics》2008,60(1):1-18
The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised
as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively
annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against
which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC
haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed,
resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have
been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence
of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets
revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line)
designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent
builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation
data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource
for future association studies of all MHC-associated diseases and transplant medicine.
Horton and Gibson contributed equally to this work. 相似文献
106.
Stephan Grosse Hélène Bergeron Akihiro Imura Jason Boyd Shaozhao Wang Kazuo Kubota Akihiko Miyadera Traian Sulea Peter C. K. Lau 《Microbial biotechnology》2010,3(1):65-73
There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50°C was outperformed by TtEST, a naturally occurring homologue with a Tm of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis. 相似文献
107.
108.
Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes. 相似文献
109.
110.
Joanneke E. Jansen Dominik Aschenbrenner Holm H. Uhlig Mark C. Coles Eamonn A. Gaffney 《PLoS computational biology》2022,18(6)
Cell-cell communication is mediated by many soluble mediators, including over 40 cytokines. Cytokines, e.g. TNF, IL1β, IL5, IL6, IL12 and IL23, represent important therapeutic targets in immune-mediated inflammatory diseases (IMIDs), such as inflammatory bowel disease (IBD), psoriasis, asthma, rheumatoid and juvenile arthritis. The identification of cytokines that are causative drivers of, and not just associated with, inflammation is fundamental for selecting therapeutic targets that should be studied in clinical trials. As in vitro models of cytokine interactions provide a simplified framework to study complex in vivo interactions, and can easily be perturbed experimentally, they are key for identifying such targets. We present a method to extract a minimal, weighted cytokine interaction network, given in vitro data on the effects of the blockage of single cytokine receptors on the secretion rate of other cytokines. Existing biological network inference methods typically consider the correlation structure of the underlying dataset, but this can make them poorly suited for highly connected, non-linear cytokine interaction data. Our method uses ordinary differential equation systems to represent cytokine interactions, and efficiently computes the configuration with the lowest Akaike information criterion value for all possible network configurations. It enables us to study indirect cytokine interactions and quantify inhibition effects. The extracted network can also be used to predict the combined effects of inhibiting various cytokines simultaneously. The model equations can easily be adjusted to incorporate more complicated dynamics and accommodate temporal data. We validate our method using synthetic datasets and apply our method to an experimental dataset on the regulation of IL23, a cytokine with therapeutic relevance in psoriasis and IBD. We validate several model predictions against experimental data that were not used for model fitting. In summary, we present a novel method specifically designed to efficiently infer cytokine interaction networks from cytokine perturbation data in the context of IMIDs. 相似文献