Inna Khozin-Goldberg

Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid

We have hypothesized that among algae of alpine environment there could be strains particularly rich in long chain polyunsaturated fatty acids (LC-PUFA). Indeed, the chlorophyte (Trebuxiophyceae) Parietochloris incisa isolated from Mt. Tateyama, Japan, was found to be the richest plant source of the pharmaceutically valuable LC-PUFA, arachidonic acid (AA, 20:4omega6). The alga is also extremely rich in triacylglycerols (TAG), which reaches 43% (of total fatty acids) in the logarithmic phase and up to 77% in the stationary phase. In contrast to most algae whose TAG are made of mainly saturated and monounsaturated fatty acids, TAG of P. incisa are the major lipid class where AA is deposited, reaching up to 47% in the stationary phase. Except for the presence of AA, the PUFA composition of the chloroplastic lipids resembled that of green algae, consisting predominantly of C(16) and C(18) PUFAs. The composition of the extrachloroplastic lipids is rare, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) as well as diacylglyceryltrimethylhomoserine (DGTS). PC and PE are particularly rich in AA and are also the major depots of the presumed precursors of AA, l8:3omega6 and 20:3omega6, respectively.

ACCUMULATION OF OLEIC ACID IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) UNDER NITROGEN STARVATION OR HIGH LIGHT IS CORRELATED WITH THAT OF ASTAXANTHIN ESTERS1

The chlorophyte Haematococcus pluvialis accumulates large quantities of astaxanthin under stress conditions. Under either nitrogen starvation or high light, the production of each picogram of astaxanthin was accompanied by that of 5 or 3–4 pg of fatty acids, respectively. In both cases, the newly formed fatty acids, consisting mostly of oleic (up to 34% of fatty acids in comparison with 13% in the control), palmitic, and linoleic acids, were deposited mostly in triacylglycerols. Furthermore, the enhanced accumulation of oleic acid was linearily correlated with that of astaxanthin. Astaxanthin, which is mostly monoesterified, is deposited in globules made of triacylglycerols. We suggest that the production of oleic acid‐rich triacylglycerols on the one hand and the esterification of astaxanthin on the other hand enable the oil globules to maintain the high content of astaxanthin esters.

EFFECT OF NITROGEN STARVATION ON OPTICAL PROPERTIES, PIGMENTS, AND ARACHIDONIC ACID CONTENT OF THE UNICELLULAR GREEN ALGA PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE, CHLOROPHYTA) 1

Spectral properties of cell suspensions, individual cells, and extracts of the unicellular green alga Parietochloris incisa (Reisigl) Shin Watan. grown under low light were studied. Long‐term nitrogen (N) deprivation resulted in a decrease of chloroplast volume, appearance of numerous large cytoplasmic oil bodies, and the deposition of triacylglycerols with a high proportion of arachidonic acid. Chlorophylls a and b underwent a synchronous decline, whereas carotenoids (Car) showed a relative increase. Simultaneously, significant qualitative changes in the spectral properties of P. incisa individual cells, cell extracts, and cell suspensions were observed. To a large extent, the spectral changes observed in cell suspension could be attributed to a decrease in overall pigment content, leading to a gradual weakening of the so‐called package effect and accumulation of additional amounts of Car over chl, most probably, in oil bodies. Several optical characteristics of cell suspensions could serve as sensitive indicators of N‐deficiency in P. incisa. Furthermore, the absorption ratios, A476/A676 and A650/A676, showed close correlations with the Car‐to‐chl ratio and relative arachidonic acid (AA) content, respectively. The latter makes it possible to suggest that the increase in AA percentage in P. incisa proceeds in parallel with a decrease in cell chl content, accounting for the weakening of the package effect. N‐replenishment resulted in complete recovery of cell optical properties. The possible significance of the changes in cell ultrastructure, pigments, lipids, and optical properties is discussed with special reference to the ability of algae to adapt to and survive under conditions of long‐term nutrient deficiency.

NITROGEN STARVATION INDUCES THE ACCUMULATION OF ARACHIDONIC ACID IN THE FRESHWATER GREEN ALGA PARIETOCHLORIS INCISA (TREBUXIOPHYCEAE)1

The Chlorophyte Parietochloris incisa comb. nov (Trebuxiophyceae) was found to be the richest plant source of the pharmaceutically valuable long‐chain polyunsaturated fatty acid (PUFA), arachidonic acid (20:4ω6, AA). Over 90% of total AA are deposited in triacylglycerols (TAG). Under nitrogen starvation, the fatty acid content constituted over 35% of dry weight and the proportion of AA exceeded 60% of total fatty acids. Consequently, we obtained an AA content of over 20%. This is, to the best of our knowledge, the highest reported content of any PUFA in algae. Increasing the biomass concentration resulted in an enhancement of both the proportion of AA and the fatty acid content. We hypothesize that one of the roles of TAG in P. incisa is to serve as a reservoir of AA that can be used for the construction of membranal lipids.

LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology

Microalgae offer potential for numerous commercial applications, among them the production of long-chain polyunsaturated fatty acids (LC-PUFAs). These valuable fatty acids are important for a variety of nutraceutical and pharmaceutical purposes, and the market for these products is continually growing. An appropriate ratio of LC-PUFA of the ω-3 and ω-6 groups is vital for “healthy” nutrition, and adequate dietary intake has strong health benefits in humans. Microalgae of diverse classes are primary natural producers of LC-PUFA. This mini-review presents an introductory overview of LC-PUFA-related health benefits in humans, describes LC-PUFA occurrence in diverse microalgal classes, depicts the major pathways of their biosynthesis in microalgae, and discusses the prospects for microalgal LC-PUFA production.

INHIBITION OF ASTAXANTHIN SYNTHESIS UNDER HIGH IRRADIANCE DOES NOT ABOLISH TRIACYLGLYCEROL ACCUMULATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) 1

Under stress conditions, Haematococcus pluvialis Flotow accumulates fatty acid–esterified astaxanthin, in extraplastidial lipid globules. The enhanced accumulation of fatty acids, mainly in triacylglycerols (TAG), among which oleic acid predominates, is linearly correlated with that of astaxanthin. We used inhibitors of either carotenoid or lipid biosynthesis to assess the interrelationship between carotenogenesis and TAG accumulation under high light irradiance as the stress factor. The two carotenogenesis inhibitors used—norflurazon, an inhibitor of phytoene desaturase, and diphenylamine (DPA), an inhibitor of β‐carotene C‐4 oxygenase—suppressed the accumulation of astaxanthin in a concentration‐dependent manner. Concurrently, the accumulation of neutral lipids was significantly less affected. The lipid biosynthesis inhibitor sethoxydim, which inhibits acetyl‐CoA carboxylase, significantly decreased de novo fatty acid synthesis and, in concert, drastically inhibited astaxanthin formation. In the presence of various concentrations of the three inhibitors, the inhibition of astaxanthin was not accompanied by a proportional decrease in oleic acid, which was used as a marker for TAG fatty acids. When astaxanthin synthesis was completely inhibited, the volumetric content of oleic acid was about 60% of the control value when the two carotenogenesis inhibitors (0.05 μM norflurazon or 20 μM DPA) were used and 27% of the control when the lipid‐synthesis inhibitor (50 μM) was used. We suggest therefore that TAG accumulation under high irradiance is not tightly coupled with astaxanthin accumulation, although the correlation between these two processes was demonstrated earlier. Furthermore, we propose that the accumulation of a certain amount of TAG is a prerequisite for the initiation of fatty acid–esterified astaxanthin accumulation in lipid globules.

Biosynthesis of arachidonic acid in the oleaginous microalga Parietochloris incisa (Chlorophyceae): Radiolabeling studies

The fresh-water green alga Parietochloris incisa is the richest plant source of the PUFA arachidonic acid (20∶4n−6, AA). To elucidate the biosynthesis of AA in this alga we labeled cultures of P. incisa with radioactive precursors. Pulse chase labeling with acetate resulted in its incorporation via the de novo biosynthesis pathway of FA. However, labeled acetate was also utilized for the elongation of C16 and C18 PUFA. Labeling with [1-14C]oleic acid has shown that the first steps of the lipid-linked FA desaturations utilize cytoplasmic lipids. PC and diacylglyceryltrimethylhomoserine are the major lipids involved as acyl carriers for the Δ12 and Δ6 desaturations of oleic acid, leading sequentially to linoleic and γ-linolenic acids. The latter is released from its lipid carrier and elongated to 20∶3n−6, which is reincorporated primarily into PF and PC and finally desaturated to AA. Galactolipids, mostly monogalctosyldiacylglycerol (MGDG), serve as substrates for the chloroplastic Δ12 desaturase and, apparently, the ω3 desaturation, common to higher plants and many green algae. The predominant sequence desaturates the 18∶1/16∶0 molecular species of MGDG stepwise to the 18∶3n−3/16∶3n−3 molecular species similar to the prokaryotic pathway of higher plants and green algae.

Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae, Chlorophyta)

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4omega6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3omega6, 20:3omega6 and AA, decreased, indicating an inhibition of the Delta12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 degrees C with [1-14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 degrees C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.

Cloning and molecular characterization of a novel acyl‐CoA:diacylglycerol acyltransferase 1‐like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum

We have identified and isolated a cDNA encoding a novel acyl‐CoA:diacylglycerol acyltransferase (DGAT)1‐like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full‐length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63‐bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid‐deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C16 and C18 fatty acids into TAG.

Differences in membrane fluidity and fatty acid composition between phenotypic variants of Streptococcus pneumoniae.

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < or = 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < or = 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < or = 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.