Fatty acid composition as an indicator of physiological condition of the cyanobacterium Microcystis aeruginosa

The present study examined the fatty acid composition of Microcystis aeruginosa grown in a batch culture and that of Microcystis-dominated plankton collected in an experimental enclosure in a shallow, eutrophic embayment of Lake Biwa (Akanoi Bay). In pure culture, we detected 16 : 0, 18 : 2ω6, 18 : 3ω3, 18 : 3ω6, and 18 : 4ω3 acids as major fatty acids of M. aeruginosa, with trace amounts of C₂₀ polyunsaturated fatty acids. In both pure culture and the field enclosure, the ratio of total fatty acid weight to dry weight decreased with decreasing availability of dissolved inorganic nitrogen. The ω3/ω6 ratios of C₁₈ polyunsaturated fatty acids [(18 : 3ω3 + 18 : 4ω3)/(18 : 2ω6 + 18 : 3ω6)] varied greatly (range, 2–5) in response to the changes in physical and chemical conditions for Microcystis growth. Most notably, the ω3/ω6 fatty acid ratios were significantly positively correlated with the growth rate of cells in a batch culture. We suggest that the fatty acid composition is a useful indicator of the physiological state of Microcystis in freshwater lakes. Received: March 2, 2001/ Accepted: December 19, 2001     

Production of ω3 fatty acids in marine cyanobacterium Synechococcus sp. strain NKBG 15041c via genetic engineering

Omega-3 fatty acids (ω3 FAs) have attracted attention because they have various health benefits for humans. Fish oils are currently major sources of ω3 FAs, but a sustainable supply of ω3 FAs based on fish oils is problematic because of the increasing demand. In this study, the production potential of a genetically engineered marine cyanobacterium, Synechococcus sp. strain NKBG 15041c, was examined as an alternative source of ω3 FAs. A change in fatty acid composition of this cyanobacterium was successfully induced by the expression of a heterologous Δ6-desaturase, and the transformants synthesized stearidonic acid, which the wild type cannot produce. As a result of optimization of culture conditions, maximal contents of stearidonic acid and total ω3 FAs reached 12.2 ± 2.4 and 118.1 ± 3.5 mg/g, respectively. The maximal ω3 FA productivity was 4.6 ± 0.7 mg/(L⋅day). These are the highest values of the contents of stearidonic acid and ω3 FAs in genetically engineered cyanobacteria reported thus far. Therefore, genetically engineered Synechococcus sp. strain NKBG 15041c may be a promising sustainable source of ω3 fatty acids.     

Isolation and Characterisation of a High-Efficiency Desaturase and Elongases from Microalgae for Transgenic LC-PUFA Production

The production of long-chain polyunsaturated fatty acids from precursor molecules linoleic acid (LA; 18:2ω6) and α-linolenic acid (ALA; 18:3ω3) is catalysed by sequential desaturase and elongase reactions. We report the isolation of a front-end Δ6-desaturase gene from the microalgae Ostreococcus lucimarinus and two elongase genes, a Δ6-elongase and a Δ5-elongase, from the microalga Pyramimonas cordata. These enzymes efficiently convert their respective substrates when transformed in yeast (39–75% conversion for ω3 substrate fatty acids), and the Δ5-elongase in particular displays higher elongation efficiency (75% for conversion of eicosapentaenoic acid (20:5ω3) to docosapentaenoic acid (22:5ω3)) than previously reported genes. In addition, the Δ6-desaturase is homologous with acyl-CoA desaturases and shows a strong preference for the ω3 substrate ALA.     

Antioxidant activity of vegetable oils with different omega-6/omega-3 fatty acids ratio

The antioxidant activity and the oxidative stability of flax, sesame, silybum oils and seed blend oils with different ω-6/ω-3 fatty acid ratios have been investigated. The antioxidant content (AO) in crude oils and their reactivity towards peroxyl radicals were studied using the kinetic method based on oil addition to the model reaction of cumene oxidation. There were correlations between the ratio polyunsaturated fatty acids (PUFA)/ω-9 and thermal stability (50°C); between the effect of γ-tocopherol content and oil resistance to oxidative changes during long-term storage at (10 ± 2)°C.     

Biosynthesis of ω-alicyclic fatty acids induced by cyclic precursors and change of membrane fluidity in thermophilic bacteria <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis> and <Emphasis Type="Italic">Meiothermus</Emphasis><Emphasis Type="Italic">ruber</Emphasis>

Two thermophilic strains belonging to Geobacillus stearothermophilus and Meiothermus ruber, which naturally do not synthesize ω-alicyclic fatty acids (ω-FAs) were cultivated with cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl carboxylic acids. Gas chromatography–mass spectrometry analysis of fatty acid methyl and picolinyl esters showed that both strains are able to synthesize ω-FAs when cultivated with the appropriate precursor. The incorporation of cyclic acids influenced the whole FA composition as well as membrane fluidity. Membrane fluidity of intact cells was studied by measuring the fluorescence polarisation of the probe l,6-diphenyl-1,3,5-hexatriene incorporated into membrane lipid bilayers. Cytoplasmic membrane became more fluid with increasing content of ω-FAs. This is caused by considerable changes in lipid packing within the membrane induced by the presence of ω-FAs not found in the natural environment of Geobacillus and Meiothermus strains.     

Seasonal dynamics of fatty acids in the lipids of water moss <Emphasis Type="Italic">Fontinalis antipyretica</Emphasis> from the Yenisei River

Identification of lipid fatty acids (FA) and studying of their seasonal dynamics in water moss Fontinalis antipyretica from the Yenisei River were carried out by means of gas chromatography-mass spectrometry. FA composition of water moss was notable for a relatively low level of saturated acids and predominance of polyunsaturated acids (PUFA) with double bonds (accounting for more than 30% of total FA) and polyunsaturated acids with double and triple bonds (acetylenic acids, accounting for more than 40% of total FA). Among PUFA, α- and γ-linolenic (18:3ω3 and 18:3ω6), arachidonic (20:4ω6), and eicosapentaenoic (20:5ω3) acids prevailed. Relative content of PUFA from ω3-family was the greatest in spring, and the level of PUFA from ω6-group was essentially the same throughout all the seasons. In the biomass of water moss, we identified seven acetylenic acids; among them octadeca-9,12-dien-6-ynoic (6a,9,12-18:3), octadeca-9,12,15-trien-6-ynoic (6a,9,12,15-18:4), and eicosa-11,14-dien-8-ynoic (8a,11,14-20:3) acids were predominant. For the first time, in the lipids of water moss we identified an acetylenic eicosa-11,14,17-trien-8-ynoic acid (8a,11,14,15-20:4). Relative content of acetylenic acids in the total FA was great throughout the entire period of investigation with the peak accumulation in summer. Owing to a steadily high level in the biomass of water moss and the lack of other producers of these acids in the ecosystem, acetylenic FA are highly specific biochemical markers useful for the investigation of trophic interactions between higher aquatic plants and zoobenthos.     

Screening of Red Algae Filaments As a Potential Alternative Source of Eicosapentaenoic Acid

Lipids were extracted by a supercritical fluid extraction method from 10 species of filamentous red algae obtained from culture collections and their fatty acid compositions were determined. The fatty acid profiles of the 10 species were similar. The major fatty acids were 16:0, 20:4ω6 and 20:5ω3 (eicosapentaenoic acid, EPA), which amounted to over 70% of the total fatty acids. The highest EPA content (29.8 mg/L), as a percentage of total fatty acids, was produced by Liagora boergesenii filaments, which has good potential for EPA mass production in pilot plants.     

Taxonomy of Pinus species based on the seed oil fatty acid compositions

 The fatty acid compositions of the seed oils from ten pine species have been established by capillary gas-liquid chromatography of the methyl esters. With regard to either normal fatty acids or Δ5-olefinic acids, the general pattern of fatty acids did not differ from that of other pine seed oils reported previously. The main fatty acid was linoleic (9,12–18:2) acid (44.4–57.1%), followed by either oleic (9–18:1) acid (13.4–24.5%) or pinolenic (5,9,12–18:3) acid (1.5–25.2%). When applying multivariate analyses to the chemometric data (13 variables) of 49 pine species (ca. 40% of the living pine species), it was possible to distinguish between several sections: Pinea, Longifolia, Halepensis, Ponderosa-Banksiana, Sylvestris, and Cembra. The latter section was clearly divided into two sub-groups. A few species that presented a low overall content of Δ5-olefinic acids, and that grow in warm-temperate regions, were isolated from the bulk of other pine species. It is hypothesized that Δ5-olefinic acids might be related to cold-acclimation. Received: 5 June 1997 / Accepted: 17 August 1997     

Chemotaxonomic differentiation of conifer families and genera based on the seed oil fatty acid compositions: multivariate analyses

 The fatty acid compositions of seed oils from 34 conifer species, mainly Pinaceae and secondarily Cupressaceae, have been determined by gas-liquid chromatography of the methyl esters. As noted in earlier studies, these oils were characterized by the presence of several Δ5-olefinic acids, i.e., 5,9-18:2, 5,9,12-18:3, 5,9,12,15-18:4, 5,11-20:2, 5,11,14-20:3, and 5,11,14,17-20:4 acids, in addition to the more common saturated, oleic, linoleic and α-linolenic acids. Based on these fatty acid compositions, and on those established in earlier systematic studies (totalling 82 species), we established a chemotaxonomic grouping of the main conifer families, i.e., of the Pinaceae, Taxodiaceae, Cupressaceae, and Taxaceae. This was achieved using multivariate analyses (principal component analysis and discriminant analysis). The fatty acids that discriminate best in this classification are the 5,11,14,17-20:4, 9,12,15-18:3 and 5,9,12-18:3 acids. Moreover, it was possible to differentiate between several genera of the Pinaceae: Pinus (including Tsuga and Pseudotsuga), Abies, Cedrus, and Picea plus Larix, represented quite distinct groups. Other fatty acids such as oleic, linoleic, and 5,9-18:2 acids were also important for this purpose. The fatty acid compositions, and particularly the Δ5-olefinic acid contents of conifer seed oils, may thus be applied to the chemosystematic distinction among conifer families as well as genera of the Pinaceae. Received: 3 January 1997 / Accepted: 17 April 1997     

Degradation and modification of fats, oils and grease by commercial microbial supplements

Fats, oils and greases (FOGs) in wastewater create problems including the production of foul odours, the blockage of sewer lines and may interfere with the proper operation of sewage treatment works. Removal of FOG from wastewater is thus critically important to ensure that wastewater is disposed of efficiently and economically. In this study, the ability of commercial microbial supplements to degrade fat/oil under laboratory conditions was investigated. One of the multi-species supplements examined was capable of significantly enhancing the degradation of several fats and oils by 37–62%, in contrast to all of the single-species supplements which had no significant effect. The multi-species supplement showed no preferential cleavage or degradation of fatty acids in a range of FOGs, whilst wastewater-associated bacteria preferentially degraded octadecatrienoic acid (18:3ω3) and octadecadienoic acid (18:2ω6). A semi-solid, sticky material, likely to cause blockages in pipework and sewer lines formed when the oil was incubated in the presence of wastewater bacteria. The sticky material was enriched in saturated and mono-unsaturated fatty acids and depleted in polyunsaturated fatty acids relative to the original oil, most likely reflecting preferential fatty acid usage by the bacteria. The production of this semi-solid material by the wastewater bacteria was significantly reduced by the presence of the multi-species product, indicating that commercial supplements have the potential to minimize FOG accumulation and blockages in grease traps and sewer lines as well as enhancing FOG degradation.     

Evolution-related amino acids play important role in determining regioselectivity of fatty acid desaturase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

ω³-fatty acid desaturase and Δ¹²-fatty acid desaturase of Pichia pastoris with distinguishable regioselectivity and high degree of sequence similarity were chosen for regioselectivity research. Chimeras were constructed in which Histidine-rich boxes 1, 2 and the carboxyl terminal region of ω³-fatty acid desaturase were replaced with corresponding region of Δ¹²-fatty acid desaturase. The replacement was found to result in a change of regioselectivity from ωy to x + 3 by functionally characterizing these chimeric enzymes in Saccharomyces cerevisae strain INVScI. Using site-directed mutagenesis, we further demonstrated that seven conserved amino acids of ω³-fatty acid desaturase within the first two Histidine-rich regions are responsible for the regioselectivity switch. Therefore, the regioselectivity of fatty acid desaturases may be better understood by investigating the evolutionary relationships of different fatty acid desaturases. Dongsheng Wei is the partake of first-author’s profits.     

Changes in fatty acids composition,hydrogen peroxide generation and lipid peroxidation of salt-stressed corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) roots

Comparative study about the salt-induced oxidative stress and lipid composition has been realised in primary root tissues for two varieties of maize (Zea mays L.) in order to evaluate their responses to salt stress. The root growth, root water content (WC), hydrogen peroxide (H₂O₂) generation, lipid peroxidation, membrane stability index and the changes in the profile of fatty acids composition were investigated. Salinity impacts in term of root growth, water content, H₂O₂ generation, lipid peroxidation and membrane destabilisation were more pronounced in primary roots of Aristo than in those of Arper indicating more sensitivity of the first variety. It was confirmed by gas chromatography that the composition of fatty acids in roots of both varieties was constituted mainly by 16:0 and 18:0 as major saturated fatty acids and 18:1ω9, 18:2ω6 and 18:3ω3 as major unsaturated fatty acids. Total lipid extracts from the roots of both varieties showed that the lipid saturation level increased under salt stress, notwithstanding the increased proportion of polyunsaturated fatty acids. The changes in lipid saturation being predominantly due to decreases in oleic acid (18:1ω9) and increases in palmitic acid (16:0). However, Arper root extracts contained a lower proportion of saturated lipids than Aristo. The enhanced proportion of highly polyunsaturated fatty acids especially linolenic and eicosapentaenoic acids was considered to be the characteristic of the relatively salt tolerance in Arper roots.     

Cytochrome P450-dependent fatty acid hydroxylases in plants

In plants, hydroxy-fatty acid production is mainly the result of enzymatic reactions catalyzed by cytochrome P450 dependent fatty acid hydroxylases. One can distinguish ω-hydroxylases that catalyze the hydroxylation of the terminal methyl of aliphatics acids (ω position) and sub-terminal or in-chain hydroxylases that oxidize carbons in the chain (ω-n position). Since both types of enzymes were discovered about three decades ago, the majority of investigations have focused on the CYP94 and CYP86 families, which mediate ω-hydroxylations. The activities of ω-hydroxylases in cutin synthesis have been clearly established, but the studies of LCR (LACERATA) and att1 (aberrant induction of type three genes), which are the first Arabidopsis thaliana mutants with alterations in coding sequences of CYP86A8 and CYP86A2, show that these types of ω-hydroxylases can be involved in many aspects of plant development. The existence of different ω-hydroxylases in plants with distinct regulation patterns suggests that these enzymes mediate diverse biological processes. Much less information concerning in-chain hydroxylases is available despite the fact that they were initially reported along with ω-hydroxylases. This lack of information might be explained by the very few examples of sub-terminal hydroxy-fatty acids described in plants. We present here the best characterized fatty acid hydroxylases and we discuss their possible roles in plant defense and development, fatty acid catabolism, plant reproduction and detoxification.     

Cloned transgenic heart-healthy pork?

Here I comment on the production and uses of swine that express a humanized fat-1 gene. The gene product is a fatty acid desaturase that converts ω-6 fatty acids to ω-3 fatty acids. Omega-3 fatty acids have been implicated as being important for reproductive success, maintaining a healthy cardiovascular system, sustaining a functional immune system, and even preventing depression and cancer. The descendants of these hfat-1 transgenic swine will be very useful as models of the human condition, and if they are permitted to enter the food chain, they may improve human health.     

Expression and characterization of two new alkane-inducible cytochrome P450s from <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>

n-Dodecane and fatty acids were good inducers of cytochrome P450 (CYP) and the ω-hydroxylase of lauric acid, which is a marker for ω-hydroxylation of n-alkanes, in Trichoderma harzianum. A cDNA, containing an ORF of 1520 bp, encoding a CYP52 of 520 amino acids, was isolated by RACE. Another n-alkane-inducible CYP was identified by LLC-MS/MS analysis of a microsomal protein band induced by n-dodecane in a library of T. harzianum. This suggests that T. harzianum has a CYP-dependent conversion of alkanes to fatty acids allowing their incorporation into lipids.     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

Thraustochytrid Marine Protists: Production of PUFAs and Other Emerging Technologies

Thraustochytrids, the heterotrophic, marine, straminipilan protists, are now established candidates for commercial production of the omega-3 polyunsaturated fatty acid (ω-3 PUFA), docosahexaenoic acid (DHA), that is important in human health and aquaculture. Extensive screening of cultures from a variety of habitats has yielded strains that produce at least 50% of their biomass as lipids, and DHA comprising at least 25% of the total fatty acids, with a yield of at least 5 g L⁻¹. Most of the lipids occur as triacylglycerols and a lesser amount as phospholipids. Numerous studies have been carried out on salinity, pH, temperature, and media optimization for DHA production. Commercial production is based on a fed batch method, using high C/N ratio that favors lipid accumulation. Schizochytrium DHA is now commercially available as nutritional supplements for adults and as feeds to enhance DHA levels in larvae of aquaculture animals. Thraustochytrids are emerging as a potential source of other PUFAs such as arachidonic acid and oils with a suite of PUFA profiles that can have specific uses. They are potential sources of asataxanthin and carotenoid pigments, as well as other lipids. Genes of the conventional fatty acid synthesis and the polyketide-like PUFA synthesis pathways of thraustochytrids are attracting attention for production of recombinant PUFA-containing plant oils. Future studies on the basic biology of these organisms, including biodiversity, environmental adaptations, and genome research are likely to point out directions for biotechnology explorations. Potential areas include enzymes, polysaccharides, and secondary metabolites.     

A comparison of suberin monomers from the multiseriate exodermis of <Emphasis Type="Italic">Iris germanica</Emphasis> during maturation under differing growth conditions

Iris germanica roots develop a multiseriate exodermis (MEX) in which all mature cells contain suberin lamellae. The location and lipophilic nature of the lamellae contribute to their function in restricting radial water and solute transport. The objective of the current work was to identify and quantify aliphatic suberin monomers, both soluble and insoluble, at specific stages of MEX development and under differing growth conditions, to better understand aliphatic suberin biosynthesis. Roots were grown submerged in hydroponic culture, wherein the maturation of up to three exodermal layers occurred over 21 days. In contrast, when roots were exposed to a humid air gap, MEX maturation was accelerated, occurring within 14 days. The soluble suberin fraction included fatty acids, alkanes, fatty alcohols, and ferulic acid, while the suberin poly(aliphatic) domain (SPAD) included fatty acids, α,ω-dioic acids, ω-OH fatty acids, and ferulic acid. In submerged roots, SPAD deposition increased with each layer, although the composition remained relatively constant, while the composition of soluble components shifted toward increasing alkanes in the innermost layers. Air gap exposure resulted in two significant shifts in suberin composition: nearly double the amount of SPAD monomers across all layers, and almost three times the alkane accumulation in the first layer. The localized and abundant deposition of C18:1 α,ω-dioic and ω-OH fatty acids, along with high accumulation of intercalated alkanes in the first mature exodermal layer of air gap-exposed roots indicate its importance for water retention under drought compared with underlying layers and with entire layers developing under water.     

The growth of external AM fungal mycelium in sand dunes and in experimental systems

We estimated the biomass and growth of arbuscular mycorrhizal (AM) mycelium in sand dunes using signature fatty acids. Mesh bags and tubes, containing initially mycelium-free sand, were buried in the field near the roots of the dune grass Ammophila arenaria L. AM fungal mycelia were detected at a distance of about 8.5 cm from the roots after 68 days of growth by use of neutral lipid fatty acid (NLFA) 16:1ω5. The average rate of mycelium extension during September and October was estimated as 1.2 mm day⁻¹. The lipid and fatty acid compositions of AM fungal mycelia of isolates and from sand dunes were analysed and showed all to be of a similar composition. Phospholipid fatty acids (PLFAs) can be used as indicators of microbial biomass. The mycelium of G. intraradices growing in glass beads contained 8.3 nmol PLFAs per mg dry biomass, and about 15% of the PLFAs in G. intraradices, G. claroideum and AM fungal mycelium extracted from sand dunes, consisted of the signature PLFA 16:1ω5. We thus suggest a conversion factor of 1.2 nmol PLFA 16:1ω5 per mg dry biomass. Calculations using this conversion factor indicated up to 34 μg dry AM fungal biomass per g sand in the sand dunes, which was less than one tenth of that found in an experimental system with Glomus spp. growing with cucumber as plant associate in agricultural soil. The PLFA results from different systems indicated that the biomass of the AM fungi constitutes a considerable part of the total soil microbial biomass. Calculations based on ATP of AM fungi in an experimental growth system indicated that the biomass of the AM fungi constituted approximately 30% of the total microbial biomass. This revised version was published online in June 2006 with corrections to the Cover Date.     

Erratum to: Characterization of a KCS-like KASII from<Emphasis Type="Italic"> Jessenia bataua</Emphasis> that Elongates Saturated and Monounsaturated Stearic Acids in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

As the world population grows, the demand for food increases. Although vegetable oils provide an affordable and rich source of energy, the supply of vegetable oils available for human consumption is limited by the “fuel vs food” debate. To increase the nutritional value of vegetable oil, metabolic engineering may be used to produce oil crops of desirable fatty acid composition. We have isolated and characterized β-ketoacyl ACP-synthase II (KASII) cDNA from a high-oleic acid palm, Jessenia bataua. Jessenia KASII (JbKASII) encodes a 488-amino acid polypeptide that possesses conserved domains that are necessary for condensing activities. When overexpressed in E. coli, recombinant His-tagged JbKASII was insoluble and non-functional. However, Arabidopsis plants expressing GFP-JbKASII fusions had elevated levels of arachidic acid (C20:0) and erucic acid (C22:1) at the expense of stearic acid (C18:0) and oleic acid (C18:1). Furthermore, JbKASII failed to complement the Arabidopsis KASII mutant, fab1-2. This suggests that the substrate specificity of JbKASII is similar to that of ketoacyl-CoA synthase (KCS), which preferentially elongates stearic and oleic acids, and not palmitic acid. Our results suggest that the KCS-like JbKASII may elongate C18:0 and C18:1 to yield C20:0 and C22:1, respectively. JbKASII may, therefore, be an interesting candidate gene for promoting the production of very long chain fatty acids in transgenic oil crops.