Inna Khozin

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acids to P. cruentum indicated the existence of two possible pathways as outlined in sequences I and II, respectively (unpublished data).

Biosynthesis of eicosapentaenoic acid in the microalgaPorphyridium cruentum. I: The use of externally supplied fatty acids

The biosynthetic pathways of eicosapentaenoic acid (20:5n-3) in microalgae, in general, and inPorphyridium cruentum, in particular, are not known. Some of the putative intermediates along the suggested pathways could not be detected probably due to their low endogenous level. In order to increase the endogenous levels of the intermediates, we provided various fatty acids in the growth medium. Exogenously supplied fatty acids were indeed incorporated into algal lipids and were further metabolized along the n-6 and n-3 pathways. In the n-6 pathway, 18:2 was desaturated to 18:3n-6, elongated to 20:3n-6, and subsequently desaturated to 20:4n-6 and then to 20:5n-3. In the n-3 pathway, 18:2 was first desaturated to 18:3n-3 which was then sequentially converted, apparently by the same enzymatic sequence of the n-6 pathway to 18:4n-3, 20:4n-3, and 20:5n-3.

Fatty acid unsaturation in the red alga Porphyridium cruentum. Is the methylene interrupted nature of polyunsaturated fatty acids an intrinsic property of the desaturases

Polyunsaturated fatty acids (PUFAs) exogenously supplied to the red microalga Porphyridium cruentum were incorporated into cellular lipids. All the C18 PUFAs studied were desaturated by a delta 6-desaturase and all the C20 PUFAs by a delta 5-desaturase. The latter enzyme desaturated even 20:2(11, 14) to 20:3(5, 11, 14) and 20:3(11, 14, 17) to 20:4(5, 11, 14, 17). We infer the existence of several fatty acid desaturases, with different chain length specificities. Furthermore, the introduction of double bonds in a methylene interrupted pattern, at least for alpha-type desaturase such as the delta 5- and delta 6-desaturases, is not an intrinsic property of the enzyme but a consequence of the arrangement of the pre-existing double bonds in the fatty acid substrate.

Isolation and Characterization of a Novel Leaf-Inhabiting Osmo-, Salt-, and Alkali-Tolerant Yarrowia lipolytica Yeast Strain

Salt‐excreting leaves of Atriplex halimus plants harvested in the central Negev Highlands of Israel were screened for yeasts inhabiting their surfaces. Several aerobic, moderately salt‐ and alkalitolerant yeasts were isolated. One of the isolates (tentatively designated S‐8) was identified as Yarrowia lipolytica (Wick.) van der Walt and Arx, on the basis of its morphological, biochemical/physiological characteristics, and of quantitative chemotaxonomic and molecular marker analyses. However, the strain is distinguished from the known members of the type Y. lipolytica strain by its pronounced osmo‐, salt‐, and pH tolerance. Cells displayed a unique capacity to grow over a wide pH range (from 3.5 to 11.5) with a pH optimum at 4.5 to 7.5. It is proposed that the S‐8 strain be assigned to a single Y. lipolytica species as its anamorpha, or as a new variety, Y. lipolytica var. alkalitolerance. The ecophysiological properties and biotechnological potentials of the new strain are discussed.

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.

Differential response of microalgae to the substituted pyridazinone, sandoz 9785, reveal different pathways in the biosynthesis of eicosapentaenoic acid

Abstract Treating the red alga, Porphyridium cruentum, with the herbicide, SAN 9785, an inhibitor of chloroplastic ω3-desaturation, resulted in a decrease in the proportion of eicosapentaenoic acid (EPA), especially in monogalactosyldiacylglycerols (MGDG). However, when the eustigmatophyte, Monodus subterraneus, was treated with this herbicide, the proportion of EPA in MGDG surprisingly increased from 54 to 81%. Molecular species analysis of MGDG, showed that the prokaryotic molecular species 20:5/16:0 was almost eliminated, while the eukaryotic 20:5/20:5 dominated. These findings indicate that EPA biosynthesis in M. subterraneous is different from that in P. cruentum. We suggest that in M. subterraneus there are at least two pathways involving ω3-desaturase activity which lead to the production of EPA. The chloroplastic pathway, which is inhibited by SAN 9785, and the cytoplasmic pathway, which presumably uses phospholipid-bound fatty acids and is not affected by this inhibitor. The product of the latter pathway is exported through the chloroplast membrane resulting in the production of 20:5/20:5 MGDG. We further suggest that, by analogy with higher plants, microalgae can be categorized according to their respective EPA biosynthetic pathways.

Spectrophotometric Analysis of Carotenoids in Plant Extracts Based on Elimination of Chlorophyll Absorption

Subtraction of the absorption spectra of chlorophyll(s) from the total spectra of plant cell extracts reveals the carotenoid absorption spectra in both the ultraviolet and visible regions, from which the total carotenoid content can be easily evaluated. The applications of this method to Spirulina platensis cells treated with the herbicide norflurazon and to Anabaena siamensis cells grown under high light irradiation are demonstrated. The possibility of subtraction of the absorption of both chlorophyll a and b is demonstrated in an extract of the leaves of a higher plant (grape).

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.