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.     

Cold adaptation of eicosapentaenoic acid-less mutant of Shewanella livingstonensis Ac10 involving uptake and remodeling of synthetic phospholipids containing various polyunsaturated fatty acids

An Antarctic psychrotrophic bacterium, Shewanella livingstonensis Ac10, produces cis-5,8,11,14,17-eicosapentaenoic acid (EPA), a long-chain polyunsaturated fatty acid (LPUFA), as a component of membrane phospholipids at low temperatures. The EPA-less mutant generated by disruption of the EPA synthesis gene becomes cold-sensitive. We studied whether the cold sensitivity could be suppressed by supplementation of various LPUFAs. The EPA-less mutant was cultured at 6°C in the presence of synthetic phosphatidylethanolamines (PEs) that contained oleic acid at the sn-1 position and various C20 fatty acids with different numbers of double bonds from zero to five or cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) at the sn-2 position. Mass spectrometric analyses revealed that all these fatty acids became components of various PE and phosphatidylglycerol species together with shorter partner fatty acids, indicating that large-scale remodeling followed the incorporation of synthetic PEs. As the number of double bonds in the sn-2 acyl chain decreased, the growth rate decreased and the cells became filamentous. The growth was restored to the wild-type level only when the medium was supplemented with phospholipids containing EPA or DHA. We found that about a half of DHA was converted into EPA. The results suggest that intact EPA is best required for cold adaptation of this bacterium.     

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°C. This is the first report of the recombinant production of DHA in a polyketide biosynthesis mode.     

Gene cloning,expression and functional characterization of a phosphopantetheinyl transferase from <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> serotype O1

Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins from fatty acid synthetases (FASs) in primary metabolism and polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NRPSs) in secondary metabolism. Bacteria typically harbor one PPTase specific for carrier proteins of primary metabolism (ACPS-type PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism (Sfp-type PPTases). Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, has been reported to be synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulent plasmid pJM1 from strain 775 of V. anguillarum serotype O1, and the PPTase, necessary for the activation of the anguibactin-NRPS, is therefore expected to lie on the pJM1 plasmid. In this work, a putative PPTase gene, angD, was first identified on pEIB1 plasmid (a pJM1-like plasmid) from a virulent strain MVM425 of V. anguillarum serotype O1. A recombinant clone carrying complete angD was able to complement an Escherichia coli entD mutant deficient in Sfp-type PPTase. angD was overexpressed in E. coli and the resultant protein, AngD, was purified. Simultaneously, two carrier proteins involved in anguibactin-NRPS, ArCP and PCP, were overproduced in E. coli and purified. The purified AngD, PCP and ArCP were used to establish an in vitro enzyme reaction, and the PPTase activity of AngD was proved through HPLC analysis to detect the conversion of inactive carrier proteins to active carrier proteins in the reaction mixture. Co-expression of AngD with PCP or ArCP showed that AngD functioned well as a PPTase in vivo in E. coli, modifying PCP and ArCP completely.     

Effect of cyanocobalamin and p-toluic acid on the fatty acid composition of Schizochytrium limacinum (Thraustochytriaceae, Labyrinthulomycota)

Changes in the fatty acid composition of docosahexaenoic acid (DHA)-producing Schizochytrium limacinum SR21 were investigated. The addition of cyanocobalamin, which is an active component of vitamin B₁₂, decreased the content of odd-chain fatty acids such as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). Cyanocobalamin may upregulate the cobalamin-dependent methylmalonyl-CoA mutase, which converts propionic acid to succinic acid, thereby decreasing the content of odd-chain fatty acids. The addition of p-toluic acid resulted in a decrease in docosapentaenoic acid (DPA, 22:5n-6) content and an increase in eicosapentaenoic acid (EPA, 20:5n-3) content in a dose-dependent manner. Two additional peaks of fatty acids, characterized as Δ4,7,10,14-eicosatetraenoic acid (20:4n-7) and Δ4,7,10,14-docosatetraenoic acid (22:4n-9), were detected.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Docosahexaenoic acid production and ultrastructure of the thraustochytrid <Emphasis Type="Italic">Aurantiochytrium mangrovei</Emphasis> MP2 under high glucose concentrations

The effect of high glucose concentrations on the ultrastructure and the production of docosahexaenoic acid (DHA) by Aurantiochytrium mangrovei MP2 was investigated at 25°C with orbital shaking. The cultured cells were separated into a floating and a bottom layer after centrifugation during harvest; therefore, the ultrastructure and DHA level were also analyzed separately. Cell size generally increased with glucose concentrations, whereas the overall DHA production (mg/l) increased 19% when the glucose concentration was raised from 6% to 10% w/v. Biomass and DHA production increased significantly, but not linearly, at the floating layer and decreased at the bottom layer in elevated glucose concentrations. Also, the lipid bodies of the cells in the floating layer were more heavily stained in osmium tetroxide than those in the bottom, suggesting that the cells in the floating layer may contain greater amount of unsaturated fatty acids.     

High content of polyunsaturated fatty acids in muscle phospholipids of a fast runner,the European brown hare (Lepus europaeus)

To investigate both seasonal changes and possible intracorporal gradients of phospholipid fatty acid composition, skeletal muscles (n=124), hearts (n=27), and livers (n=34) from free-living brown hares (Lepus europaeus) were analyzed. Phospholipids from both skeletal muscles and heart had a high degree of unsaturation with 66.8±0.63% and 65.7±0.5% polyunsaturated fatty acids, respectively. This is the highest proportion of polyunsaturated fatty acids reported in any mammalian tissue. Polyunsaturated fatty acid content in skeletal muscles was 2.3% greater in winter compared to summer (F_1,106=17.7; P=0.0001), which may reflect thermoregulatory adjustments. Arachidonate (C20:4n-6) showed the greatest seasonal increase (+2.5%; F=7.95; P=0.0057). However, there were no pronounced differences in polyunsaturated fatty acid content between skeletal muscles from different locations in the body (m. iliopsoas, m. longissimus dorsi and m. vastus). Total muscle phospholipid polyunsaturated fatty acid content was correlated with polyunsaturated fatty acid content in triacyglycerols from perirenal white adipose tissue depots (r²=0.61; P=0.004). Polyunsaturated fatty acids were enriched in muscle phospholipids (56.8–73.6%), compared to white adipose tissue lipids (20.9–61.2%), and liver phospholipids (25.1–54.2%). We suggest that the high degree of muscle membrane unsaturation is related to hare-specific traits, such as a high maximum running speed.Abbreviations BMR basal metabolic rate - DPA docosapentaenoic acid - DHA docosahexaenoic acid - FA fatty acid - MUFA monounsaturated fatty acid - PC principal component - PUFA polyunsaturated fatty acid - SFA saturated fatty acid - UI unsaturation index - WAT white adipose tissueCommunicated by: G. Heldmaier     

Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production

The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas–liquid volumetric mass transfer coefficient k _L a positively. The k _L a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k _L a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r _DHA (9.75 mg l⁻¹ h⁻¹) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.     

Coexpression of Elo-like enzyme and Δ5, Δ4-desaturases derived from Thraustochytrium aureum ATCC 34304 and the production of DHA and DPA in Pichia pastoris

Thraustochytrium aureum ATCC 34304 produces a high level of polyunsaturated fatty acids (PUFAs), which are typically synthesized by strings of reactions catalyzed by desaturase and elongase enzymes. In this study, the genes related to the biosynthesis of PUFAs were investigated and targeted to enable optimization of the production of PUFAs. To the best of our knowledge, this is first study to evaluate the co-expression of genes TaElo, Tad5, and Tad4genes derived from T. aureum. We found that C22 PUFAs such as docosapentaenoic acid (DPA, C22:5n–6) and docosahexaenoic acid (DHA, 22:6n–3) were synthesized from γ-linolenic acid (GLA, C18:3n–6) and stearidonic acid (SDA, C18:4n–3), respectively, as exogenous substrates via a series of reactions catalyzed by an Elo-like enzyme and Δ5, Δ4-desaturase enzymes. In addition, the results of this study revealed that the TaElo gene could synthesize the Δ6-and Δ5-elongation products. Taken together, these results confirmed that the Elo-like enzyme was involved in multiple reactions leading to the production of PUFAs and that the TaElo, Tad5, and Tad4 genes were capable of functioning together to produce DPA and DHA using GLA and SDA.     

A New Labyrinthulid Isolate That Produces Only Docosahexaenoic Acid

We show here that a new labyrinthulid strain, L72, isolated from a fallen leaf in the Seto Inland Sea of Japan, produced only docohexaenoic acid (DHA) among all the long-chain polyunsaturated fatty acids (LCPUFAs). The main fatty acid composition was 16:0 (28.9%), 18:0 (7.2%), 18:1 (5.7%), 18:2 (10.4%), and DHA (45.9%) without any other LCPUFA. The lipid content of the strain was 27.4%. The cells had many lipid bodies, which were densely located in all of the cells. On phylogenetic analysis using the 18S rDNA sequence, the strain was located in the labyrinthulids group, forming a monophyletic group with Labyrinthula sp. (strain s) and Labyrinthuila sp. (strain L59). We further tested the culture optimization of strain L72 to evaluate the ability of the strain to produce DHA. The optimum salt concentration and the temperature of the strain were 100% of artificial seawater and 20°C. Strain L72 could grow well on soybean oil (SBO) or soybean lecithin (SBL) as the carbon source. When 20 g/l of SBL was added to the medium, DHA production reached the maximum amount at 0.67 g/l for 14 d. The two important facts, that the strain can use SBL as the main nutrient and contains only DHA among the LCPUFAs, will be of great advantage for industry.     

Fatty acid composition of lipids from mushrooms belonging to the family Boletaceae

The non-polar lipid content and fatty acid (FA) composition of 11 mushroom species of the family Boletaceae were determined. The non-polar lipid content ranged from 2.0 (Leccinum aurantiacum and Boletus erythropus) to 5.4 % (w/w) d.w. (Suillus grevillei) with an average value of 2.9 %. More than 25 different FAs were found in the mushroom lipids. Unsaturated FAs, mainly linoleic and oleic acids, accounted for about 83 % of the total FAs, while palmitic acid was the main saturated FA. Some FAs are identified for the first time in Boletaceae and in higher Basidiomycetes (cis-11,12-methyleneoctadecanoic acid, 7-cis,10-cis hexadecadienoic) or in fungi (cis-11,12-methyleneoctadecanoic acid). There were significant differences (P < 0.05) in the contents of specific FAs between mushroom species.     

Isolation and Characterisation of a Δ5-fatty Acid Elongase from the Marine Microalga Pavlova salina

The marine microalga Pavlova salina (Haptophyta, Pavlovophyceae) produces lipids containing approximately 50% n-3 long-chain polyunsaturated fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). A full-length cDNA sequence, designated PsElo5, was isolated from P. salina. Sequence alignment showed that the gene was homologous to corresponding ELO-type elongases from other microalgae. Heterologous expression of PsElo5 in yeast and in higher plants confirmed that it encodes a specific Δ5-elongase activity as predicted and, furthermore, within the n-3 pathway, the elongation activity was confined exclusively to EPA.     

Production of structured triacylglycerols by acidolysis catalyzed by lipases immobilized in a packed bed reactor

The aim of this work was to produce structured triacylglycerols (STAGs), with caprylic acid located at positions 1 and 3 of the glycerol backbone and docosohexaenoic acid (DHA) at position 2, by acidolysis of tuna oil and caprylic acid (CA) catalyzed by lipases Rd, from Rhizopus delemar, and Palatase 20000L from Mucor miehei immobilized on Accurel MP1000 in a packed bed reactor (PBR), working in continuous and recirculation modes. First, different lipase/support ratios were tested for the immobilization of lipases and the best results were obtained with ratios of 0.67 (w/w) for lipase Rd and 6.67 (w/w) for Palatase. Both lipases were stable for at least 4 days in the operational conditions. In the storage conditions (5 °C) lipases Rd and Palatase maintained constant activity for 5 months and 1 month, respectively.These catalysts have been used to obtain STAGs by acidolysis of tuna oil and CA in a PBR operating with recirculation of the reaction mixture through the lipase bed. Thus, STAGs with 52–53% CA and 14–15% DHA were obtained. These results were the basis for establishing the operational conditions to obtain STAGs operating in continuous mode. These new conditions were established maintaining constant intensity of treatment (IOT, lipase amount × reaction time/oil amount). In this way STAGs with 44–50% CA and 17–24% DHA were obtained operating in continuous mode. Although the compositions of STAGs obtained with both lipases were similar, Palatase required an IOT about four times higher than lipase Rd.To separate the acidolysis products (free fatty acids, FFAs, and STAGs) an extraction method of FFAs by water–ethanol solutions was tested. The following variables were optimized: water/ethanol ratio (the best results were attained with a water/ethanol ratio of 30:70, w/w), the solvent/FFA–STAG mixture ratio (3:1, w/w) and the number of extraction steps (3–5). In these conditions highly pure STAGs (93–96%) were obtained with a yield of 85%. The residual FFAs can be eliminated by neutralization with a hydroethanolic KOH solution to obtain pure STAGs. The positional analysis of these STAGs, carried out by alcoholysis catalyzed by lipase Novozym 435, has shown that CA represents 55% of fatty acids located at positions 1 and 3 and DHA represents 42% of fatty acids at position 2.     

Supercritical fluid extraction of lipids from the heterotrophic microalga Crypthecodinium cohnii

Microalgae biomass can be a feasible source of ω‐3 fatty acids due to its stable and reliable composition. In the present study, the Crypthecodinium cohnii growth and docosahexaenoic acid (DHA, 22:6ω3) production in a 100 L glucose‐fed batch fermentation was evaluated. The lipid compounds were extracted by supercritical carbon dioxide (SC‐CO₂) from C. cohnii CCMP 316 biomas, was and their fatty acid composition was analysed. Supercritical fluid extraction runs were performed at temperatures of 313 and 323 K and pressures of 20.0, 25.0 and 30.0 MPa. The optimum extraction conditions were found to be 30.0 MPa and 323 K. Under those conditions, almost 50% of the total oil contained in the raw material was extracted after 3 h and the DHA composition attained 72% w/w of total fatty acids. The high DHA percentage of total fatty acids obtained by SC‐CO₂ suggested that this extraction method may be suitable for the production of C. cohnii value added products directed towards pharmaceutical purposes. Furthermore, the fatty acid composition of the remaining lipid fraction from the residual biomass with lower content in polyunsaturated fatty acids could be adequate for further uses as feedstock for biodiesel, contributing to the economy of the overall process suggesting an integrated biorefinery approach.     

Production and identification of a novel compound, 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-hexadecenoic acid from palmitoleic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids are considered as important value-added product for industrial application because of their special properties such as higher viscosity and reactivity. Microbial production of the hydroxy fatty acids from various fatty acid substrates have been actively studied using several microorganisms. The new bacterial isolate Pseudomonas aeruginosa (PR3) had been reported to produce mono-, di-, and tri-hydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) were produced from oleic acid and ricinoleic acid, respectively. Based on the postulated common metabolic pathway involved in DOD and TOD formation by PR3, it was assumed that palmitoleic acid containing a singular 9-cis double bond, common structural property sharing with oleic acid and ricinoleic acid, could be utilized by PR3 to produce hydroxy fatty acid. In this study, we tried to use palmitoleic acid as substrate for production of hydroxy fatty acid by PR3 and firstly confirmed that PR3 could produce 7,10-dihydroxy-8(E)-hexadecenoic acid (DHD) with 23% yield from palmitoleic acid. DHD production was peaked at 72 h after the substrate was added to the 24-h-culture.     

Analysis of whole cellular fatty acids and anastomosis relationships of binucleate Rhizoctonia spp. associated with Ceratobasidium cornigerum

Previous research has demonstrated that whole cellular fatty acids analysis is a useful tool for identifying and establishing taxonomic relationships between anastomosis groups (AGs) and related Rhizoctonia isolates. In this experiment, the composition of fatty acid of 28 isolates of teleomorph genus Ceratobasidium cornigerum, consisting of binucleate Rhizoctonia, AG-A, AG-B(o), AG-C, AG-P, and AG-Q, was evaluated using gas chromatography. Eleven fatty acids identified, i.e., myristic, pentadecanoic, palmitic, 2-hydroxypalmitic, palmitoleic, heptadecanoic, 9-heptadecenoic, stearic, oleic, linoleic, and linolenic acids, were present in isolates of AG-A, AG-B(o), AG-C, AG-P, and AG-Q. The major fatty acids, palmitic, oleic, and linoleic acids, were common in all isolates, constituting 87.1% to 94.7% of the whole cellular fatty acids identified. Isolates within the same AG were closely clustered, whereas isolates from different AGs were clearly and distinctly clustered based on average linkage cluster analysis of whole cellular fatty acids. Principal-component analysis generated from all fatty acids also confirmed the divergent separation of the 5 AGs of binucleate Rhizoctonia.     

Lipid accumulation in <Emphasis Type="Italic">Schizochytrium</Emphasis> G13/2S produced in continuous culture

Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations, nitrogen-growth-limiting conditions induced the accumulation of 30–28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h⁻¹, both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h⁻¹, carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism.     

Functional analysis of a fatty acid elongase from arachidonic acid-producing <Emphasis Type="Italic">Mortierella alpina</Emphasis> 1S-4

We describe the isolation and characterization of a gene (MAELO) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4. Although the homologous MAELO gene had already been isolated from M. alpina ATCC 32221, its function had not yet been identified. The MAELO gene from M. alpina 1S-4 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae. Analysis of the fatty acid composition of the yeast transformant revealed the accumulation of 22-, 24-, and 26-carbon saturated fatty acids. On the other hand, RNA interference of the MAELO gene in M. alpina 1S-4 was carried out. The gene-silenced strain obtained on RNA interference exhibited low contents of 20-, 22-, and 24-carbon saturated fatty acids and a high content of stearic acid (18 carbons), compared with those in the wild strain. The enzyme encoded by the MAELO gene was demonstrated to be involved in the biosynthesis of 20-, 22-, and 24-carbon saturated fatty acids in M. alpina 1S-4.