Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Diet-induced changes in lipid and fatty acid composition of Artemia salina

The influence of diet on lipid and fatty acid composition of the brine shrimp Artemia salina nauplii was investigated. Various diets with different lipid composition and fatty acid profiles were fed to nauplii for 2 weeks. The lipid composition of microalgal diets, Isochrysis galbana, Phaeodactylum tricornutum and Nannochloropsis oculata and baker's yeast was analyzed. Newly hatched nauplii were examined before the feeding experiment. It was shown that Artemia was able to incorporate and selectively concentrate some dietary lipids. Depot lipids were more sensitive to changes in the dietary lipid composition than the main structural lipids, polar lipids and sterols. Variations in the content of the lipid classes correlated with stage of development of the animal. The fatty acid composition of the animal varied with that of diet. The concentrations of saturated fatty acids were apparently supported in the nauplii by biosynthesis de novo. The acid 16:1(n-7) originated from the food. The concentration range of n-6 polyunsaturated fatty acids (PUFAs) remained constant through the accumulation from the diet. The proportion of n-3 PUFAs varied with their level in the diet. The dynamics of alteration of 20:5(n-3) content in Artemia fed on Isochrysis, which is poor in this acid, suggested a limited capacity for elongation and desaturation of 18:3(n-3) to 20:5(n-3). None of the diets provided dietary input of 22:6(n-3). (C) 1998 Elsevier Science Inc. All rights reserved.

Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: Identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii

Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal Delta 12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae).

Study on the fatty acid composition of jellyfish Rhopilema esculentum

The fatty acids composition in different parts of full-grown Rhopilema esculentum jellyfish from Yellow Sea was investigated. The lipids, extracted from the umbrella and oral arms and gonads of R. eculentum jellyfish, respectively were analysed by combined capillary gas chromatography/mass spectrometry. The results show that there are more than thirty kinds of fatty acids in jellyfish, and the fatty acid compositions of three parts of R. esculentum are almost the same. In the three parts, percentages of polyunsaturated fatty acids (PUFA) are high, and range from 36.23% to 38.74%. Docosahexaenoic acid (DHA), eicosatetraenoic acid (AA) and eicosapentaenoic acid (EPA) are three major PUFA.

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentration on the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) was cultured in media with different nitrogen sources and concentrations. During the pre-logarithmic phase, the alga grew faster with ammonium as N source than with nitrate, but the reverse applied during the post-logarithmic phase. The alga grew poorly in N-free medium or medium with urea as the sole N source. In the same growth phase, ammonium medium resulted in higher yield of total lipid, but the EPA yield did not differ significantly different from that using nitrate medium. The maximum growth rate occurred in medium containing 1.28 mmol L-1 sodium nitrate, while maximum EPA and total lipid contents were reached at 1.92 mmol L-1, when EPA accounted for 27.9% total fatty acids. The growth rate kept stable when NH4Cl ranged from 0.64 to 2.56 mmol L-1, and the maximum content of total lipid and EPA occurred in the medium with 2.56 mmol L-1 NH4Cl. The EPA content was higher in the pre- than post-logarithmic phase, though the total lipid content was lower. The highest EPA content expressed as percent total fatty acid was 27.9% in nitrate medium and and 39.0% in ammonium medium.

Investigations into selective metabolic aspects of bifidobacteria: carbohydrate metabolism, fatty acid biosynthesis and plasmid biology

The gastrointestinal tract (GIT) is a diverse ecosystem, and is colonised by a diverse array of bacteria, of which bifidobacteria are a significant component. Bifidobacteria are Gram-positive, saccharolytic, non-motile, non-sporulating, anaerobic, Y-shaped bacteria, which possess a high GC genome content. Certain bifidobacteria possess the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA) by a biochemical pathway that is hypothesised to be achieved via a linoleic isomerase. In Chapter two of this thesis it was found that the MCRA-specifying gene is not involved in CLA production in B. breve NCFB 2258, and that this gene specifies an oleate hydratase involved in the conversion of oleic acid into 10-hydroxystearic acid. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon. Key to the development of such novel prebiotics is to understand which carbohydrates support growth of bifidobacteria and how such carbohydrates are metabolised. In Chapter 3 of this thesis we describe the identification and characterisation of two neighbouring gene clusters involved in the metabolism of raffinose-containing carbohydrates (plus related carbohydrate melibiose) and melezitose by Bifidobacterium breve UCC2003. The fourth chapter of this thesis describes the analysis of transcriptional regulation of the raf and mel clusters. In the final experimental chapter two putative rep genes, designated repA7017 and repB7017, are identified on the megaplasmid pBb7017 of B. breve JCM 7017, the first bifidobacterial megaplasmid to be reported. One of these, repA7017, was subjected to an in-depth characterisation. The work described in this thesis has resulted in an improved understanding of bifidobacterial fatty acid and carbohydrate metabolism, Furthermore, attempts were made to develop novel genetic tools.

Fatty acid composition of wild anthropoid primate milks.

Fatty acids in milk reflect the interplay between species-specific physiological mechanisms and maternal diet. Anthropoid primates (apes, Old and New World monkeys) vary in patterns of growth and development and dietary strategies. Milk fatty acid profiles also are predicted to vary widely. This study investigates milk fatty acid composition of five wild anthropoids (Alouatta palliata, Callithrix jacchus, Gorilla beringei beringei, Leontopithecus rosalia, Macaca sinica) to test the null hypothesis of a generalized anthropoid milk fatty acid composition. Milk from New and Old World monkeys had significantly more 8:0 and 10:0 than milk from apes. The leaf eating species G. b. beringei and A. paliatta had a significantly higher proportion of milk 18:3n-3, a fatty acid found primarily in plant lipids. Mean percent composition of 22:6n-3 was significantly different among monkeys and apes, but was similar to the lowest reported values for human milk. Mountain gorillas were unique among anthropoids in the high proportion of milk 20:4n-6. This seems to be unrelated to requirements of a larger brain and may instead reflect species-specific metabolic processes or an unknown source of this fatty acid in the mountain gorilla diet.