Waxes

To my knowledge, there is no satisfactory definition of the word "wax" in chemical terms. It is derived from the Anglo-Saxon word "weax" for beeswax, so a practical definition of a wax may therefore be "a substance that resembles beeswax in composition and physical properties". Those of best characterized are often chemically inert, non-polar lipids that are secreted onto the surface of organisms as a defence against biotic and abiotic stresses. Technologists use the term for a variety of commercial products of mineral, marine, plant and insect origin that contain fatty materials of various kinds, and synthetic waxes are now manufactured as lubricants to replace those formerly obtained from whales. Here, only those from living organisms are described, and waxes of mineral origin, such as montan wax, are not discussed. For practical convenience, alkylresorcinols are treated below, although their connection to main-stream waxes is tenuous.

1. Wax Components

Biochemists often link waxes with the thin layer of fatty constituents that cover the leaves of plants or provide a surface coating for insects or the skin of animals for which the primary requirements are hydrophobicity, spreadability, and chemical and metabolic stability. Such surface waxes are produced by specialized cells or glands, and all tend to contain wax esters as major and perhaps defining components, i.e., esters of long-chain fatty alcohols with long-chain fatty acids.

Formula of a wax ester

The nature of the other lipid constituents can vary greatly with the source of the waxy material, but they include hydrocarbons (e.g., alkanes and squalene), sterol esters, aliphatic aldehydes, primary and secondary alcohols, 1,2-, 2,3- and α,ω-diols, ketones, β-diketones, triacylglycerols and many more.

Formulae of common wax constituents

The chain-length and degree of unsaturation and branching of the fatty acids and the other aliphatic constituents, varies with the origin of the wax, but other than in some waxes of marine origin or from some higher animals, the aliphatic moieties tend to be saturated or monoenoic.

2. Plant Waxes

In the epidermal cells of plant leaves, the plasma membrane is covered by the cell wall structure, then a cutin layer and finally a coating of a thin layer of hydrophobic waxy material that is microcrystalline in structure and forms the outermost boundary of the cuticular membrane, i.e., it is the interface between the plant and the atmosphere. This wax layer serves many purposes, for example to limit the diffusion of water and solutes and control gas exchange, while permitting a controlled release of volatiles that may deter pests or attract pollinating insects. It provides protection from ultraviolet light, disease and insects, and it helps the plants to resist drought and other environmental stresses. Most analytical studies have dealt with the epicuticular layer, i.e., on the surface of the cutin scaffold, as this is easily extracted for analysis, but there is also an intracuticular wax, i.e., embedded in the cutin polymer, that is less well studied but is reported to be the more important transpiration barrier. As plants cover much of the earth's surface, it seems likely that plant waxes are among the most abundant of all natural lipids.

For practical reasons, cutin per se is discussed in another web page; it is a complex polyester with linear and branched chains that consist mainly of mono-, di- and trihydroxy fatty acids together with α,ω‑dicarboxylic fatty acids; the last may be linked via glycerol moieties.

The range of lipid types in plant waxes is highly variable, both in nature and in composition, and Table 1 illustrates some of this diversity in the main components.

Table 1. The major constituents of plant leaf waxes.
n-Alkanes	CH₃(CH₂)_xCH₃	21 to 35C - odd numbered
Alkyl esters	CH₃(CH₂)_xCOO(CH₂)_yCH₃	34 to 62C - even numbered
Fatty acids	CH₃(CH₂)_xCOOH	16 to 32C - even numbered
Fatty alcohols (primary)	CH₃(CH₂)_yCH₂OH	22 to 32C - even numbered
Fatty aldehydes	CH₃(CH₂)_yCHO	22 to 32C - even numbered
Ketones	CH₃(CH₂)_xCO(CH₂)_yCH₃	23 to 33C - odd numbered
Fatty alcohols (secondary)	CH₃(CH₂)_xCHOH(CH₂)_yCH₃	23 to 33C - odd numbered
β-Diketones	CH₃(CH₂)_xCOCH₂CO(CH₂)_yCH₃	27 to 33C - odd numbered
Triterpenols	sterols, α-amyrin, β-amyrin, uvaol, lupeol, erythrodiol, etc.
Triterpenoid acids	ursolic acid, oleanolic acid, etc.

In addition to those listed, there may be hydroxy-β-diketones, oxo-β-diketones, alkenes, branched alkanes, acids, esters, acetates and benzoates of aliphatic alcohols, methyl, phenylethyl and triterpenoid esters, polyketides and many more, some of which have specialized functions. Of these, triterpenes tend to be more abundant in the intracuticular layer. Phytol released by the catabolism of chlorophyll in plants is stored in leaves and fruit of some plant species and in mosses and algae in the form of an ester with fatty acids. In this form, phytol cannot disrupt membranes, but when required it can be released and utilized as a biosynthetic precursor for tocopherol and perhaps fresh chlorophyll. Polar complex lipids are rarely encountered in other than trace amounts.

The amount of each lipid class and the nature and proportions of the various molecular species within each class vary greatly according to the plant species and the site of wax deposition (leaf, flower, fruit, etc.), and some data for some well-studied species are listed in Table 2. It should be noted that these compositions can vary with developmental stage and with abiotic and biotic stresses, such as drought or insect predation.

	Arabidopsis leaf	Rape leaf	Apple fruit	Rose flower	Pea leaf	Sugar cane stem
Table 2. Relative proportions (wt %) of the common wax constituents in some plant species.

Hydrocarbons	73	33	20	58	40-50	2-8
Wax esters	4	16	18	11	5-10	6
Aldehydes	14	3	2	-	5	50
Ketones	-	20	3		-	-
Secondary alcohols	1	8	20	9	7	-
Primary alcohols	8	12	6	4	20	5-25
Acids	1	8	20	5	6	3-8
Other components present include various diol types and triterpenoid acids

Carnauba - The leaves of the carnauba palm, Copernicia cerifera that grows in Brazil, have a thick coating of wax, which can be harvested from the dried leaves. It contains mainly wax esters (85%), which constitute C₁₆ to C₂₀ fatty acids linked to C₃₀ to C₃₄ alcohols to give C₄₆ to C₅₄ molecular species, and these are accompanied by small amounts of free acids and alcohols, hydrocarbons and resins. This is the only leaf wax available in sufficient quantities to be of commercial value.

Jojoba - The jojoba plant (Simmondsia chinensis), which grows in the semi-arid regions of Mexico and the U.S.A., is unique in producing wax esters rather than triacylglycerols as the storage lipids in its seeds, and it has become a substantial crop. These consist mainly of 18:1 (6%), 20:1 (35%) and 22:1 (7%) fatty acids linked to 20:1 (22%), 22:1 (21%) and 24:1 (4%) fatty alcohols. Therefore, it contains C₃₈ to C₄₄ wax esters with one double bond in each alkyl moiety. As methylene-interrupted double bonds are absent, the wax is relatively resistant to oxidation.

Bayberry (Myrica pensylvanica) fruits are covered with a thick layer of crystalline wax (30% of the dry weight), which consists of triacylglycerols and diacylglycerols esterified exclusively with saturated fatty acids, mainly 14:0 and 16:0. It is believed that this may attract birds as an aid to seed dispersal. Biosynthesis and secretion differ from conventional plant waxes and are more closely related to cutin production with distinct pools of acyl donors and the final assembly of triacylglycerols outside of cells. The Japanese sumac tree (Rhus verniciflua) produces a similar wax ('Japan wax').

Biosynthesis of Plant Waxes

Because of their biochemical significance and relative ease of study, the waxes of the plant cuticle have received most study. All the aliphatic components of plant waxes are synthesised in the epidermal cells from saturated very-long-chain fatty acids (commonly C₂₀ to C₃₄). 16:0 and 18:0 Fatty acids are first synthesised in the stroma of plastids by the soluble enzymes forming the fatty acid synthase complex. The acyl-ACP products are hydrolysed by thioesterases to free fatty acids, which are esterified to coenzyme A before translocation to the endoplasmic reticulum. In the second stage, multiple elongation steps are catalysed by membrane-associated multi-enzyme complexes, known as fatty acid elongases, (see our web page on the biosynthesis of saturated fatty acids), requiring CER2-LIKE proteins (related to acyltransferases) to change the chain-length specificity of the elongation machinery. As in fatty acid synthesis de novo, each two-carbon extension of the chain involves four reactions: condensation between a CoA-esterified fatty acyl substrate and malonyl-CoA, followed by a β‑keto reduction, dehydration and an enoyl reduction to produce saturated very-long-chain fatty acids. These can be released as the free acids for export directly as cuticular waxes, or they can be further processed.

There are then two main pathways for biosynthesis of wax components in the endoplasmic reticulum of plastids of the epidermal cells: an acyl reduction pathway, which yields primary alcohols and wax esters, and a decarbonylation pathway that results in synthesis of aldehydes, alkanes, secondary alcohols and ketones. In the reductive pathway, acyl-CoA esters produced by chain elongation are reduced in a two-step process via a transient aldehyde intermediate, catalysed by acyl-CoA reductases in the endoplasmic reticulum that use NAD(P)H as reducing equivalents. This family of enzymes is necessary for wax production in seeds and the accumulation of leaf surface waxes and suberin. Other reductases are soluble plastid proteins that can use both acyl-ACP and acyl-CoA as substrate and are mainly involved in the biosynthesis of sporopollenin.

Biosynthesis of fatty alcohols and wax esters

Primary alcohols can be exported directly to the surface of the plant or are first esterified by acyl-CoAs to from wax esters. In A. thaliana, this reaction is catalysed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (WSD1). An enzyme with some sequence similarity to this has been characterized from microsomal fractions of seeds of the jojoba plant, and this is responsible for production of the storage wax in lipid droplets like those containing triacylglycerols in oil seeds, including a surface layer of oleosins. Analogous mechanisms have been revealed in studies with insects, algae and birds (uropygial glands).

A few only of the enzymes involved in wax synthesis have been fully characterized. In the decarbonylation pathway for the synthesis of wax constituents, the first step is again believed to be the reduction of acyl-CoA ester to an aldehyde by means of an acyl-CoA reductase. Removal of the carbonyl group by an aldehyde decarbonylase yields an alkane, with one fewer carbon atom than the fatty acid precursor. Further metabolism of the hydrocarbon is then possible by two consecutive reactions catalysed by the cytochrome P450 enzyme CYP96A15, referred to as mid-chain alkane hydroxylase MAH1, to insert a hydroxyl group to form a secondary alcohol and thence a ketone, with the position of the substitution depending on the species and the specificities of the enzymes involved. Secondary alkanols can in turn be esterified to form wax esters. An associated pathway leads to the formation of 2-alkanols, β-diketones, diols and other wax constituents with two or more functional groups. For example, the common C₃₁ β-diketone is synthesised from two C₁₆ groups via 3-ketoacid intermediates in reactions by a thioesterase and a polyketide synthase located in the plastids and endoplasmic reticulum, respectively, implying substrate transfer between these sub-cellular compartments. cis-9-Alkenes in leaf waxes can degrade spontaneously by oxidative cleavage to generate volatile aldehydes with a potential signalling role. Again, these processes have been studied in plants mostly, but comparable biochemical reactions appear to occur in insects and birds.

Biosynthesis of hydrocarbons, secondary alcohols and ketones

Once synthesised, the wax components must be exported from the sites of lipid synthesis in the plastid and the endoplasmic reticulum to the Golgi- and trans-Golgi network, where a protein termed ECHIDNA regulates transport in secretory vesicles through the plasma membrane. They must then pass in this form into the cutin layer, mainly a polyester of hydroxy fatty acids, that provides a matrix, within and upon which the waxes are deposited. The regulatory mechanisms behind cuticular wax biosynthesis are complex and must strike a balance between optimal growth and development and defence against abiotic and biotic stresses. In Arabidopsis, wax synthesis is induced by wounding and is dependent upon jasmonyl-isoleucine concentrations with increased gene expression dependent on abscisic acid.

3. Waxes from Animal Tissues

Waxes associated with skin

In mammals, the main wax production is associated with the sebaceous glands of the skin, most of which are associated with hair follicles, although there are related structures on the eyelids termed Meibomian glands. Sebaceous glands secrete mainly non-polar lipids in the form of sebum onto the skin surface, where they are easily recovered for analysis. In humans, sebaceous glands are distributed throughout the body except for the palm and sole of the foot. They consist of three type of cells, peripheral undifferentiated cells, cells that produce lipid bodies, and lysing cells loaded with lipid that empty their contents into the lumen. Although relatively few species have been studied in real detail, it is evident that a wide range of lipid classes are present and that these vary greatly in amount and nature between species with some variation with age. The composition of human sebum differs appreciably from that of other species in the high content of triacylglycerols and in the nature of the fatty acids. Some typical data are listed in Table 3.

	Squalene	Sterols	Sterol esters	Wax esters	Diesters	Glyceryl ethers	Triacyl- glycerols	Free acids	Free alcohols
Table 3. Relative composition (wt % of the total) of the non-polar lipids of from the skin surface of various species.

Human	12	1	3	25			41	16
Sheep		12	46	10	21				11
Rat	1	6	27	17	21	8		1
Mouse		13	10	5	65		6
Adapted from Downing, D.T. Mammalian waxes. In: Chemistry and Biochemistry of Natural Waxes. pp. 18-48 (Ed. P.E. Kolattukudy, Elsevier, Amsterdam) (1976).

Formula of squalene The triterpene hydrocarbon squalene (2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene) is produced ubiquitously in nature but is present in most animal tissues as a minor lipid component only, other than in some marine waxes and especially those of sharks (Squalus sp. - hence the name). Sebaceous waxes are the only mammalian tissue where it accumulates in appreciable amounts. Squalene can move readily through cellular and subcellular membranes, and it can act as an antioxidant by scavenging or quenching free radicals, breaking lipid peroxidation chains both in the initial and propagation phases. As the biosynthetic precursor of cholesterol and other sterols, it has special biological significance, but it may have its own biological properties, for example in the immune system, and there is increasing interest in it as a functional food. It may be a factor in non-alcoholic fatty liver disease and aberrant metabolism may be important in other health conditions, but potential beneficial effects against oxidative stress disorders such as cardiovascular disease appear to be unproven.

The alcohol components of sebaceous wax esters are C₂₄ to C₂₇ iso- and anteiso-methyl-branched, while the fatty acids are mainly C₁₂ to C₂₉ saturated and monounsaturated with a relatively high proportion being branched chain, although the last tend to be trace components only in other organs. Very-long-chain ω‑hydroxy acids are also present, and in some the hydroxyl group is esterified with a conventional fatty acid. The biosynthesis of long-chain and very-long-chain fatty acids by elongases is discussed in our web page dealing with saturated fatty acids. Fatty alcohols are synthesised from the corresponding acids by the action of fatty acyl-CoA reductases with NADPH as the reductant, apparently without formation of an aldehyde as an intermediate. Finally, fatty acids and alcohols are coupled by multifunctional wax synthases (acyl-CoA wax alcohol acyltransferases), designated AWAT1 and AWAT2, although an enzyme involved in triacylglycerol biosynthesis, i.e., an acyl CoA:diacylglycerol acyltransferase (DGAT1), is used to synthesise wax and retinol esters in some tissues.

Human sebum is unique in containing cis-6-hexadecenoic acid (6-16:1 or sapienic acid), which is the single most abundant fatty acid component and is accompanied by an elongation and desaturation product 5,8-octadecadienoic acid (‘sebaleic’ acid), likewise unique to human skin. Sapienic acid is formed in the sebaceous glands by the Δ6-desaturase FADS2 involved in the formation of polyunsaturated fatty acids; it has powerful antibacterial properties against the organisms responsible for acne and the opportunist pathogen Staphylococcus aureus.

Skin contains a wide range of distinctive but more polar lipids based on the ceramide backbone. They have been most studied in the skin of the pig and human, where a range of unusual ceramides have been identified, some of which contain linoleic acid (a) esterified to a hydroxy acid (b) that is in turn linked to a long-chain base (c), and several molecular forms of glucosylceramide, based on similar structures, have been characterized.

Formula of a skin ceramide

Their compositions depend on the particular layer of the skin (epidermis, stratum corneum, etc.). Whether they should truly be called waxes is debatable, and there is much more information on these lipids in our web page on ceramides.

Vernix caseosa is a waxy material that coats the skin of the human foetus and new-born and is produced during the third trimester of gestation. In utero, it acts as waterproofing to control the flux of water across the skin and as a protective agent during the final stages of development of the skin. Following birth, it protects the skin against bacterial attack and aids the neonate to adapt to exposure to air. The lipid component includes a high proportion of triacylglycerols and wax esters (with some 1‑O‑acyl ceramides).. The latter have a rather unusual pattern of fatty acids in a range of chain-lengths (C₁₀ to C₁₆) with methyl substituents in different even-numbered positions in the alkyl chain, presumably formed during biosynthesis by the replacement of malonyl CoA with a molecule of methyl malonyl CoA at irregular intervals on the growing alkyl chain.

Wool wax (lanolin) - The grease obtained from the wool of sheep during the cleaning or refining process and presumably derived from the sebaceous glands and/or stratum corneum of the skin is rich in wax esters (of 1- and 2-alkanols and of 1,2-diols), sterol esters, triterpene alcohols, free fatty acids and sterols. The nature of the product varies with the degree and type of processing involved, but it can contain up to 50% wax esters and 33% sterol esters. A high proportion of the sterol component is lanosterol, while the fatty acid components are mainly saturated with straight and iso- and anteiso-methyl-branched-chains. This is the only animal wax of commercial value. although others have crucial biological functions in the producing organs.

Other animal waxes

Meibomian glands are holocrine glands that are located in the upper and the lower eyelids of humans and most animals, and they produce an oily, lipid-enriched secretion, often termed meibum, that is very different in composition from other tissue lipids. This is excreted onto the eye, mixing with tears to form the outermost surface layer as a protection from desiccation and bacterial infection, while preventing dry-eye disease. The lipids constitute a mixture of many different classes, including wax esters, cholesterol esters, acylglycerols, diacylated diols, free fatty acids and cholesterol; more than 600 components have been identified by modern lipidomics methodology. A multitude of different fatty acid components are present, including very-long chain (C₁₆ to C₃₆), branched-chain, mono-unsaturated (often with a double bond in position 6), and ω-hydroxy and (O‑acyl)‑ω‑hydroxy fatty acids, some of which are unesterified while others are linked to cholesterol or triacylglycerols.

While many aspects of the biosynthesis remain to be elucidated, it is evident that mice and humans have similar gene products and that AWAT1 and AWAT2 are required, both for the synthesis of (O-acyl)-ω-hydroxy fatty acids and wax diesters. Cholesterol synthesis de novo occurs, and there are cholesterol ester synthases. In mice, two isoenzymes of the fatty acyl-CoA reductases (FAR1 and FAR2) have been identified with different substrate specificities: FAR1 generates C₁₆ and C₁₈ fatty alcohols, while FAR2 produces those >C₂₂.

Harderian glands are exocrine glands located deep in the orbit of the eyeball in those terrestrial vertebrates that possess a nictitating membrane, i.e., a protective translucent third eyelid. These are found in many fish, amphibians, reptiles, birds and mammals, but are rare in primates. The gland secretes lipids onto this membrane, and in rodents these consist mainly of alkyldiacylglycerols and wax esters with many different fatty acid constituents from isovaleric up to C₃₀ in chain length and often with hydroxyl groups in position 2. In the rat, the main molecular species of wax ester consists of a 24:1(n-7) alcohol attached to a 20:1(n-7) fatty acid.

Scottish thistle Marine waxes. Many marine animals from invertebrates to whales contain appreciable amounts of waxes in the form mainly of hydrocarbons and wax esters. In addition, glycerol ethers and sterols could be classified as wax components in some species. They are found in a variety of tissues from fish roe to liver and muscle tissues. The wax esters consist of the normal range of saturated, monoenoic and polyunsaturated fatty acids typical of fish, esterified to mainly saturated and monoenoic alcohols often with the 18:1 fatty alcohol as the main component. The orange roughy, a fish found predominantly in the seas around New Zealand, has more than 90% of its total lipid in the form of wax esters. In the hydrocarbon fraction, squalene and other terpenoid hydrocarbons are frequently major constituents, and they can be accompanied by saturated (straight-chain and methyl-branched), monoenoic and polyenoic components.

Waxes have a variety of functions in fish, from serving as an energy source to insulation, buoyancy and even echo location, but only rarely are they encountered in depot fats. Zooplankton can produce large amounts of wax esters at some stages of their life cycle, and they make a major contribution to the marine food chain, although in general these are broken down in the intestines so their components can be utilized in tissues. Swordfish produce an oily secretion consisting largely of methyl esters of fatty acids from glands below the skin in the head, the function of which appears to be to reduce friction drag and increase swimming efficiency.

Spermaceti or sperm whale oil (wax esters, 76%; triacylglycerols, 23%) was once in great demand as a lubricant but now is proscribed. In toothed whales and dolphins, the wax esters (and triacylglycerols) tend to contain large amounts of branched-chain fatty acids, including short-chain component such as isovaleric acid, synthesised from amino acid precursors. It appears that the relative concentrations of the two main lipid classes in an organ in the head of the animals (termed the ‘melon’) are arranged anatomically in a three-dimensional topographical pattern to enable them to focus sound waves for echolocation.

Bird waxes. The uropygial or preen glands of birds are similar structurally to sebaceous glands, and they secrete waxes that consist largely of wax esters. With monoesters, the fatty alcohol components are usually relatively simple in nature, consisting largely of normal C₁₆ and C₁₈ saturated compounds, although those with branched chains can make up an appreciable proportion in some species. The chicken and related species contain fatty acid esters of 2,3-alkanediols. Depending on species, the fatty acids can be highly complex and are often shorter chain than usual with up to four methyl branches (see our web page on branched-chain fatty acids). Mono- and bifunctional wax ester synthases have been characterized, the latter catalysing both wax ester and triacylglycerol synthesis, and both forms differ in their substrate specificities for branched-chain alcohols and acyl-CoA thioesters. The main purpose of the waxes is presumed to be to give a water-proof layer to the feathers, but other functions have been suggested.

Insect surface waxes and related lipids. The external surface of insects is covered by a layer of wax that serves to restrict movement of water across the cuticle and prevent desiccation, while providing a physical barrier to protect against abiotic stresses, such as the penetration of insecticides, chemicals and toxins, and biotic stresses such as attack by microorganisms, parasitic insects and other predators. The nature of this lipid is dependent on species, but in general a high proportion tends to be saturated alkanes, often with one or two methyl branches (C₂₃ to C₃₁), but wax esters, sterol esters, and free fatty alcohols and acids may be present. Some species of insect secrete triacylglycerols in their waxes together with free sterols and other terpenoid components; aphids are distinctive in that they secrete triacylglycerols containing sorbic (2,4-hexadienoic) acid. Shellac is a polymer of unusual hydroxy fatty acids secreted by the female lac bug, Laccifer lacca.

Some of these lipids can have a further role as pheromones in sexual signalling, and most moth species utilize Type I pheromones derived from fatty acids (C₁₀ to C₁₈) but with a functional group of a primary alcohol, aldehyde or acetate ester and usually with several double bonds. The best known is bombykol (E10,Z12‑hexadecadien-1-ol), which is synthesised in pheromone glands of adult female silk moths.

Beeswax: the archetypal wax. Glands under the abdomen of bees secrete a wax, which they use to construct the honeycomb, and this is recovered as a by-product when the honey is harvested and refined. It contains a high proportion of wax esters (35 to 80%), which consist of C₄₀ to C₄₆ molecular species, based on 16:0 and 18:0 fatty acids some with hydroxyl groups in the ω-2 and ω-3 positions, while some diesters with up to 64 carbons may be present, together with triesters, hydroxy-polyesters and free acids (which are different in composition and nature from the esterified acids). The hydrocarbon content is highly variable, and much may be "unnatural" in commercial production as beekeepers may feed some to bees to improve the yield of honey.

Nematode waxes. The dauer-larvae of the nematode Pristionchus pacificus (see or web page on ascarosides) are coated with an extremely long-chain highly unsaturated wax ester (‘nematoil’) that covers the surface of the animal and promotes congregation of numerous individuals into stable 'dauer towers' that can reach a beetle host more easily. Both the acid and alcohol components have C₃₀ chains and cis double bonds in positions 7, 15, 18, 21, 24 and 27.

Formula of nematoil

Digestion and absorption of wax esters: Wax esters in mammalian food are poorly hydrolysed in the digestive system by pancreatic lipase, so they have little nutritional value. However, fish are obviously well adapted to their diet of wax-rich zooplankton, and they have little difficulty in hydrolysing wax esters and assimilating their components into their tissues, presumably with the aid of so-far uncharacterized lipases or esterases.

4. Microbial Waxes

Waxes, often accompanied by triacylglycerols, are known to be produced for storage purposes by many species of prokaryotes, including both Gram-negative and Gram-positive bacteria, when carbon is plentiful but other essential nutrients such as nitrogen are limited. In the Gram-negative Acinetobacter baylyi, biosynthesis of wax esters consists of three enzymatic steps; a fatty acid CoA ester is reduced by an NADPH-dependent fatty acyl-CoA reductase to a long-chain aldehyde, which is further reduced by an aldehyde reductase to a fatty alcohol for esterification with a fatty acyl-CoA by the well characterized bifunctional enzyme wax ester synthase/diacylglycerol acyltransferase to produce a wax ester. Some species of this genus can synthesise wax esters from long-chain alkanes as the precursors. Cyanobacteria of the Prochlorococcus and Synechococcus genera produce appreciable amounts of the hydrocarbon pentadecane (C₁₅) by decarboxylation of palmitic acid in nutrient-poor regions of the oceans, but the function of this is not known.

The mycobacteria produce waxes, termed 'mycoserosates', based on branched-chain alcohols or 'phthiocerols'. These are C₃₄ or C₃₆ branched-chain aliphatic compounds with hydroxyl groups in positions 9 and 11, or 11 and 13, which are esterified with long-chain fatty acids varying in chain length from C₁₈ to C₂₆ and with two to four methyl branches, often at the 2, 4, 6 and 8 positions. In pathogenic mycobacteria such as Mycobacterium tuberculosis, the phthiocerol dimycocerosate esters are major virulence factors. They are present on the outermost surface of the cell wall as part of the mycobacterial outer capsule-like layer and are transferred to macrophage membranes during infections, where their conical structures cause changes in the organization of lipids in the host membranes and enhance phagocytosis. By masking the pattern recognition receptors of macrophages, they enable the organisms to escape immune recognition. Biosynthesis involves chain-elongation of an existing fatty acid by a polyketide synthase.

Formulae of a phthiocerol ester and phenylglycolipid from M. leprae

Structurally related phenylglycolipids that have a p-hydroxyphenyl group at the terminus of the phthiocerol chain are highly pathogenic, especially when the hydroxyl group of this is attached to carbohydrate moieties of varying complexity, depending on species, such as trisaccharides with several methoxyl groups in some of the more virulent strains of M. leprae.

Mycobacteria are unusual in a number of other ways in that their cell walls can contain many unique lipids, which include complex trehalose-containing lipids and phosphatidylinositol mannosides amongst others, and links to these are available in the web page describing the distinctive mycolic acids, where there is a more extensive discussion of the cell wall structure and lipid composition of M. tuberculosis.

5. Alkylresorcinols

5-Alkylresorcinols or resorcinolic lipids are minor wax or cell wall constituents of higher plants, although they are present in other plant tissues, including roots, and some forms are found in insects, fungi, algae, slime moulds, mosses and bacteria. They are part of a wider class of phenolic lipids that include the anacardic acids. In structure, they are 5-alkyl-1,3-dihydroxybenzenes in which the alkyl moiety is linear (non-isoprenoid), and they exist in a wide range of chain lengths with varying numbers and positions of double bonds. Species with fully saturated chains tend to be more abundant in cereals, where alkylresorcinols can occur in significant amounts. In rye, 74 forms have been characterized with 5-alkyl chain lengths of 14 to 27 carbon atoms and zero to four double bonds, together with forms with oxo groups or 2‑methyl-substituents in the ring. The wax layer of Scilla bifolia, a herbaceous perennial, contains five classes of alkylphenyl lipids, including resorcinols and structurally related methoxy- and methyl-benzene derivatives. Sorgoleone or 2-hydroxy-5-methoxy-3-[8Z,11Z,14-pentadecatriene]-p-benzoquinone and related molecules are major components of the oily substance exuding from the roots of sorghum (Sorghum bicolor). 2-Methyl-alkylresorcinols are only present in significant amounts in the pseudo-cereal quinoa.

Alkylresorcinols

The two components of alkylresorcinols are synthesised in different subcellular compartments. In the biosynthesis of sorgoleone in Sorghum bicolor, a type III polyketide synthase can repeatedly condense a fatty acid substrate, derived from plastids, with malonyl CoA-linked building blocks to produce a tetraketide intermediate, which is cyclized and decarboxylated to produce an aromatic ring with a long alkyl tail; other functional groups are then added by other enzyme systems. It seems likely that comparable mechanisms are involved in the biosynthesis of other alkylresorcinols, which in general display a wide range of biological activities in plants. They are strongly amphiphilic and serve as antioxidants, with antimicrobial, antiparasitic, cytotoxic and growth regulation properties. Sorgoleone is a growth inhibitor, urushiols (related structurally to the anacardic acids) are the active ingredients in poison ivy, and olivetolic acid is a precursor to cannabinoids.

In cereal grains, they are present in bran, the hard outer cell wall comprising pericarp, testa and aleurone layers, but they are not found in the endosperm used to make flour; they have been detected in rice, but not in the edible parts. They are consumed in the human diet in significant amounts with cereal grains, and while they are reported to exert biological effects in animal cells in vitro, it is not known whether these are observed in vivo, although alkylresorcinols in general are reported to have beneficial effects upon metabolic and immune processes. They are catabolized by oxidation in the liver by a mechanism like that of tocopherols, commencing with ω‑oxidation of the alkyl tail of the molecule catalysed by CYP4F2, followed by β-oxidation to form 3,5‑dihydroxybenzoic acid ultimately, which can be eliminated as the glucuronide or sulfate.

Dialkylresorcinols are produced by many bacterial species, including insect and human pathogens of the genus Photorhabdus, where they may have structural roles in membranes. Some may function as quorum sensing molecules in the same way as homoserine lactones. They are the main constituents of the outer shell of the cyst of Azotobacter sp., while 2-n-hexyl-5-n‑propylresorcinol is an antibiotic isolated from cultures of Pseudomonas sp. Resorcinolic lipids from Cyanobacteria are structurally distinct from those of heterotrophic bacteria in that they are often halogenated or glycosylated, and they may exhibit intra- or inter-molecular cyclizations.

6. Analysis

Thin-layer and high-performance liquid chromatography have been used to isolate individual classes of waxes for more detailed analysis. On the other hand, much of the more recent published work has made use of high-temperature gas chromatography following trimethylsilylation, often in combination with mass spectrometry (electron impact ionization), so that simultaneous identification and quantification of the various molecular species can be achieved.

	© Author: William W. Christie
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