Reconstitution of polyunsaturated fatty acid synthesis enzymes in mammalian cells to convert LA to DHA / 生物工程学报

DHA (22:6n-3) is a Ω-3 polyunsaturated fatty acid with 22 carbon atoms and 6 double bonds, which has important biological functions in human body. Human and other mammals synthesize only limited amounts of DHA, more requirements must be satisfied from food resources. However, the natural resources of DHA (Mainly deep-sea fish and other marine products) are prone to depletion. New resources development is still insufficient to satisfy the growing market demand. Previous studies have revealed that the mammals can increase the synthesis of DHA and other long-chain polyunsaturated fatty acids after transgenic procedures. In this study, mammalian cells were transfected with Δ6, Δ5 desaturase, Δ6, Δ5 elongase, Δ15 desaturase (Isolated from nematode Caenorhabditis elegans) and Δ4 desaturase (Isolated from Euglena gracilis), simultaneously. Results show that the expression or overexpression of these 6 enzymes is capable of conversion of the o-6 linoleic acid (LA, 18:2n-6) in DHA (22:6n-3). DHA content has increased from 16.74% in the control group to 25.3% in the experimental group. The strategy and related technology in our research provided important data for future production the valuable DHA (22:6n-3) by using genetically modified animals.

Cloning of delta8-fatty acid desaturase gene from Euglena gracilis and its expression in Saccharomyces cerevisiae / 生物工程学报

Delta8 desaturase pathway, different from common delta6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Delta8-fatty acid desaturase is one of the key enzymes in delta8 desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn't intron in this delta8-fatty acid desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning delta8-fatty acid desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates omega6-eicosadienoic acid and omega3-eicosatrienoic acid for the expression of delta8-fatty acid desaturase gene. The results indicated that high level expressed As-fatty acid desaturase could convert omega6-eicosadienoic acid and omega3-eicosatrienoic acid to dihomo-gamma-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.

New record of two genera of trypanorhynch cestodes infecting red sea fishes in Egypt

Callitetrarhynchus and Floriceps [Lacistorhynchidae: Trypanorhyncha] belong to the less host specific genera with a wide host range. The present study reported plerocerci of Callitetrarhynchus speciosus Linton, 1897 from Conger cinereus, Longfine African Conger [Family: Congridae] and C. gracilis Pintner, 1931 [Family: Lacistorhynchidae] from five new teleost hosts of 2 different families: Lethrinus mehsena, Sky emperor and L. variegatus, Variegated Emperor [Lethrinidae]; Cephalopholis miniata, Coral grouper, Epinephe-us polyphekadion, Small toothed grouper or camouflage grouper and Epinephelus summana, Summana grouper [Serranidae]. C. gracilis is characterized by an elongated scolex, 2 bothria, lacking a pars post bulbosa, elongated bulbs, a poeciloacanthous atypical metabasal armature with 7 principal hooks, and a chainette. Tegumental surface is covered with polymorphic microtriches: palmate [tetra, penta, hexadigitate], tricuspidate, bifurcate and filiform. For the first time, tufts of filamentous microtriches surrounding a cilium are recorded from C. gracilis proximal bothrial surface obtained from L. variegatus. Seven new host are recorded for the related lacistorhynchid Floriceps minacanthus, Lethrinus nebulosus Spangled Emperor, Lutjanus bohar 2 red spots Snapper, Lutjanus fulviflamma Dory snapper, Epinephelus polyphekadion Small toothed grouper, E. summana Summana grouper, Variola louti Lunartail grouper, and Spharaena qenie Blackfine Barracuda

Effects of cadmium on Euglena gracilis membrane lipids

The toxic effects of cadmium (2 mug/ml) on membrane lipids and growth of Euglena gracilis were studied using autotrophic (AUTO), heterotrophic (DARK) and mixotrophic (LIGHT) cells. Cadmium caused inhibition of cellular proliferation (IC50 1.2 mug/ml) and morphological alterations which were most pronounced in chloroplasts. The chlorophyll content of LIGHT cadmium-treated cells was reduced 42.5 per cent. Cadmium also caused an increase in protein and total lipid content per cell in all three cell types. Among the membrane lipids, cholesterol content was lower in cadmium-treated cells cultivated under illumination (AUTO: 0.40 ñ 0.02 vs 0.64 ñ 0.08 and LIGHT: 0.40 ñ 0.09 vs 0.53 ñ 0.01 mug/l0(5) cells). There were no changes in total phospholipid content, although cardiolipin content was altered in all three cell types, and in mixotrophic cells there was an increase in phosphatidylglycerol, a phospholipid typically found in chloroplasts. These results suggest that cadmium has an overall toxic effect on Euglena gracilis and that part of the effect can be ascribed to defects in the structure of chloroplasts and mitochondrial membranes.

Differences in phospholipid composition of autotrophic and heterotrophic Euglena gracilis

We have determined the phospholipid composition of E. gracillis under different environmental conditions. Half of the phosphoglycerides was phosphatidylcholine, regardless of growth conditions, but differences were noted in the distribution of the remaining lipids. Autotrophic, green, light-grown euglenas had 31% more amino-containing lipids (phosphatidylethanolamine and phosphatidylserine) than the heterotrophic, white, dark-grown cells. In contrast, dark-grow cells had 47% more anionic lipids, such as the ones belonging to the "phosphatidynositol cycle" (phosphatidylinositol, phosphatidylinositol-phosphate phosphatidylinositol-bis-phosphate and phosphatidic acid) plus cardiolipin. The results suggest an adaptation of some biosynthetic and degradative phospholipid pathways to autotrophic and to heterotophic growth