Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 microg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p(R) promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.     

High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.     

Cyclic AMP delays neutrophil apoptosis via stabilization of Mcl-1

Human neutrophils underwent spontaneous apoptosis, which was accompanied by degradation of Mcl-1, but not other anti-apoptotic molecules (cIAP1, cIAP2, A1, survivin and Bcl-2). Spontaneous neutrophil apoptosis and Mcl-1 degradation were prevented by cyclic AMP (cAMP) agonists (dibutyryl cAMP and prostaglandin E(1)), and the effects of cAMP agonists on neutrophils were highly resistant to cycloheximide, a protein synthesis inhibitor, although slight increase in Mcl-1 mRNA expression was induced by cAMP agonists. Proteasome inhibitors (epoxomicin and lactacystin) also prevented spontaneous neutrophil apoptosis and Mcl-1 degradation to the same extent as cAMP agonists, and no additive effect was obtained by combination of cAMP agonists and proteasome inhibitors. These findings suggest that cAMP agonists, like proteasome inhibitors, delay neutrophil apoptosis primarily via stabilization of Mcl-1.     

Endocrine secretory granule production is caused by a lack of REST and intragranular secretory content and accelerated by PROX1

Journal of Molecular Histology - Endocrine secretory granules (ESGs) are morphological characteristics of endocrine/neuroendocrine cells and store peptide hormones/neurotransmitters. ESGs contain...     

Synthesis and anti-HIV-1 evaluation of phosphonates which mimic the 5′-monophosphate of 4′-branched 2′,3′-didehydro-2′,3′-dideoxy nucleosides

Synthesis of the 4′-ethynyl and 4′-cyano phosphonates 8–11, which mimic the 5′-monophosphate of 4′-branched 2′,3′-didehydro-2′,3′-dideoxy nucleosides, was investigated by employing the 3′,4′-unsaturated nucleosides (13 and 28) as the starting material. The synthesis was initiated by the electrophilic addition of NIS/(EtO)₂P(O)CH₂OH to these unsaturated nucleosides. After introduction of the 2′,3′-double bond, the 4′-hydroxylmethyl group of the resulting adducts was transformed into the ethynyl or cyano group. While the 4′-cyano phosphonates 9 and 11 were not sufficiently stable to be isolated, the 4′-ethynyl counterparts (8 and 10) were obtained as their mono-ammonium salts. The adenine derivative 8 showed almost comparable anti-HIV-1 activity to that of d4T.     

Variable relationships between the hydrophobic fraction of dissolved organic matter and metals in Scottish freshwater before the estuarine mixing zone

Limnology - The organic-Fe association in Scottish freshwater rivers has received little attention compared with in the estuarine mixing zone. We collected 201 water samples from rivers and lakes...