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Rhamnolipids, Sophorolipids and Related Glycolipids

Scottish thistleInnumerable simple glycolipids comprising simply fatty acids esterified to a carbohydrate moiety have been described in nature from animals, plants and microorganisms, and it is impossible to discuss more than a few representative examples here. They can vary in structure from monosaccharides with one or more fatty acyl substituents to complex carbohydrates, which can in turn be linked to terpenoids, aromatic compounds or nucleosides, as well as having multiple points of attachment to fatty acids via ester or glycosidic linkages. Some are integral or structural components of tissues, while others produced by microorganisms are secreted into the growth medium. Because of their amphipathic nature, simple glycolipids are natural surfactants, which are biodegradable and have low ecotoxicity. In addition, some are reputed to have valuable pharmaceutical properties, for example as antibiotic, anti-fungal or even anticancer agents. A number of these lipids are major products of certain organisms, and they have appreciable commercial importance although costs still restrict their use.

Substantial amounts of simple fatty acyl derivatives of sugars, e.g. sucrose esters, are produced in industry by chemical synthesis, but the discussion here is restricted to natural glycolipids.

1. Simple Carbohydrate-Fatty Acid/Alcohol Conjugates

Simple conjugates of mono- and disaccharides with fatty acids via glycosidic or ester bonds (alkyl or acyl glycosides) are common in nature, but especially in marine organisms and in plants, and a few examples only are described here. Little or nothing is known of the biological functions or biosynthesis of most, but the reviews by Dembitsky cited below cover the chemical literature thoroughly. In contrast, a glucopyranosyl derivative of tuberonic acid is known to induce tuber formation in potatoes (see our web page on plant oxylipins).

Leukotoxin glucuronides: Linoleic acid is oxidized in the human liver by a cytochrome P450 mono-oxygenase to a mixture of 9,10- and 12,13-epoxides, which are converted to the corresponding diols, known as leukotoxin and isoleukotoxin, by an epoxide hydrolase (note that the term 'leukotoxin' is used also in a wider sense for many non-lipid compounds toxic to leukocytes) (see our web page on hydroxy-eicosatetraenoic acids (HETE)). Specific enantiomers of each of the four possible hydroxyl groups can then be converted to glucuronides by the action of a UDP-glucuronosyltransferase. The products from 9,10-dihydroxyoctadec-12-enoate are illustrated.

Fatty acid glucuronide

As the precursor monoepoxides of linoleic acid are produced at high levels during acute inflammation and in patients with adult respiratory distress syndrome or suffering from severe burns, it is believed that glucuronidation may be a detoxification mechanism to facilitate excretion. However, there are also suggestions that some fatty acid glucuronides, for example of phytanic and docosahexaenoic acids, may be ligands for hormone receptors in the nucleus or have signalling functions.

Trichome sugar esters: Plants in the Solanacea family have glandular trichomes, i.e. secretory organs often associated with hairs. They secrete mixtures of sugar esters onto the plant aerial surfaces that act as protective agents against insect herbivores and pathogenic fungi. In the tomato, for example, these metabolites consist of a carbohydrate backbone, usually glucose or sucrose, to which two to five fatty acids are esterified. The aliphatic acyl chains vary in length from C2 to C12 and are straight chain or have iso- or anteiso-methyl-branches. Biosynthesis involves a series of reactions catalysed by four acyl-sugar acyltransferases in which fatty acids as the CoA esters are transferred sequentially to the sugar unit.

Formula of a diacyl-sucrose

Heterocyst glycolipids: Many cyanobacterial species contain distinctive organelles termed heterocysts that are capable of fixing nitrogen. The cell walls of these maintain a micro-anaerobic environment to enable the reaction to occur, and they consist of three extra layers external to the normal cell envelope, the innermost of which is comprised of unusual glycolipids, i.e. very-long-chain fatty alcohols (up to 32 carbons) linked to a carbohydrate moiety, such as the 1-(O-α-D-glucopyranosyl)-3R,25R-hexacosanediol illustrated.

Formula of 1-(O-alpha-D-glucopyranosyl)-3R,25R-hexacosanediol

Other forms exist differing in the number of carbon atoms, and the number and position of hydroxyl and/or keto groups, but they usually have C26 or C28 carbon-chains with hydroxyl groups at the C-3, omega-1 or omega-3 positions. Keto-ols and keto-diols have their carbonyl moieties mainly in the C-3 position. Marine endosymbiotic cyanobacteria contain related heterocyst glycolipids with longer-chain alcohols (C30 and C32) linked to the C5 sugar ribose.

Other simple glycolipids: Nematodes, including a number of human parasites, contain unusual glycolipids termed ascarosides in the eggs and ovaries especially. These consist of α-L-3,6-dideoxymannose or ascarylose, which occurs in few other organisms, linked glycosidically to the hydroxyl group of a 2-hydroxy alcohol or of an (ω-1)-hydroxy fatty acid. The free hydroxyl groups of the ascarylose moiety may be acetylated, and the chain length of the alkyl component can vary from 6 to 29 and can contain further hydroxyl groups or double bonds. For example, the eggs of Ascaris sp. have a four-layer shell, the innermost layer of which consists of 75% of ascarosides and is responsible for the impermeability of the shell, which protects the contents from the harsh conditions in the intestines. In addition, certain ascarosides in the nematode Caenorhabditis elegans function as pheromones as well as signalling molecules that regulate development and behaviour. For example a short-chain (ω-1)-hydroxy acid linked to ascarylose (and often then to more complex entities) is part of a signally mechanism that controls the entry and exit of nematodes from a dormant or 'dauer' stage. Two representative examples are illustrated.

Formulae of two representative ascarosides

Myxomycetes (slime moulds) produce unusual glycolipids containing glucose, mannose or rhamnose linked to a multimethylbranched-polyunsaturated fatty acid (quite different from the more conventional fatty acids). Similarly, Mycobacteria produce simple 6-O-acylglucosides of mycolic acids in addition to the more complex trehalose lipids described below. They are produced in vivo from host glucose and are recognized as a glycolipid antigens.

A glycolipid from myxomycetes

2. Rhamnolipids

Pseudomonads are rod-shaped gram-negative bacteria and opportunistic human pathogens that are found in soils and produce extracellular lipids known as rhamnolipids, which are the most intensively studied biosurfactants because they have high surface activities and are produced in high yields after relatively short incubation periods by a well-understood microorganism. The name is indicative of the fact that these lipids contain one or two L-(+)-rhamnose units, linked glycosidically to a 3-(R)-hydroxy acid, thence by an ester bond to a further 3-(R)-hydroxy acid as illustrated. Thus, the monorhamnolipid from Pseudomonas aeruginosa grown on hydrocarbons is 2-O-α-L-rhamnopyranosyl-α-L-3-hydroxydecanoyl-3-hydroxydecanoic acid. Forms that lack the second fatty acid or have a third occur also. While 3- or β-hydroxydecanoic acid is the most common fatty acid constituent, others may be found depending on the Pseudomonas species or growth conditions, including 12:0, 12:1, 12:2 and 8:2 (each with a 3-hydroxyl group), resulting in a number of distinct molecular species. Rhamnolipids are essential for the growth of P. aeruginosa on hydrophobic carbon sources.

Formulae of rhamnolipids

Rhamnolipids are considered one of the most effective biosurfactants, mainly due to the reduction of surface tension in water from 72 to <30 mN/m with critical micellar concentration values between 20 and 225 mg/L, depending largely on the nature of the acyl groups. Because of these potent detergent properties, they are produce commercially as soil remediation agents to remove hydrocarbon and heavy metal pollutants and to combat marine oil pollution. Although the exact mechanism is not clear, it is evident that rhamnolipids are able to bind to substrates with low degrees of aqueous solubility including hydrophobic pollutants. Rhamnolipids are also used as a source of L-rhamnose for the chemical industry, and specific genetically modified Pseudomonas species can produce as much as 100g/L of culture medium under optimum conditions. All of these lipids have potent antifungal properties, especially to Fusarium species, and they exhibit antiviral and bactericidal properties to Gram-positive bacteria. On the other hand, they are one of the virulence factors in human infections by Pseudomonas sp. While the wild organisms are pathogenic and must be cultured in a strictly regulated environment, the recombinant Pseudomonads used in industry appear to be safe.

Some non-pathogenic thermophilic bacteria of the genera Thermus and Meiothermus contain rhamnolipids in a wide range of molecular forms, including mono- and di-rhamnolipid homologues containing one or two 3-hydroxy-fatty acids with zero to two double bonds and up to 24 carbon atoms. Similarly, the nitrogen-fixing soil bacterium Burkholderia kururiensis produces appreciable amounts of rhamnolipids. Two unusual rhamnolipids, designated myxotyrosides A and B, have been isolated from a Myxococcus sp. (gliding bacteria); these have a rhamnose unit linked to tyrosine and thence to a fatty acid such as (Z)-15-methyl-2-hexadecenoic and (Z)-2-hexadecenoic acid.

The biosynthesis of rhamnolipid in Pseudomonas species occurs during the late exponential and stationary phase of growth under limiting concentrations of iron and nitrogen. It involves two sequential glycosyl-transfer reactions catalysed by specific rhamnosyltransferases, in which the estolide 3-hydroxydecanoyl-3-hydroxydecanoate is linked to an activated rhamnose moiety (thymidine diphospho-rhamnose), generated from glucose-1-phosphate. The monoacyl-rhamnolipids are produced by hydrolysis of the diacyl forms, not my release of the partially acylated intermediate. The mono- and di-rhamnolipids appear to have separate functions in the swarming motility of the organisms, leading to the formation of biofilms that facilitate colonization of new environmental niches.

3. Sophorolipids

Some yeast species, and in particular the non-pathogenic Candida (Torulopsis or Starmerella) bombicola, secrete extracellular glycolipids known as sophorolipids (or sophorosides), as they contain the sugar sophorose (β-D-Glc-(1→2)-D-Glc). This is linked glycosidically to the hydroxyl group of a 17-(L)-hydroxy-C18 saturated or monoenoic (cis-9) fatty acid, the carboxyl group of which is usually linked to the 4’-hydroxyl group of the second glucose unit to form a lactone though it can also remain in free form; one or both of the 6-hydroxyl groups on the glucose units are acetylated. With the organism C. bogoriensis, the fatty acid is 13-hydroxydocosanoate, while in C. batistae it is 18-hydroxy-stearic acid (and the acidic form of the lipid predominates).

Formulae of sophorolipids

Biosynthesis involves hydroxylation of a fatty acid by the cytochrome P450 monooxygenase enzyme CYP52M1, followed by sequential transfer of activated glucose molecules, UDP-glucose (see our web page on glycosyldiacylglycerols), to the hydroxy acid in processes catalysed by two different glycosyltransferases. After the molecule is acetylated by an acetyltransferase, lactonization of the acidic form is effected by a specific lactone esterase. The fatty acid constituents can be synthesised de novo from acetate or by modifying alkanes in the growth medium. In so-called 'bolaform surfactants', the free acid group is attached to a separate sophorose unit. Although these are present in trace amounts only under normal conditions, they can accumulate in genetically modified organisms.

While the physiological role of sophorolipids in yeast species is uncertain, it seems likely that they serve for extracellular carbon storage (reducing the cellular sugar content) and as a defence against competing microorganisms.

These lipids are produced on a commercial scale when the organism is cultured on substrates containing glucose and a source of alkyl moieties, such as alkanes or seed oils, which influence the nature of the fatty acid constituent; genetic modification of the organism is used to improve yields and modify the nature of the products. Yields can be as much as 300g/L from organisms in the stationary phase. As a generality, sophorolipid lactones have better surface tension lowering and antimicrobial activity, whereas the acidic forms display better foam production and solubility. They are being used increasingly as detergents and 'eco-surfactants'. In addition, sophorolipids are used in commerce in cosmetics as deodorant, anti-dandruff and bacteriostatic agents, and they are also known to possess antifungal, antiviral and spermicidal properties. They induce apoptosis of cancer cells specifically in vitro, so they have some clinical potential. The hydroxy acid constituents are in demand for lactonization and thence for use in perfumes.

4. Mannosylerythritol and other Mannose-containing Lipids

Formula - mannosylerythritol lipidsMannosylerythritol lipids are produced abundantly by yeast strains of the genus Pseudozyma; they consist of fatty acids linked to either 4-O-β-D-mannopyranosylerythritol or 1-O-β-D-mannopyranosylerythritol as hydrophilic head group. For example, the yeast Pseudozyma (Candida) antarctica secretes an extracellular mannosylerythritol lipid (4-O-(2’,6’-di-O-acyl-β-D-mannopyranosyl)-D-erythritol) in high yield with biosurfactant properties when grown on a vegetable oil or alkane substrate. When grown on glucose, the same lipid accumulates intra-cellularly as an energy store until it amounts to 10% or more of the dry weight of the cell.

One or two of the hydroxyls (4' and/or 6') on the mannose residue are acetylated, and there are one to three esterified fatty acids, which are both odd- and even-numbered from C8 to C12 in chain-length (longer in related species). Several other species of the genus Pseudozyma are now known to produce similar lipids in which the nature, number and positions of the acyl groups vary. For example, P. aphidis produces a tri-acylated molecule in which the third fatty acid chain is attached to the primary alcohol of the erythritol moiety and is comprised mainly of oleic and linoleic acids, which are not found in conventional di-acylated mannosylerythritol lipids. Some of these lipids are finding commercial applications in food, pharmaceuticals and cosmetics.

As with other biosurfactants, these compounds are believed to facilitate dissolution of organic hydrophobic compounds so that they can be consumed by the organism. Mannosylerythritol lipids have been shown to have a number of profound biological effects in animals, but especially to induce the differentiation of certain cancer cells. They have anti-inflammatory properties in that they inhibit the secretion of inflammatory mediators from mast cells.

While Pseudozyma species give the greatest yields of these lipids, they were first found in the fungus Ustilago maydis and termed ustilipids. In this instance, the 2-hydroxyl group of the mannose residue is esterified with a C2 to C8 fatty acid, while the 3-hydroxyl group is esterified by a C12 to C20 fatty acid.

Liamocins are a class of mannitol-lipids produced by the black yeast Aureobasidium pullulans, which is used commercially to produce pullulan polysaccharide for breath-freshener films. The liamocins have a mannitol head group ester linked to 3,5-dihydroxydecanoate acyl chains, several of which are joined by 1,5-polyester linkages. Four liamocins were characterized from Aureobasidium strain NRRL 50380 with the A series containing three acyl groups and the B series containing four; liamocins A1 and B1 are non-acetylated, whereas A2 and B2 each contain a single 3-O-acetyl group. Some forms are showing promise as anticancer agents and others have antibacterial activity with a specificity for Streptococcus species.

Liamocins and exophilins

The exophilins are related molecules lacking the mannitol residue, i.e. they are unesterified fatty acids, which were first found in the marine yeast Exophiala pisciphila but have also been detected in Aureobasidium. They have pronounced antibacterial properties.

5.  Polymeric Exolipopolysaccharides

Emulsan is a lipopolysaccharide, produced by Acinetobacter calcoaceticus and the less toxic A. venetianus, consisting of a trisaccharide backbone of D-galactosamine, D-galactosaminouronic acid, and a deoxyaminohexose, to which fatty acid groups, ranging in chain-length from C10 to C22, are linked via ester and amide bonds. It is variable in composition depending on growth conditions, both in the nature of the fatty acyl constituents and the degree of branching of the carbohydrate backbone, with an approximate molecular weight of 1,000 kDa.

Structural formula for emulsan

Rather than reducing surface tension, it is a very strong emulsifier, especially for hydrocarbons. It is already in use for commercial applications in oil recovery, transportation and bioremediation, and it is approved for use as an adjuvant to enhance the human immune response.

6. Trehalose Lipids

Trehalose is a non-reducing disaccharide in which two glucopyranosyl units are linked in an α,α-1,1-glycosidic linkage; it is is a non-reducing disaccharide with high thermostability and resistance to acid hydrolysis, while not partaking in Maillard reaction. In nature, it is considered to be a universal stress-protector molecule with effects upon cryoprotection, and growth regulation and osmoregulation in plants. In those bacteria of the Actinomycetales that degrade alkanes, such as Mycobacteria and Corynebacteria, it is a basic component of a number of cell wall glycolipids. Of these trehalose lipids, cord factor is the best known (the name comes from its supposed influence on the arrangement of M. tuberculosis cells into long slender formations). These are components of the cell wall lipid of M. tuberculosis and comprise distinctive branched and very-long-chain mycolic acids esterified to the 6-hydroxyl group of each glucose to give trehalose 6,6’-dimycolate. In this esterified form, the mycolic acid residues are believed to adopt a singular 'W' conformation. Such is the complexity of mycolic acids, which differ in chain length and the presence of cyclopropane rings and various oxygenated functional groups, that 500 distinct molecular species are possible. In addition to being major pathogenic components of the cell wall, adversely influencing many aspects of the immune system and metabolism in host animals via a specific receptor, trehalose diesters are believed to be responsible for the low permeability of the Mycobacterial membranes conferring appreciable drug resistance to the organisms.

Formula of cord factor

During biosynthesis, trehalose is first esterified with mycolic acids synthesised in the cytoplasm to form the trehalose monomycolate, which is believed to be the precursor to the dimycolate, and via the action of a mycolyl transferase it also may translocate to the periplasm or outer membrane to serve as the donor of mycolic acid residues to the cell wall arabinogalactan to produce the mycolyl-arabinogalactan-peptidoglycan complex.

Among the other antigenic glycolipids in the mycobacterial cell wall based upon trehalose, there are acylated trehaloses with various di- and trimethyl fatty acids attached to the 2‑ and 3‑hydroxyl groups of the same glucose together with tri- and penta-acyltrehaloses, which have important structural functions in the cell envelope. They are synthesised sequentially by adjacent enzyme systems. The fatty acids include n-C16‑19 saturated fatty acids, C21‑25 α-methyl-branched fatty acids and C24‑28 α-methyl-branched, β-hydroxy fatty acids, as well as the mycolic acids. In addition, sulfoglycolipids consisting of 2,3,6,6′-tetraacyl-α-α’-trehalose-2′-sulfates are present in M. tuberculosis (see our web page on sulfolipids). As tuberculosis is one of the most ancient infectious diseases known to man and is still responsible for over a million deaths world-wide every year, there is an intense interest in the molecular biology of the lipids of the organism with the hope of finding a suitable drug that disrupts cell wall biosynthesis.

Di- and penta-acyltrehalose lipids from M. tuberculosis

Trehalose lipids produced by Corynebacteria and Nocardia are similar in structure but contain the corynomycolic or norcardomycolic acids, respectively, which are related in structure to the mycolic acids.

Strains of Rhodococcus erythropolis produce many different trehalose-containing lipids, including trehalose-6-monocorynomycolates, trehalose-6,6'-dicorynomycolates, and other species containing up to eight acyl groups, which can comprise corynomycolic acids, long-chain dibasic acids and methyl-branched acids, such as 10-methyloctadecanoic acid. Some of these lipids contain succinic acid, e.g.. 2,3,4,2''-di-O-succinoyl-di-O-alkanoyl-α,α-trehalose and 2,3,4-mono-O-succinoyl-di-O-alkanoyl-α,α-trehalose, while a 3,4-di-O-alkanoyl-2-O-succinoyl-α-D-glucopyranosyl-2'-O-succinoyl-α-D-glucopyranoside produced by Rhodococcus sp. SD-74 is illustrated; they are powerful surfactants. More complex sulfated trehalose lipids are known. These lipids are located in the cell envelope of Rhodococcus species, and they are produced mainly when the organisms are grown on hydrocarbons. As they have immunomodulatory properties, they are attracting some biomedical interest.

A succinoyl trehalose lipid

Maradolipids are the only known trehalose-containing lipids isolated from an animal source. They are present in the dauer larva of the nematode C. elegans, and are structurally similar to the 'cord factor' trehalose lipids discussed above, i.e. they are 6,6'-di-O-acyltrehalose but with two C15 to C19 fatty acid constituents (monoacyl-maradolipids have been characterized also). C. elegans produces the glycolipid ascarosides discussed above.

7. Other Important Biosurfactants

Formula of ustilagic acidIn addition to the mannose-containing lipid described above, Ustilago maydis contains distinctive cellobiose lipids termed ustilagic acids, consisting of the disaccharide cellobiose linked O-glycosidically to the ω‑hydroxyl group of the unusual long-chain fatty acids 15,16‑dihydroxyhexadecanoic or 2,15,16‑trihydroxyhexadecanoic acid, produced by sequential oxidations of palmitic acid by cytochrome P450 enzymes. The remaining hydroxyl groups are esterified either to acetate or a medium-chain 3-hydroxy fatty acid. Rapid and substantial production of the mannosyl ustilipids (see above) and ustilagic acid in this organism occurs under conditions of nitrogen starvation, the yield and ratio of each class depending on the available carbon source.

A further unusual cellobiose lipid is produced by the fungal biocontrol agent, Pseudozyma flocculosa, and has been show to be 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl)octadecyl cellobioside (flocculosin), the compound responsible for the antifungal activities of the organism.

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Lipid listings Credits/disclaimer Updated: May 8th, 2017 Author: William W. Christie LipidWeb icon