Production of lipid compounds in the yeast Saccharomyces cerevisiae

This review describes progress using the yeast Saccharomyces cerevisiae as a model organism for the fast and efficient analysis of genes and enzyme activities involved in the lipid biosynthetic pathways of several donor organisms. Furthermore, we assess the impact of bakers yeast on the production of novel, high-value lipid compounds. Yeast can be genetically modified to produce selected substances in relatively high amounts. A major advantage in choosing yeast as an object for metabolic engineering is the fact that the lipid pathways in this organism have been described in detail and are well characterized. We focus on the de novo production of three major families of lipid products. These are: (1) sterols, providing some previously known and some novel applications as examples of the lipid pathway enhancement that occurs naturally in yeast, (2) the reconstitution of the biosynthetic pathway of steroid hormones and (3) the biosynthesis of polyunsaturated fatty acids, leading to the biosynthesis of different omega-3 and omega-6 fatty acids which do not occur naturally in yeast. We utilize the current knowledge and point out perspectives and problems for future biotechnological applications in the field of lipid compounds.     

Lipid biosynthesis in the marine flatworm Convoluta roscoffensis and its algal symbiont Platymonas convoluta.

As a part of an investigations on the lipid metabolism in Platyhelminthes, the acoel Convoluta roscoffensis, which harbors the green alga Platymonas convoluta as a symbiont, was studied. Isotopic tracer experiments established that the acoel lacks the ability to synthesize de novo long-chain saturated and unsaturated fatty acids and depends on its algal symbiont for these compounds. The acoel's fatty acid composition closely resembles that of the alga but differs from those of other animals; the acoel's polyunsaturated fatty acids are of the plant type (omega 3 family) rather than of the animal type (omega 6 family). The acoel also lacks the ability to synthesize sterols de novo. It contains 24-methylenecholesterol synthesized by the algae and, in addition, significant amounts of cholesterol, which is probably a host modification product of the algal sterol. With fatty acids provided by the symbiont, the acoel has the ability to synthesize its own complex lipids. The acoel contains relatively large amounts of triglyceride, phosphatidylcholine, and ethanolamine plasmalogen. These compounds are either not present at all or present only in minute amounts in the symbiotic alga. Since acoels belong to the most primitive forms of the present-day flatworms, the observed metabolic defects in this organism suggest that mechanisms for the biosynthesis of fatty acids and sterols were lost early during the evolution of the Platyhelminthes, and that this phenomenon is widespread within the phylum.     

Molecular complexes of amino acids with porphyrins as possible precursors of pigment-protein systems

The present communication gives experimental results of the studies of catalytic and photochemical properties of peptide-like compounds containing metalloporphyrins (hemoproteinoids and molecular complexes obtained through adsorption of porphyrins and amino acids on volcanic ash). The data suggest that molecular complexes of amino acids with porphyrins could have evolved in the course of chemical evolution and were intermediated between abiogenically synthesized molecules of amino acids and porphyrins and pigment-protein systems of living organisms.     

The Deoxyxylulose Phosphate Pathway for the Biosynthesis of Plastidic Isoprenoids: Early Days in Our Understanding of the Early Stages of Gibberellin Biosynthesis

The identification of a novel pathway for isopentenyl diphosphate synthesis by Rohmer, Arigoni and colleagues in the early 1990's has led to a reappraisal of terpenoid biosynthesis in many organisms. It is now apparent that in plants there are two biosynthetic routes to isopentenyl diphosphate-the classical mevalonate pathway in the cytosol and the deoxyxylulose phosphate pathway in plastids. Sesquiterpenoids and sterols are predominantly synthesized in the cytosol by the mevalonate pathway whereas monoterpenoids, diterpenoids, the phytol side-chain of chlorophyll, carotenoids, and the nonaprenyl side-chain of plastoquinone-9 are synthesized within plastids by the deoxyxylulose phosphate pathway. Our assumptions that the early stages of gibberellin biosynthesis are plastid-localized has led to several attempts to demonstrate that the deoxyxylulose phosphate pathway is the biosynthetic route to gibberellins. Although definitive evidence is still not available there is a growing body of evidence, mostly from transgenic plants and from the use of the inhibitor, fosmidomycin, that gibberellins are synthesized from deoxyxylulose phosphate-derived isopentenyl diphosphate. However, there is evidence that a small amount of cross-talk between the two pathways may occur, implying that the pathways are not totally autonomous. Implications for the regulation of the early stages of gibberellin biosynthesis are discussed.     

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.     

Identification of unusual fatty acids of four alpine plant species from the Pamirs

Fatty acid composition and structure in total lipids from the green above-ground parts of four alpine plants, Oxygraphis glacialis, Primula macrophylla, Rhodiola pamiroalaica, and Swertia marginata, were established by GC and GC-MS. A total of 55 fatty acids was detected, and 48 of them were identified. Ubiquitous palmitate, linoleate, and linolenate predominated in the lipids accounting for about 72-90% of the total fatty acids. At the same time, the latter contained numerous species, which were unusual for higher plants and included saturated odd-numbered n-acids (six C15-C25 species, 0.26-1.40%), saturated even-numbered very-long-chain n-acids (six C20-C30 species, 1.00-2.49%), iso-acids (nine C15-C26 species, 0.64-1.53%), anteiso-acids (four C15-C20 species, 0.08-1.57%), certain uncommon mono- and dienoic acids, as well as 16:3omega3, 18:3omega6, and 18:4omega3 acids that are absent from the most higher plants. Nine fatty acids were found here for the first time in higher plants and two may be new to science. The evidence on the unusual fatty acids is discussed with respect to their distribution in living organisms, pathways of biosynthesis, and chemotaxonomic role.     

Studies to determine the role rates of chain elongation and desaturation play in regulating the unsaturated fatty acid composition of rat liver lipids.

Rat liver microsomes were used to measure the rates of chain elongation and desaturation of acids in the linoleate, oleate and palmitoleate biosynthetic pathways. These studies were designed to determine whether there is a relationship between rates of conversion and the types of unsaturated fatty acids found in rat liver lipids. In some cases rates of conversion correlate well with the types of unsaturated fatty acid found inrat liver lipids. In other cases, rates of conversion must be correlated with other controls such as competitive interactions, retroconversion, and specificities for incorporating given acids into lipids in order to explain the unsaturated fatty acid composition of rat liver lipids. The roles and interrelationships of these various metabolic processes are discussed relative to the control of polyunsaturated fatty acid biosynthesis.     

Occurrence and biosynthesis of carotenoids in phytoplankton

Naturally occurring carotenoids are important sources of antioxidants, anti-cancer compounds and anti-inflammatory agents and there is thus considerable market demand for their pharmaceutical applications. Carotenoids are widely distributed in marine and freshwater organisms including microalgae, phytoplankton, crustaceans and fish, as well as in terrestrial plants and birds. Recently, phytoplankton-derived carotenoids have received much attention due to their abundance, rapid rate of biosynthesis and unique composition. The carotenoids that accumulate in particular phytoplankton phyla are synthesized by specific enzymes and play unique physiological roles. This review focuses on studies related to the occurrence of carotenoids in different phytoplankton phyla and the molecular aspects of their biosynthesis. Recent biotechnological advances in the isolation and characterization of some representative carotenoid synthases in phytoplankton are also discussed.     

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.     

Metabolites from algae with economical impact

In order to survive in a highly competitive environment, freshwater or marine algae have to develop defense strategies that result in a tremendous diversity of compounds from different metabolic pathways. Recent trends in drug research from natural sources have shown that algae are promising organisms to furnish novel biochemically active compounds. The current review describes the main substances biosynthesized by algae with potential economic impact in food science, pharmaceutical industry and public health. Emphasis is given to fatty acids, steroids, carotenoids, polysaccharides, lectins, mycosporine-like amino acids, halogenated compounds, polyketides and toxins.     

Sterol-content lowering action of o-chlorobenzylchloride in yeast

o-Chlorobenzylchloride, a simple aromatic halogen compound, was found to inhibit the growth of Saccharomyces cerevisiae and to lower the contents of sterols and fatty acids. The growth inhibition was considerably alleviated by the presence of sterols such as ergosterol and cholesterol and of unsaturated fatty acids such as oleate and linolenate. Inspection of effect of the inhibitor on the electron transport system related to the biosyntheses of these compounds revealed that the cytochrome contents and some enzyme activities in the system of the inhibited cells were much lower than those of the control cells. The features of the inhibition were similar to those of inhibition for other organisms by the hypocholesterolemic compounds such as triparanol and benzmalecene.     

Lipid synthesis and metabolism in the plastid envelope

Plastid envelope membranes play a major role in the biosynthesis of glycerolipids. In addition, plastids are characterized by the occurrence of plastid-specific membrane glycolipids (galactolipids, a sulfolipid). Plant lipid metabolism therefore has unique features, when compared to that of other eukaryotic organisms, such as animals and yeast. However, the glycerolipid biosynthetic pathway in chloroplasts is almost identical to that found in cyanobacteria, and reflects the prokaryotic origin of the chloroplast. Fatty acids generated in the plastid stroma are substrates for a whole set of enzymes involved in the synthesis of polar lipids of plastid membranes such as galactolipids, the sulfolipid, the phosphatidylglycerol. In addition, fatty acids are exported outside the plastid where they are used for extraplastidial polar lipid synthesis (phosphatidylcholine, phosphatidylethanolamine, etc.). Various desaturation steps leading to the formation of polyunsaturated fatty acids occur in various cell compartments, especially in chloroplasts, using fatty acids esterified to polar lipids as substrates. Furthermore, plant glycerolipids can be metabolized by a series of very active envelope enzymes, such as the galactolipid:galactolipid galactosyltransferase and the acyl-galactolipid forming enzyme. The physiological significance of these enzymes is however largely unknown. One of the most active pathways involved in lipid metabolism and present in envelope membranes is the oxylipin pathway: polyunsaturated fatty acids that are released from polar lipids under various conditions (injury, pathogen attack) are converted to oxylipin. Thus, the plastid envelope membranes are also involved in the formation of signalling molecules.     

Microalgae metabolites: A rich source for food and medicine

Microalgae are one of the important components in food chains of aquatic ecosystems and have been used for human consumption as food and as medicines. The wide diversity of compounds synthesized from different metabolic pathways of fresh and marine water algae provide promising sources of fatty acids, steroids, carotenoids, polysaccharides, lectins, mycosporine-like amino acids, halogenated compounds, polyketides, toxins, agar agar, alginic acid and carrageenan. This review discusses microalgae used to produce biological substances and its economic importance in food science, the pharmaceutical industry and public health.     

Neuronal models to study amyloid precursor protein expression and processing in vitro

Most organisms obtain their fatty acids through their diet or by de novo synthesis, but human blood flukes belonging to the genus Schistosoma lack the oxygen-dependent pathways required for the synthesis of sterols and fatty acids so they are entirely dependent on their hosts for these and other complex lipids. Fatty acid binding proteins (FABPs) of the FABP/P2/CRABP/CRBP family of beta-barrel cytosolic lipid binding proteins (cLBP) appear to be particularly important to schistosomes in the uptake, transport and compartmentalisation of host-derived fatty acids and may provide important targets for immuno- and chemotherapy. Here we describe the isolation of a set of cDNAs prepared from the Asiatic schistosome, Schistosoma japonicum, which encode two groups of cLBPs based on sequence homology and unique cDNA restriction sites. Representative clones from the two groups, one encoding a complete Sj-FABP (F10), and the other encoding a deletion mutant (F25) were characterised at the nucleic acid level by Southern and Northern hybridisation analysis, and at the protein level by immunoblotting. The presence and size of introns in the genes encoding F10 and F25 were determined and, because of the interest in the Schistosoma mansoni FABP homologue (Sm14) as a putative vaccine candidate, the immunogenicity and protective efficacy of the two proteins were also evaluated. A particularly interesting finding was the degree of Sj-FABP amino acid sequence polymorphism found to occur within the S. japonicum worm population, which appears to be greater than that described from cLBPs from vertebrates or, indeed, any other group of organisms investigated to date.     

Fatty acids and other lipids of corals: Composition, distribution, and biosynthesis

This paper reviews the literature data on the composition, structure, and distribution of the main lipid classes and their fatty acids in reef-building and soft corals and in hydrocorals of the genus Millepora. The review presents information about more than 150 coral species from tropical and cold waters of the World Ocean, as well as data on the influence of environmental factors on the lipid compositions of corals. The possible pathways of polyunsaturated fatty acid biosynthesis in corals are discussed.     

Oxylipins and oxylipin synthesis pathways in fungi

Oxylipins are a family of oxygenated fatty acids that are very diverse with regard to origin, structure, and functions. These compounds are found in almost all living beings and serve both as autoregulators of the development of organisms and as communication molecules. The autoregulatory role of oxylipins in fungi is to control the development, reproduction, synthesis of secondary metabolites (including mycotoxins), and adaptive responses. The role of oxylipins in the regulation of pathogenesis accounts for an important aspect of research on the biological activity of these compounds. The synthetic pathways and functions of oxylipins of fungi, the differences between fungal oxylipins and oxylipins from bacteria, higher plants, and mammals, and the role of oxylipins in the interaction of fungi with other organisms are considered in the present review.     

Genomic and biochemical analysis of lipid biosynthesis in the unicellular rhodophyte Cyanidioschyzon merolae: lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis

The acyl lipids making up the plastid membranes in plants and algae are highly enriched in polyunsaturated fatty acids and are synthesized by two distinct pathways, known as the prokaryotic and eukaryotic pathways, which are located within the plastids and the endoplasmic reticulum, respectively. Here we report the results of biochemical as well as genomic analyses of lipids and fatty acids in the unicellular rhodophyte Cyanidioschyzon merolae. All of the glycerolipids usually found in photosynthetic algae were found, such as mono- and digalactosyl diacylglycerol, sulfolipid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. However, the fatty acid composition was extremely simple. Only palmitic, stearic, oleic, and linoleic acids were found as major acids. In addition, 3-trans-hexadecanoic acid was found as a very minor component in phosphatidylglycerol. Unlike the case for most other photosynthetic eukaryotes, polyenoic fatty acids having three or more double bonds were not detected. These results suggest that polyunsaturated fatty acids are not necessary for photosynthesis in eukaryotes. Genomic analysis suggested that C. merolae lacks acyl lipid desaturases of cyanobacterial origin as well as stearoyl acyl carrier protein desaturase, both of which are major desaturases in plants and green algae. The results of labeling experiments with radioactive acetate showed that the desaturation leading to linoleic acid synthesis occurs on phosphatidylcholine located outside the plastids. Monogalactosyl diacylglycerol is therefore synthesized by the coupled pathway, using plastid-derived palmitic acid and endoplasmic reticulum-derived linoleic acid. These results highlight essential differences in lipid biosynthetic pathways between the red algae and the green lineage, which includes plants and green algae.     

Efficiency of transfer of essential polyunsaturated fatty acids versus organic carbon from producers to consumers in a eutrophic reservoir

One of the central paradigms of ecology is that only about 10% of organic carbon production of one trophic level is incorporated into new biomass of organisms of the next trophic level. Many of energy-yielding compounds of carbon are designated as ‘essential’, because they cannot be synthesized de novo by consumers and must be obtained with food, while they play important structural and regulatory functions. The question arises: are the essential compounds transferred through trophic chains with the same efficiency as bulk carbon? To answer this question, we measured gross primary production of phytoplankton and secondary production of zooplankton and content of organic carbon and essential polyunsaturated fatty acids of ω-3 family with 18–22 carbon atoms (PUFA) in the biomass of phytoplankton and zooplankton in a small eutrophic reservoir during two summers. Transfer efficiency between the two trophic levels, phytoplankton (producers) and zooplankton (consumers), was calculated as ratio of the primary production versus the secondary (zooplankton) production for both carbon and PUFA. We found that the essential PUFA were transferred from the producers to the primary consumers with about twice higher efficiency than bulk carbon. In contrast, polyunsaturated fatty acids with 16 carbon atoms, which are synthesized exclusively by phytoplankton, but are not essential for animals, had significantly lower transfer efficiency than both bulk carbon, and essential PUFA. Thus, the trophic pyramid concept, which implicitly implies that all the energy-yielding compounds of carbon are transferred from one trophic level to the next with the same efficiency of about on average 10%, should be specified for different carbon compounds.     

The Fatty Acid Composition of Paramecium aurelia Cells and Cilia: Changes with Culture Age

SYNOPSIS.
The fatty acids of whole cells and cilia from Paramecium tetraurelia strains 51s and d,95 and from Paramecium octaurelia strain 299s were identified. Ciliates were grown axenically in 3 types of culture media. More than 30 fatty acid species were identified and their structures determined by gas chromatography, mass spectrometry, argentation chromatography, hydro-genation, and fragmentation technics. The major fatty acids were hexadecanoic, octadecanoic, 9-octadecenoic, 9,12-octadecadi-enoic, 6,9,12-octadecatrienoic, and 5,8,11,14-eicosatetraenoic acids. Minor variations in fatty acid compositions were observed in cells grown in the different culture media as well as among the 3 strains. Major changes in fatty acid compositions occurred with culture age and cell density. The cells accumulated exogenous lipids in cytoplasmic vesicles. These lipids were utilized as culture age progressed. Both cellular volume and lipid content were greater in young than in older cultures. Fatty acid compositions of both whole cells and cilia changed with age and had a relative decrease in saturated, short-chained and odd-numbered carbon acids. Cilia lipids were enriched in long-chained, polyunsaturated acids as compared to lipids in whole cell extracts. Eicosatetra-enoic acid (arachidonic acid) increased to the greatest extent with age in both cellular and ciliary lipids, accounting for 20–60% of the total fatty acids in cilia. The age-related change in fatty acid composition in Paramecium is among the largest observed in eukaryotic organisms. It was concluded that some minor fatty acids found in Paramecium lipids were incorporated directly from certain culture media and that Paramecium had w 3, 6, and 9 pathways for polyunsaturated fatty acid biosynthesis.