Mammalian wax biosynthesis. I. Identification of two fatty acyl-Coenzyme A reductases with different substrate specificities and tissue distributions

The conversion of fatty acids to fatty alcohols is required for the synthesis of wax monoesters and ether lipids. The mammalian enzymes that synthesize fatty alcohols have not been identified. Here, an in silico approach was used to discern two putative reductase enzymes designated FAR1 and FAR2. Expression studies in intact cells showed that FAR1 and FAR2 cDNAs encoded isozymes that reduced fatty acids to fatty alcohols. Fatty acyl-CoA esters were the substrate of FAR1, and the enzyme required NADPH as a cofactor. FAR1 preferred saturated and unsaturated fatty acids of 16 or 18 carbons as substrates, whereas FAR2 preferred saturated fatty acids of 16 or 18 carbons. Confocal light microscopy indicated that FAR1 and FAR2 were localized in the peroxisome. The FAR1 mRNA was detected in many mouse tissues with the highest level found in the preputial gland, a modified sebaceous gland. The FAR2 mRNA was more restricted in distribution and most abundant in the eyelid, which contains wax-laden meibomian glands. Both FAR mRNAs were present in the brain, a tissue rich in ether lipids. The data suggest that fatty alcohol synthesis in mammals is accomplished by two fatty acyl-CoA reductase isozymes that are expressed at high levels in tissues known to synthesize wax monoesters and ether lipids.     

Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily

The esterification of alcohols such as sterols, diacylglycerols, and monoacylglycerols with fatty acids represents the formation of both storage and cytoprotective molecules. Conversely, the overproduction of these molecules is associated with several disease pathologies, including atherosclerosis and obesity. The human acyl-CoA:diacylglycerol acyltransferase (DGAT) 2 gene superfamily comprises seven members, four of which have been previously implicated in the synthesis of di- or triacylglycerol. The remaining 3 members comprise an X-linked locus and have not been characterized. We describe here the expression of DGAT2 and the three X-linked genes in Saccharomyces cerevisiae strains virtually devoid of neutral lipids. All four gene products mediate the synthesis of triacylglycerol; however, two of the X-linked genes act as acyl-CoA wax alcohol acyltransferases (AWAT 1 and 2) that predominantly esterify long chain (wax) alcohols with acyl-CoA-derived fatty acids to produce wax esters. AWAT1 and AWAT2 have very distinct substrate preferences in terms of alcohol chain length and fatty acyl saturation. The enzymes are expressed in many human tissues but predominate in skin. In situ hybridizations demonstrate a differentiation-specific expression pattern within the human sebaceous gland for the two AWAT genes, consistent with a significant role in the composition of sebum.     

Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.     

Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray analysis

Plant epidermal wax forms a hydrophobic layer covering aerial plant organs which constitutes a barrier against uncontrolled water loss and biotic stresses. Wax biosynthesis requires the coordinated activity of a large number of enzymes for the formation of saturated very-long-chain fatty acids and their further transformation in several aliphatic compounds. We found in the available database 282 candidate genes that may play a role in wax synthesis, regulation and transport. To identify the most interesting candidates, we measured the level of expression of 204 genes in the aerial parts of 15-day-old Arabidopsis seedlings by performing microarray experiments. We showed that only 25% of the putative candidates were expressed to significant levels in our samples, thus significantly reducing the number of genes which will be worth studying using reverse genetics to demonstrate their involvement in wax accumulation. We identified a beta-keto acyl-CoA synthase gene, At5g43760, which is co-regulated with the wax gene CER6 in a number of conditions and organs. By contrast, we showed that neither the fatty acyl-CoA reductase genes nor the wax synthase genes were expressed in 15-day-old leaves and stems, raising questions about the identity of the enzymes involved in the acyl-reduction pathway that accounts for 20% of the total wax amount.     

Differences in substrate specificities of five bacterial wax ester synthases

Wax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria: Marinobacter aquaeolei VT8, Acinetobacter baylyi, Rhodococcus jostii RHA1, and Psychrobacter cryohalolentis K5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. These in vitro results are compared to the wax ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final wax ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular from M. aquaeolei VT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production.     

Acyl-CoA reductase specificity and synthesis of wax esters in mouse preputial gland tumors.

Long-chain alcohols are synthesized in the mouse preputial gland tumor (ESR-586) by NADPH:acyl-CoA oxidoreductase. In this study, a series of labeled acids was tested as substrates for the oxidoreductase in a cell-free system from the tumor, and the distribution of label into alcohols, waxes, and other products was determined. The system contained the labeled acid, an acyl-CoA-generating system, an NADPH-generating system, and tumor homogenate. The highest rates of alcohol synthesis were obtained with palmitic (16:0), heptadecanoic (17:0), stearic (18:0), myristic (14:0), elaidic (18:1 trans), and linoleic (18:2) acids, which yielded, respectively, 151, 124, 102, 76, 65, and 35 pmol alcohol/min per mg protein. Decanoic (10:0), lauric (12:0), oleic (18:1 cis), linolenic (18:3), arachidonic (20:4), and behenic (22:0) acids all gave lower activities. Acyl-CoA formation did not appear to be rate limiting with any of the substrates tested except behenic acid. In addition to the fatty alcohol product, a small amount of fatty aldehyde was formed in the system. Incorporation of the labeled fatty acids into wax esters was examined and the distribution of label between the alcohol and acid components of the waxes was determined. Incubation of [1-(14)C]palmitic acid yielded 3.4% free alcohol, 8.3% alcohol esterified in waxes, and 7.7% palmitoyl groups esterified into waxes, whereas, at the other extreme, [1-(14)C]linolenic acid yielded 0.8%, 0.6%, and 38%, respectively, into the homologous components.-Wykle, R. L., B. Malone, and F. Snyder. Acyl-CoA reductase specificity and synthesis of wax esters in mouse preputial gland tumors.     

Production of wax esters in plant seed oils by oleosomal cotargeting of biosynthetic enzymes

Wax esters are neutral lipids exhibiting desirable properties for lubrication. Natural sources have traditionally been whales. Additionally some plants produce wax esters in their seed oil. Currently there is no biological source available for long chain length monounsaturated wax esters that are most suited for industrial applications. This study aimed to identify enzymatic requirements enabling their production in oilseed plants. Wax esters are generated by the action of fatty acyl-CoA reductase (FAR), generating fatty alcohols and wax synthases (WS) that esterify fatty alcohols and acyl-CoAs to wax esters. Based on their substrate preference, a FAR and a WS from Mus musculus were selected for this study (MmFAR1 and MmWS). MmWS resides in the endoplasmic reticulum (ER), whereas MmFAR1 associates with peroxisomes. The elimination of a targeting signal and the fusion to an oil body protein yielded variants of MmFAR1 and MmWS that were cotargeted and enabled wax ester production when coexpressed in yeast or Arabidopsis. In the fae1 fad2 double mutant, rich in oleate, the cotargeted variants of MmFAR1 and MmWS enabled formation of wax esters containing >65% oleyl-oleate. The data suggest that cotargeting of unusual biosynthetic enzymes can result in functional interplay of heterologous partners in transgenic plants.     

A fatty acyl-CoA reductase highly expressed in the head of honey bee (Apis mellifera) involves biosynthesis of a wide range of aliphatic fatty alcohols

Honey bees (Apis mellifera) are social insects which have remarkable complexity in communication pheromones. These chemical signals comprise a mixture of hydrocarbons, wax esters, fatty acids, aldehydes and alcohols. In this study, we detected several long chain aliphatic alcohols ranging from C18-C32 in honey bees and the level of these alcohols varied in each body segment. C18:0Alc and C20:0Alc are more pronounced in the head, whereas C22:0Alc to C32Alc are abundant in the abdomen. One of the cDNAs coding for a fatty acyl-CoA reductase (AmFAR1) involved in the synthesis of fatty alcohols was isolated and characterized. AmFAR1 was ubiquitously expressed in all body segments with the predominance in the head of honey bees. Heterologous expression of AmFAR1 in yeast revealed that AmFAR1 could convert a wide range of fatty acids (14:0–22:0) to their corresponding alcohols, with stearic acid 18:0 as the most preferred substrate. The substrate preference and the expression pattern of AmFAR1 were correlated with the level of total fatty alcohols in bees. Reconstitution of the wax biosynthetic pathway by heterologous expression of AmFAR1, together with Euglena wax synthase led to the high level production of medium to long chain wax monoesters in yeast.     

Biosynthesis of isoprenoid wax ester in Marinobacter hydrocarbonoclasticus DSM 8798: identification and characterization of isoprenoid coenzyme A synthetase and wax ester synthases

Marinobacter hydrocarbonoclasticus DSM 8798 has been reported to synthesize isoprenoid wax ester storage compounds when grown on phytol as the sole carbon source under limiting nitrogen and/or phosphorous conditions. We hypothesized that isoprenoid wax ester synthesis involves (i) activation of an isoprenoid fatty acid by a coenzyme A (CoA) synthetase and (ii) ester bond formation between an isoprenoid alcohol and isoprenoyl-CoA catalyzed, most likely, by an isoprenoid wax ester synthase similar to an acyl wax ester synthase, wax ester synthase/diacylglycerol acyltransferase (WS/DGAT), recently described from Acinetobacter sp. strain ADP1. We used the recently released rough draft genome sequence of a closely related strain, M. aquaeolei VT8, to search for WS/DGAT and acyl-CoA synthetase candidate genes. The sequence information from putative WS/DGAT and acyl-CoA synthetase genes identified in this strain was used to clone homologues from the isoprenoid wax ester synthesizing Marinobacter strain. The activities of the recombinant enzymes were characterized, and two new isoprenoid wax ester synthases capable of synthesizing isoprenoid ester and acyl/isoprenoid hybrid ester in vitro were identified along with an isoprenoid-specific CoA synthetase. One of the Marinobacter wax ester synthases displays several orders of magnitude higher activity toward acyl substrates than any previously characterized acyl-WS and may reflect adaptations to available carbon sources in their environments.     

A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters

Acyl-CoA-dependent O-acyltransferases catalyze reactions in which fatty acyl-CoAs are joined to acyl acceptors containing free hydroxyl groups to produce neutral lipids. In this report, we characterize a human multifunctional O-acyltransferase (designated MFAT) that belongs to the acyl-CoA:diacylglycerol acyltransferase 2/acyl-CoA:monoacylglycerol acyltransferase (MGAT) gene family and is highly expressed in the skin. Membranes of insect cells and homogenates of mammalian cells overexpressing MFAT exhibited significantly increased MGAT, acyl-CoA:fatty acyl alcohol acyltransferase (wax synthase), and acyl-CoA:retinol acyltransferase (ARAT) activities, which catalyze the synthesis of diacylglycerols, wax monoesters, and retinyl esters, respectively. Furthermore, when provided with the appropriate substrates, intact mammalian cells overexpressing MFAT accumulated more waxes and retinyl esters than control cells. We conclude that MFAT is a multifunctional acyltransferase that likely plays an important role in lipid metabolism in human skin.     

蜡酯合成途径及关键酶的研究进展

蜡酯对于生物的生命活动具有重要意义,研究表明植物和动物的蜡酯合成存在保守途径。即脂酰辅酶A（fatty acyl-CoA）在脂酰辅酶A还原酶（fatty acyl-CoAreductase,FAR）的作用下还原成脂肪醇,脂肪醇和脂酰辅酶A在蜡酯合酶（wax synthase,WS）的作用下生成酯,FAR和WS是该途径的关键酶,这两个酶的结构和功能在不同物种之间表现出很大差异,目前对于这两个酶缺乏系统的归纳分析。该文综述了蜡酯合成途径及FAR和WS的序列特征、生化特性及参与的生理功能,分析了这两种酶相关研究存在的问题,旨在为昆虫的蜡酯合成研究提供参考。     

The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters

The final step of triacylglycerol biosynthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. The two known DGATs, DGAT1 and DGAT2, are encoded by unrelated genes. Although both DGAT1 and DGAT2 knockout mice have reduced tissue triacylglycerol contents, they have disparate phenotypes, prompting us to investigate whether the two enzymes have unrecognized functional differences. We now report that DGAT1 exhibits additional acyltransferase activities in vitro, including those of acyl CoA:monoacylglycerol acyltransferase (MGAT), wax monoester and wax diester synthases, and acyl CoA:retinol acyltransferase (ARAT), which catalyze the synthesis of diacylglycerols, wax esters, and retinyl esters, respectively. These activities were demonstrated in in vitro assays with membranes from insect cells or homogenates from COS7 cells overexpressing DGAT1. Wax synthase and ARAT activities were also demonstrated in intact COS7 cells expressing DGAT1. Additionally, cells and tissues from DGAT1-deficient mice exhibited reduced ARAT activity, and the mice had increased levels of unesterified retinol in their livers on a high-retinol diet. Our findings indicate that DGAT1 can utilize a variety of acyl acceptors as substrates in vitro and suggest that these activities may be relevant to the in vivo functions of DGAT1.     

Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana

In a future bio-based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de novo fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl reductases (FAR) and wax ester synthases (WS) were transiently expressed in Nicotiana benthamiana leaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts.     

Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.     

Manufacturing specialized wax esters in plants

Biologically produced wax esters can fulfil different industrial purposes. These functionalities almost drove the sperm whale to extinction from hunting. After the ban on hunting, there is a niche in the global market for biolubricants with properties similar to spermaceti. Wax esters can also serve as a mechanism for producing insect sex pheromone fatty alcohols. Pheromone-based mating disruption strategies are in high demand to replace the toxic pesticides in agriculture and manage insect plagues threatening our food and fiber reserves. In this study we set out to investigate the possibilities of in planta assembly of wax esters, for specific applications, through transient expression of various mix-and-match combinations of genes in Nicotiana benthamiana leaves. Our synthetic biology designs were outlined in order to pivot plant lipid metabolism into producing wax esters with targeted fatty acyl and fatty alcohols moieties. Through this approach we managed to obtain industrially important spermaceti-like wax esters enriched in medium-chain fatty acyl and/or fatty alcohol moieties of wax esters. Via employment of plant codon-optimized moth acyl-CoA desaturases we also managed to capture unusual, unsaturated fatty alcohol and fatty acyl moieties, structurally similar to moth pheromone compounds, in plant-accumulated wax esters. Comparison between outcomes of different experimental designs identified targets for stable transformation to accumulate specialized wax esters and helped us to recognize possible bottlenecks of such accumulation.     

Regulation of acyl coenzyme A reductase by a heat-stable cytosolic protein during preputial gland development

A low-molecular-weight protein located in the cytosol of mouse preputial glands has been shown to stimulate the activity of a microsomal acyl coenzyme A (CoA) reductase in the gland. This cytoplasmic protein was stable to heating and lyophilization, but was destroyed by trypsin digestion. It was able to bind palmitoyl-CoA and gel elution behavior indicated it had a molecular weight of 10,000–12,000. The level of this stimulatory cytosolic protein and the activity of acyl-CoA reductase were shown to correlate with differentiation of the preputial gland during development of puberty in male mice; the acyl-CoA reductase activity first appeared at 4 weeks of age and increased dramatically up to 6 weeks of age. By 8 weeks, when sexual maturity was attained, the reductase activity decreased to that level found in mature male mice. The cytosol from the preputial glands of the youngest mice (3 weeks) contained sufficient heat-stable acyl-CoA binding protein to stimulate acyl-CoA reduction; however, the 3-week-old preputial gland microsomes had little or no acyl-CoA reductase activity. As the animal matured, the stimulatory capacity in the heat-treated cytosol increased, reaching a maximum at 6 weeks; by 8 weeks, the stimulatory capacity of the soluble fraction had decreased to that found in mature male mouse. Results of this study suggest that the concentration of acyl-CoA, cytoplasmic acyl-CoA binding protein, and acyl-CoA reductase activity regulate the level of fatty alcohols in vivo and that the reductase activity and binding protein have similar patterns of development during puberty.     

植物超长链脂肪酸及角质层蜡质生物合成相关酶基因研究现状

超长链脂肪酸(very long chain fatty acids, VLCFAs)在生物体中具有广泛的生理功能, 它们参与种子甘油酯、生物膜膜脂及鞘脂的合成, 并为角质层蜡质的生物合成提供前体物质。角质层是覆盖在植物地上部分最表层的保护层, 由角质和蜡质组成, 其中蜡质又分为角质层表皮蜡和内部蜡, 在植物生长发育、适应外界环境方面起重要作用。VLCFAs的合成由脂肪酰-CoA延长酶催化, 该酶是由b-酮脂酰-CoA合酶、b-酮脂酰-CoA还原酶、b-羟脂酰-CoA脱水酶和反式烯脂酰-CoA还原酶组成的多酶体系。合成后的VLCFAs通过脱羰基与酰基还原作用进入角质层蜡质合成途径, 形成各种蜡质组分。文章就VLCFAs及角质层蜡质合成代谢途径中相关酶基因研究进展方面做了综述, 并对植物蜡质基因研究中存在的问题提出一些看法。     

The age-related occurrence of wax esters in the mouse preputial gland tumour.

The mouse preputial gland tumour (ESR-586) accumulates wax esters from between 20 and 25 days after transplantation until they become the most abundant lipid class. Prior to this time wax esters are not detectable. The process occurs in both male and female host mice and appears to be determined by a response of the host to the tumour, rather than by a property of the tumour itself. The most abundant fatty alcohol present in the wax esters is hexadecanol. This contrasts with the greater proportions of the C20 to C24 chains found for the alkyl portion of the alkyldiacylglycerols, for which the precursors are fatty alcohols.     

Use of TLC‐FID and GC‐MS/FID to examine the effects of migratory state,diet and captivity on preen wax composition in White‐throated Sparrows Zonotrichia albicollis

Preen wax is important for plumage maintenance and other functions. Its chemical composition is complex, and separating and quantifying its components, commonly by gas chromatography (GC), can be challenging. We present a simple analytical system consisting of thin‐layer chromatography/flame ionization detection (TLC‐FID) using a solvent system of 100% toluene to analyse the complex compound classes present in preen wax. We used GC and TLC‐FID to investigate the effects of migratory status, diet and captivity on the preen wax composition of White‐throated Sparrows Zonotrichia albicollis, and to measure the quantity of preen wax on the head, primary and tail feathers. White‐throated Sparrows produced preen wax containing only monoesters regardless of migratory state. The monoesters contained several isomers consisting of homologous series of fatty alcohols (C10–C20) and fatty acids (C13–C19) esterified together in different combinations to form monoesters with total carbon numbers ranging from C23 to C38. Weighted average monoester carbon number was greater in captive birds than in wild birds and was greater in captives fed a formulated diet enriched with sesame oil than in birds fed the same diet enriched with fish oil. Captivity and migratory state also affected the complexity of the mixture of monoesters. There was significantly more preen wax on head feathers compared with primary and tail feathers. We suggest that among its many functions, preen wax may play a role in drag reduction by affecting the physical properties of feathers, and/or the fluid flow at their surfaces.     

A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1

Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatis mc(2)155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinant Pseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.