Rhamnolipids, Sophorolipids, and Other Glycolipid Biosurfactants

Innumerable 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 components of tissues, while others produced by microorganisms are secreted into the growth medium. Because of their amphipathic nature, simple glycolipids are natural biodegradable detergents. In addition, some are reputed to have valuable pharmaceutical properties, for example as antibiotic, antifungal or even anticancer agents. A number of these lipids are major products of certain organisms, and have appreciable commercial importance. 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. Little or nothing is known of their biological functions or biosynthesis and the reviews by Dembitsky cited below cover the 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). Mycobacteria produce 6-O-acylglucosides of mycolic acids in addition to the more complex trehalose lipids described below.

Linoleic acid is oxidized in the human liver by a P450 mono-oxygenase to a mixture of 9,10 and 12,13 epoxides, which are converted to the corresponding diols, termed leukotoxin and isoleukotoxin, by an epoxide hydrolase. 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

A small proportion of the dihydroxy metabolites are also converted to glucuronide esters. 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, facilitating 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.

Many cyanobacterial species contain distinctive organelles termed heterocysts that are capable of fixing nitrogen. The cell walls of these maintain a micro-aerobic 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 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.

Nematodes, including a number of human parasites, contain unusual glycolipids termed ascarosides especially in the eggs and ovaries. 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. It protects the contents from the harsh conditions in the intestines. Two representative examples are illustrated.

formulae of two representative ascarosides

In addition, certain ascarosides in the nematode Caenorhabditis elegans function as pheromones as well as regulating development and behaviour.

 

2. Rhamnolipids

Pseudomonads are rod-shaped gram-negative bacteria found in soils that produce extracellular lipids known as rhamnolipids. The term is indicative of the fact that these lipids contain one or two rhamnose units, linked glycosidically to a 3-hydroxy acid, thence by an ester bond to a further 3-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.

formulae of rhamnolipids

3- or β-Hydroxydecanoic acid is the most common fatty acid constituent, but other fatty acids 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. All of these lipids have antifungal and antiviral properties, and they exhibit bactericidal properties to gram-positive bacteria. On the other hand, they are considered as one of the virulence factors in Pseudomonas sp. Because of their potent detergent properties, they are produce commercially as soil remediation agents 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. Specific genetically modified Pseudomonas species can produce as much as 100 g/L of culture medium under optimum conditions. While the wild organisms are pathogenic so must be cultured in a strictly regulated environment, the recombinant Pseudomonads used in industry appear to be safe.

Some nonpathogenic 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. Two unusual rhamnolipids, designated myxotyrosides A and B, have been isolated from a Myxococcus sp. (Myxobacteria are 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 monorhamnolipid in Pseudomonas species involves two sequential glycosyl-transfer reactions catalysed by specific rhamnosyltransferases, in which 3-hydroxydecanoyl-3-hydroxydecanoate is linked to an activated rhamnose moiety (thymidine diphospho-rhamnose). The lipid intermediate in rhamnolipid biosynthesis has a separate function in the swarming motility of the organisms.

 

3. Sophorolipids

Some yeast species, and in particular Candida (Torulopsis) 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-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 and then have more powerful detergency properties. 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 sequential transfer of activated glucose molecules, UDP-glucose (see our web page on glycosyldiacylglycerols), to a hydroxy acid in processes catalysed by two different glycosyltransferases. Finally, the molecule is acetylated by an acetyltransferase. The fatty acid constituents can be synthesised de novo from acetate or by modifying alkanes in the growth medium.

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. Yields can be as much as 300 g/L from organisms in the stationary phase. Sophorolipids are used in commerce in cosmetics as deodorant, antidandruff and bacteriostatic agents, and they are also known to possess antifungal, antiviral and spermicidal properties. The hydroxy acid constituents are in demand for lactonization for use in perfumes

 

4. Mannosylerythritol and Cellobiose Lipids

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

formulae - mannosylerythritol and cellobiose lipids

One or two of the hydroxyls on the mannose residue are acetylated, and there are one to three esterified fatty acids, which are both are 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. Some of these 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. Ustilago maydis also contains distinctive cellobiose lipids (or ‘ustilagic acid’), consisting of the disaccharide cellobiose linked O-glycosidically to the ω-hydroxyl group of the unusual long-chain fatty acid 15,16-dihydroxyhexadecanoic acid or 2,15,16-trihydroxyhexadecanoic acid. Others of the hydroxyl groups are esterified either to acetate or a medium-chain 3-hydroxy fatty acid. 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.

 

5. Trehalose Lipids

Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an α,α-1,1-glycosidic linkage. It is the basic component of a number of cell wall glycolipids in Mycobacteria and Corynebacteria. Of these trehalose lipids, cord factor is the best known (the name comes from its 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-chain mycolic acids esterified to the 6-hydroxyl group of each glucose to give trehalose 6,6’-dimycolate. 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 membranes conferring appreciable drug resistance to the organisms.

structure - cord factor

During biosynthesis, trehalose is first esterified to form the monomycolate, which is believed to be the precursor to the dimycolate, although via the action of a mycolyl transferase it also may be the donor of mycolic acid residues to the cell wall arabinogalactan to produce the mycolyl-arabinogalactan-peptidoglycan complex.

a succinoyl trehalose lipidAmong the other antigenic glycolipids in the mycobacterial cell wall based upon trehalose, there are acylated trehaloses with various fatty acids attached to the 2- and 3-hydroxyl groups of the same glucose. These 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. 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). Trehalose lipids produced by Corynebacteria and Nocardia are similar in structure but contain the corynomycolic or nocardomycolic 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 also known. These lipids are located in the cell envelope of Rhodococcus species, and they are produced mainly when the organisms are grown on hydrocarbons.

The only known trehalose-containing lipids isolated from an animal source are ‘maradolipids’, i.e. trehalose esterified to two C15 to C19 fatty acids, present in the dauer larva of the nematode Caenorhabditis elegans.

 

6.  Polymeric Exolipopolysaccharides

Emulsan is a lipopolysaccharide, produced by Acinetobacter calcoaceticus, 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.

 

7.  Liamocins and Exophilins

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 nonacetylated, whereas A2 and B2 each contain a single 3-O-acetyl group. The exophilins are related molecules (unesterified fatty acids) lacking the mannitol residue, first found in the marine yeast Exophiala pisciphila but also detected in Aureobasidium. They have pronounced antibacterial properties.

Figure 10

 

Suggested Reading

  • Bauersachs, T., Compaore, J., Hopmans, E.C., Stal, L.J., Schouten, S. and, Damste, J.S.S. Distribution of heterocyst glycolipids in cyanobacteria. Phytochemistry, 70, 2034-2039 (2009) (DOI: 10.1016/j.phytochem.2009.08.014).
  • Boulton, C.A. Extracellular microbial Lipids. In: Microbial Lipids. Volume 2. pp. 669-694 (Ed. C. Ratledge & S.G. Wilkinson, Academic Press, London) (1989).
  • Brennan, P.J. Mycobacterium and other actinomycetes. In: Microbial Lipids. Volume 1. pp. 203-298 (Ed. C. Ratledge & S.G. Wilkinson, Academic Press, London) (1988).
  • Dembitsky, V.M. Astonishing diversity of natural surfactants: 1. Glycosides of fatty acids and alcohols. Lipids, 39, 933-953 (2004) (there are six further reviews by this author in Lipids that are also relevant) (DOI: 10.1007/s11745-004-1316-1).
  • Franzetti, A., Gandolfi, I., Bestetti, G., Smyth, T.J.P. and Banat, I.M. Production and applications of trehalose lipid biosurfactants. Eur. J. Lipid Si. Technol., 112, 617-627 (2010) (DOI: 10.1002/ejlt.200900162).
  • Jude, A.R., Little, J.M., Freeman, J.P., Evans, J.E., Radominska-Pandya, A. and Grant, D.F. Linoleic acid diols are novel substrates for human UDP-glucuronosyltransferases. Arch. Biochem. Biophys., 380, 294-302 (2000) (DOI: 10.1006/abbi.2000.1933).
  • Morita, T., Fukuoka, T., Imura, T. and Kitamoto, D. Production of mannosylerythritol lipids and their application in cosmetics. Appl. Microbiol. Biotechnol., 97, 4691-4700 (2013) (DOI: 10.1007/s00253-013-4858-1).
  • Price, N.P.J., Manitchotpisit, P., Vermillion, K.E., Bowman, M.J. and Leathers, T.D. Structural characterization of novel extracellular liamocins (mannitol oils) produced by Aureobasidium pullulans strain NRRL 50380. Carbohydr. Res., 370, 24-32 (2013) (DOI: 10.1016/j.carres.2013.01.014).
  • Soberón-Chávez, G., Lépine, F. and Déziel, E. Production of rhamnolipids by Pseudomonas aeruginosa. Appl. Microbiol. Biotechn., 68, 718-725 (2005) (DOI: 10.1007/s00253-005-0150-3).
  • Van Bogaert, I.N.A., Zhang, J.X. and Soetaert, W. Microbial synthesis of sophorolipids. Process Biochem., 46, 821-833 (2011) (DOI: 10.1016/j.procbio.2011.01.010).

 

Updated July 1, 2013

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