Dual Emission and Mechanofluorochromism of a V-Shaped π-System Composed of Disulfonyl-Substituted Dibenzocyclooctatetraenes.

A series of dibenzocyclooctatetraenes 6 bearing phenylethynyl and phenylsulfonyl groups were synthesized from bromo-substituted formylbenzyl sulfone 4 via cyclic dimerization of 4 and Sonogashira coupling of the resulting dibromocyclooctatetraene 3 with terminal acetylenes. The diamino derivative 6b exhibited dual emission with emission maxima at 436 and 547 nm. Furthermore, in the fluorescence of 6b, solvatofluorochromism was observed in response to solvent polarity, whereas in the solid states, mechanofluorochromism was observed.

One-Shot Double Amination of Sondheimer-Wong Diynes: Synthesis of Photoluminescent Dinaphthopentalenes.

Photoluminescent diamino-substituted dinaphthopentalenes were synthesized successfully by the treatment of in situ prepared dinaphthocyclooctadiyne with lithium amide. This reaction involves a series of transformations including the nucleophilic addition of the lithium amide to a triple bond of the cyclooctadiyne moiety, transannulation, protonation of the resulting pentalene anion, and the nucleophilic substitution of the pentalene core with the lithium amide. In this procedure, a novel double amination step plays a key role. When the diamino-substituted dinaphthopentalenes were irradiated with UV light in toluene, fluorescence was observed at around 580 nm (ΦF < 0.03).

Hydrophilic and Hydrophobic Compounds Antithetically Affect the Growth of Eicosapentaenoic Acid-Synthesizing Escherichia coli Recombinants.

The growth of Escherichia coli DH5α recombinants producing eicosapentaenoic acid (EPA) (DH5αEPA+) and those not producing EPA (DH5αEPA-) was compared in the presence of hydrophilic or hydrophobic growth inhibitors. The minimal inhibitory concentrations of hydrophilic inhibitors such as reactive oxygen species and antibiotics were higher for DH5αEPA+ than for DH5αEPA-, and vice versa for hydrophobic inhibitors such as protonophores and radical generators. E. coli DH5α with higher levels of EPA became more resistant to ethanol. The cell surface hydrophobicity of DH5αEPA+ was higher than that of DH5αEPA-, suggesting that EPA may operate as a structural constituent in the cell membrane to affect the entry and efflux of hydrophilic and hydrophobic inhibitors.

Membrane eicosapentaenoic acid is involved in the hydrophobicity of bacterial cells and affects the entry of hydrophilic and hydrophobic compounds.

Eicosapentaenoic acid (EPA)-producing Shewanella marinintestina IK-1 (IK-1) and its EPA-deficient mutant IK-1Delta8 (IK-1Delta8) were grown on microtitre plates at 20 degrees C in a nutrient medium that contained various types of growth inhibitors. The minimal inhibitory concentrations of hydrogen peroxide and tert-butyl hydroxyl peroxide were 100 microM and 1 mM, respectively, for IK-1 and 10 and 100 microM, respectively, for IK-1Delta8. IK-1 was much more resistant than IK-1Delta8 to the four water-soluble antibiotics (ampicillin sodium, kanamycin sulphate, streptomycin sulphate, and tetracycline hydrochloride) tested. In contrast, IK-1 was less resistant than IK-1Delta8 to two hydrophobic uncouplers: carbonyl cyanide m-chloro phenylhydrazone (CCCP) and N,N'-dicyclohexylcarbodiimide (DCCD). The hydrophobicity of the IK-1 and IK-1Delta8 cells grown at 20 degrees C was determined using the bacterial adhesion to hydrocarbon method. EPA-containing ( approximately 10% of total fatty acids) IK-1 cells were more hydrophobic than their counterparts with no EPA. These results suggest that the high hydrophobicity of IK-1 cells can be attributed to the presence of membrane EPA, which shields the entry of hydrophilic membrane-diffusible compounds, and that hydrophobic compounds such as CCCP and DCCD diffuse more effectively in the membranes of IK-1, where they can fulfil their inhibitory activities, than in the membranes of IK-1Delta8.

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis.

When pDHA4, a vector carrying all five pfaA-pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA-pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.

The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1.

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Delta8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H(2)O(2)), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01 mM H(2)O(2) under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H(2)O(2) for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1 mM H(2)O(2) resulted in much lower intracellular concentrations of H(2)O(2) being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2) in S. marinintestina IK-1.

In vivo conversion of triacylglycerol to docosahexaenoic acid-containing phospholipids in a thraustochytrid-like microorganism, strain 12B.

The thraustochytrid-like microorganism, strain 12B, cultivated in peptone, yeast extract, and 8% (w/v) glucose in 50% (v/v) seawater, accumulated docosahexaenoic acid (DHA)-rich triacylglycerol (TAG) at 67% of total lipid. When these TAG-accumulated cells were cultivated in glucose-deficient medium, dry cell weight (3 mg per ml culture) increased approximately 3-fold relative to baseline but the TAG/total lipid decreased to 5%. At the same time, the amount of phospholipid (5 mg) per whole culture also increased 3-fold. Hence, phospholipid/total lipid increased from 13% to 67%. High levels of DHA (more than 50% of total) were maintained in phosphatidylcholine.

Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA.

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5alpha, a strain that carries EPA biosynthesis genes (pEPADelta1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.

The cell membrane-shielding function of eicosapentaenoic acid for Escherichia coli against exogenously added hydrogen peroxide.

The colony-forming ability of catalase-deficient Escherichia coli mutant genetically modified to produce eicosapentaenoic acid (EPA) showed less decrease than in a control strain producing no EPA, when treated with 0.3mM hydrogen peroxide (H(2)O(2)) under non-growth conditions. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The amount of fatty acids was decreased more significantly for cells lacking EPA than for those producing EPA. Much lower intracellular concentrations of H(2)O(2) were detected for cells with EPA than those lacking EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2).

Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli.

The docosahexaenoic acid (DHA) biosynthesis gene cluster (pDHA3) from the DHA-producing Moritella marina strain MP-1 includes the genes pfaA, pfaB, pfaC, and pfaD, which are similar to the genes of polyketide biosynthesis. When this cluster was co-expressed in Escherichia coli with M. marina MP-1 pfaE, which encodes phosphopantetheinyl transferase, DHA was biosynthesized. The maximum production of DHA (5% of total fatty acids) was observed at 15 degrees C. This is the first report of the recombinant production of DHA in a polyketide biosynthesis mode.

A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1.

A phosphopantetheinyl transferase (PPTase) gene (pfaE), cloned from the docosahexaenoic acid (DHA)-producing bacterium Moritella marina strain MP-1, has an open reading frame of 861 bp encoding a 287-amino acid protein. When the pfaE gene was expressed with pfaA-D, which are four out of five essential genes for biosynthesis of eicosapentaenoic acid (EPA) derived from Shewanella pneumatophori SCRC-2738 in Escherichia coli, the recombinant produced 12% EPA of total fatty acids. This suggests that pfaE encodes a PPTase required for producing n-3 polyunsaturated fatty acids, which is probably involved in the synthesis of DHA in M. marina strain MP-1.

Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages.

The colony-forming ability of Escherichia coli genetically engineered to produce eicosapentaenoic acid (EPA) grown in 3mM hydrogen peroxide (H(2)O(2)) was similar to that of untreated cells. It was rapidly lost in the absence of EPA. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The fatty acid composition of the transformants was unaffected by H(2)O(2) treatment, but the amount of fatty acids decreased in cultures of cells lacking EPA and increased in cultures of cells producing EPA, suggesting that cellular EPA is stable in the presence of H(2)O(2) in vivo and may protect cells directly against oxidative damage. We discuss the possible role of EPA in partially blocking the penetration of H(2)O(2) into cells through membranes containing EPA.

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria.

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 microg cerulenin ml-1, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the content of DHA in the total fatty acids increased from 5.9-19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l-1 by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 microg cerulenin ml-1, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l-1. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.