Chiara Bigogno

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.

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.

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.

Elucidation of the Biosynthesis of Eicosapentaenoic Acid (EPA) in the Microalga Porphyridium Cruentum

The biosynthetic pathways in algae that produce PUFAs of no more than 18 carbon atoms are presumed to be similar to those of higher plants (1). However, the biosynthesis of C20 PUFAs in various algae appears to be different. The few detailed studies suggest that the biosynthesis of eicosapentaenoic acid (EPA, 20:5ω3) from C18 precursors is entirely cytoplasmatic (2 – 4). Feeding external fatty acids to P. cruentum indicated the existence of two possible pathways as outlined in sequences I and II, respectively (unpublished data).

THE EFFECT OF GROWTH TEMPERATURE AND CULTURE DENSITY ON THE MOLECULAR SPECIES COMPOSITION OF THE GALACTOLIPIDS IN THE RED MICROALGA PORPHYHDIUM CRUENTUM (RHODOPHYTA)1

The effects of biomass concentration and growth temperature on the molecular species composition of the major galactolipids of the red microalga Porphyridium cruentum Nag. were detmined. At lower biomass concentrations, the Δ17 desaturation of arachidonic acid to eicosapentaenoic acid (20:5) was enhanced in both prokaryotic‐type and eukalyotic‐type molecular species of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol. We suggest that chloroplastic Δ17 desaturation is correlated with the availability of light. A reduction in the growth temperature led to an increase in the proportion of the eukalyotic molecular species of MGDG, especially for 20:5/ 20:5 MGDG, These results suggest that this molecular species, and perhaps eukaryotic molecular species in general, may play a role in the adaptation of cells to low growth temperatures.

Salicylhydroxamic acid inhibits delta6 desaturation in the microalga Porphyridium cruentum.

Treatment of the microalga Porphyridium cruentum with salicylhydroxamic acid (SHAM) inhibited growth and affected fatty acid composition. At a relatively low concentration (40 microM) SHAM predominantly inhibits Delta6 desaturation. The effect of the inhibitor was most intense in phosphatidylcholine (PC) and phosphatidylethanolamine, in which the proportions of the downstream products of the Delta6 desaturase were reduced, whereas that of the substrate, 18:2, increased. As a result of the availability of 18:2, 18:3omega3, which under normal conditions is not observed, appeared predominantly in chloroplastic lipids. Pulse labeling with linoleic acid has shown that SHAM inhibits Delta6 desaturation almost immediately, suggesting an apparent inhibition of the activity of the desaturase, rather than its synthesis or that of its cofactors. Furthermore, the addition of gamma-linolenic acid to SHAM-inhibited cultures relieved the inhibition. Following exposure to the inhibitor, 18:3omega3 appeared first in chloroplastic glycolipids and only later in PC, indicating that the former are the substrates for the first dedicated step of the proposed omega3 pathway in this alga.

Triacylglycerols Participate in the Eukaryotic Pathway of PUFAs Biosynthesis in the Red Microalga Porphyridium Cruentum

Wada and Murata (1) selected desaturase-deficient mutants of the cyanobacterium Synechocystis based on their chill sensitivity. Wada et al. (2) have further shown that PUFAs are necessary for growth and tolerance to photoinhibition at low temperatures. In the red alga P. cruentum, the PUFA eicosapentaenoic acid (EPA, 20:5ω3) apparently fulfills a role similar to that of 18:3ω3 in cyanobacteria and Arabidopsis (3). Since the EPA content increases at low temperatures we have hypothesized that by selection and screening of chill-sensitive mutants of this alga it would be possible to obtain EPA-deficient mutant which may aid in the elucidation of EPA biosynthesis. Based on the data we have obtained we suggest that triglycerides participate in the eukaryotic pathway of EPA biosynthesis in P. cruentum. The HZ3 mutant of P. cruentum is the first mutant of any, higher or lower, plant which appear to be deficient in its ability to hydrolyze TAGs.

Effect on Environmental Conditions on the Molecular Species Composition of Galactolipids in the Alga Porphyridium Cruentum

The lipid composition in higher plants and algae can be modulated by changing growth conditions such as temperature and light intensity. One of the major effects is on the level of fatty acid desaturation. Polyunsaturated fatty acids (PUFAs), especially those of the ω3 family appear to be essential for survival at low temperatures (Wada and Murata, 1989; Browse and Somerville, 1991). While there is an abundance of information pertaining to the effect of environmental conditions on the fatty acid composition of algae (Cobelas, 1989), very little is known concerning the effects on the molecular species composition of the individual lipids. The lipids of higher plants and algae are further divided according to their molecular species composition. The diacylglycerol (DAG) moieties of the prokaryotic species of galactolipids are assembled in the chloroplast and are characterized by the presence of C16 acyl groups at their sn-2 position, while the DAGs of eukaryotic species originate in the cytoplasm and contain C18 acyl groups in their sn-2 position. The aim of the present work was to study the effect of cultivation temperature and biomass density on the molecular species composition of the major galactolipids, MGDG and DGDG, in Porphyridium cruentum.