Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli

For metabolic engineering it is advantageous in terms of stability, genetic regulation, and metabolic burden to modulate expression of relevant genes on the chromosome rather than relying on over-expression of the genes on multi-copy vectors. Here we have increased the production of beta-carotene in Escherichia coli by replacing the native promoter of the chromosomal isoprenoid genes with the strong bacteriophage T5 promoter (P(T5)). We recombined PCR fragments with the lambda-Red recombinase to effect chromosomal promoter replacement, which allows direct integration of a promoter along with a selectable marker that can subsequently be excised by the Flp/FRT site-specific recombination system. The resulting promoter-engineered isoprenoid genes were combined by serial P1 transductions into a host strain harboring a reporter plasmid containing beta-carotene biosynthesis genes allowing a visual screen for yellow color indicative of beta-carotene accumulation. Construction of an E. coli P(T5)-dxs P(T5)-ispDispF P(T5)-idi P(T5)-ispB strain resulted in producing high titers (6mg/g dry cell weight) of beta-carotene. Surprisingly, over-expression of the ispB gene, which was expected to divert carbon flow from the isoprenoid pathway to quinone biosynthesis, resulted in increased beta-carotene production. We thus demonstrated that chromosomal promoter engineering of the endogenous isoprenoid pathway yielded high levels of beta-carotene in a non-carotenogenic E. coli. The high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes.     

Identification of essential amino acid residues in valine dehydrogenase from Streptomyces albus

Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase (ValDH) were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydroganase sequences. To ascertain the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This residue was chosen, because it has been proposed to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic analysis of the V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed less activity than the wild type enzyme toward all aliphatic and aromatic amino acids tested.     

Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling

Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades.     

Adaptive stress response in segmental progeria resembles long-lived dwarfism and calorie restriction in mice

How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPD^G602D/R722W/XPA^−/−) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80^−/− mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage.     

Ligand profiling and identification technology for searching bioactive ligands

We introduce a new methodology named ligand profiling and identification for effective discovery of bioactive ligands such as peptide hormones. This technology was developed from a new concept of parallel column chromatography and active fraction profiling by nano-LC MS. Traditional methods use sequential column chromatography, and thus are inevitably limited by the low abundance of the peptide of interest and by a low yield due to the many column steps. Using this new technology, insulin was successfully identified and diarginylinsulin, a minor intermediate form of insulin, was unexpectedly also identified simultaneously from 100 mg of porcine pancreatic tissue. This integrative technology could be used to search for various low-abundance peptides (or bioactive molecules) rapidly and simultaneously, by applying this to the later stages of traditional sequential purification.     

A yeast clade near Candida kruisii uncovered: nine novel Candida species associated with basidioma-feeding beetles

Yeasts similar to Candida kruisii were isolated repeatedly from the digestive tracts of basidioma-feeding beetles, especially nitidulids inhabiting and feeding on a variety of agarics in the southeastern USA and Barro Colorado Island, Panama. Based on the identical sequences of the D1/D2 domains of the LSU rRNA gene (rDNA) and host beetle information, the isolates were grouped into 19 genotypes which varied from C. kruisii by up to 38 nucleotide differences in the D1/D2 region. Phylogenetic analysis of rDNA sequences and phenotypic traits placed the isolates in C. kruisii and in nine undescribed taxa. The new species and type strains are designated as Candida pallodes (NRRL Y-27653^T), C. tritomae (NRRL Y-27650^T), C. panamensis (NRRL Y-27657^T), C. lycoperdinae (NRRL Y-27658^T), C. atbi (NRRL Y-27651^T), C. barrocoloradensis (NRRL Y-27934^T), C. aglyptinia (NRRL Y-27935^T), C. stri (NRRL Y-48063^T), and C. gatunensis (NRRL Y-48064^T). A phylogeny based on analysis of a combined database of sequences of SSU and LSU rDNA and the ITS region showed that the nine new species formed a novel sister clade to C. kruisii that was strongly supported by bootstrap analysis. Candida pallodes, C. tritomae, C. panamensis, and C. lycoperdinae formed one subclade, while C. atbi, C. barrocoloradensis, C. aglyptinia, C. stri, and C. gatunensis formed a second distinct subclade within the larger clade. Candida pallodes and C. atbi showed a strong host specificity to beetle species in the genus Pallodes (Coleoptera: Nitidulidae) collected from a variety of agarics. On the other hand, C. panamensis, C. tritomae, and C. lycoperdinae were associated with several unrelated beetles in Erotylidae, Scarabaeidae, Tenebrionidae, and Curculionidae as well as Lycoperdina ferruginea (Nitidulidae). Candida pallodes, C. tritomae, C. lycoperdinae, and C. atbi have been isolated repeatedly in the USA, while the other five new species have been found only at Barro Colorado Island, Panama.     

Three new asexual arthroconidial yeasts, Geotrichum carabidarum sp. nov., Geotrichum histeridarum sp. nov., and Geotrichum cucujoidarum sp. nov., isolated from the gut of insects

Twenty arthroconidial yeasts were isolated from the digestive tract of basidiome-feeding beetles and lepidopteran larvae. All of the yeasts reproduced only asexually by arthroconidia and some by endo- or blastoconidia as well. Based on the comparisons of sequences in ribosomal RNA genes and other taxonomic characteristics, the yeasts were identified as three unknown Geotrichum species. The three new species are described as Geotrichum carabidarum (NRRL Y-27727^T), G. histeridarum (NRRL Y-27729^T), and G. cucujoidarum (NRRL Y-27731^T). Phylogenetic analyses from ribosomal DNA sequences showed that members of the genus Geotrichum and related arthroconidial yeast taxa were divided into two major clades: (1) Dipodascus and Galactomyces with Geotrichum anamorphs including all the new species; and (2) Magnusiomyces with Saprochaete anamorphs. G. cucujoidarum formed a subclade with G. fermentans and Geotrichum sp. Y-5419, while the two closely related species, G. carabidarum and G. histeridarum, represent a new basal subclade in the clade of Geotrichum and its teleomorphs.     

The structure-activity relationships of mansonone F, a potent anti-MRSA sesquiterpenoid quinone: SAR studies on the C6 and C9 analogs

For the systematic SAR study on mansonone F, a series of C6 and C9 analogs of mansonone F have been synthesized and their anti-MRSA activities were evaluated. Most of the analogs exhibited good or excellent anti-MRSA activities. In particular, the 6-n-butylmansonone F showed fourfold higher antibacterial activities compared to that of vancomycin.     

Identification of a new functional target of haloperidol metabolite: implications for a receptor-independent role of 3-(4-fluorobenzoyl) propionic acid

Haloperidol, a dopamine D2 receptor blocker, is a classical neuroleptic drug that elicits extrapyramidal symptoms. Its metabolites include 3-(4-fluorobenzoyl) propionic acid (FBPA) and 4-(4-chlorophenyl)-4-piperidinol (CPHP). Until now, the biological significance of these metabolites has remained largely unknown. Here, we report that the administration of FBPA to mice effected a suppression of locomotor activity and induced catalepsy in a manner similar to that observed with haloperidol, whereas CPHP had no significant effects. Neither of these two metabolites, however, exhibited any ability to bind to the dopamine D2 receptor. FBPA blocked dopamine-induced extracellular signal-regulated kinase 1/2 phosphorylation, and it specifically affected mitogen-activated protein kinase kinase (MEK)1/2 activity in hippocampal HN33 cells. Moreover, FBPA was capable of direct interaction with MEK1/2, and inhibited its activity in vitro. We demonstrated the generation of haloperidol metabolites within haloperidol-treated cells by mass spectrometric analyses. Collectively, our results confirm the biological activity of FBPA, and provide initial clues as to the receptor-independent role of haloperidol.