A Novel Bifunctional Wax Ester Synthase/Acyl-CoA:Diacylglycerol Acyltransferase Mediates Wax Ester and Triacylglycerol Biosynthesis in Acinetobacter calcoaceticus ADP1*

Rainer Kalscheuer and Alexander SteinbüchelDagger

From the Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, D-48149 Münster, Germany

Received for publication, October 15, 2002, and in revised form, December 19, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 mc2155. 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.

    INTRODUCTION
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The capability for biosynthesis of neutral lipids is widely distributed in nature and is found in animals and plants as well as microorganisms. In bacteria, the most abundant class of neutral lipids are polyhydroxyalkanoic acids serving as intracellular carbon and energy storage compound (1), but also few examples of substantial triacylglycerol (TAG)1 accumulation have been reported for species mainly belonging to the actinomycetes genera: Mycobacterium (2), Nocardia, and Rhodococcus (3) and Streptomyces (4). Furthermore, biosynthesis of wax esters (oxoesters of long-chain primary fatty alcohols and long-chain fatty acids) has been frequently reported for members of the genus Acinetobacter (5).

TAGs are the dominating storage lipid in animals, plants, and eukaryotic microorganisms. TAG biosynthesis is involved in animals in numerous processes such as regulation of plasma TAG concentration, fat storage in adipocytes, and milk production (6). In plants, TAG synthesis is mainly important for the generation of seed oils (7). Using diacylglycerol (DAG) as a substrate, three different classes of enzymes are known that mediate TAG formation (reviewed in Ref. 8). Acyl-CoA:DAG acyltransferase (DGAT) catalyzes the acylation of DAG using acyl-CoA as a substrate. Two DGAT families designated as DGAT1 and DGAT2 are known that exhibit no sequence homologies to each other. Members of the DGAT1 gene family occur in animals and plants (9-12), whereas members of the DGAT2 gene family were found in animals (13), plants (14), and yeast (15). In human, one DGAT1-related gene and five DGAT2-related genes were identified (13). Recently, DGAT has attracted great interest since it is a potential therapeutical target for obesity treatment (16). Acyl-CoA-independent TAG synthesis is mediated by a phospholipid:DAG acyltransferase found in yeast and plants that uses phospholipids as acyl donors for DAG esterification (17). A third alternative mechanism present in animals and plants is TAG synthesis by a DAG-DAG-transacylase, which uses DAG as acyl donor and acceptor yielding TAG and monoacylglycerol (18, 19), although no gene coding such a transacylase could be identified as yet.

Wax esters have diverse and important biological functions including coating of aerial surfaces of plants as epicuticular waxes to provide protection against desiccation, ultraviolet light, and attack of pathogens; regulation of buoyant density as the principal component of the spermaceti oil of sperm whales; and serving as energy storage materials in the seeds of the jojoba plant (20). The latter is the main natural source of wax esters for commercial applications since the world-wide ban on whale hunting. However, the high price of jojoba oil has limited its use. Wax esters have a multitude of important technical applications in a variety of areas, including medicine, cosmetics, and food industries as well as their more traditional usage as lubricants. Acinetobacter calcoaceticus accumulates wax esters intracellularly as insoluble inclusions under growth-limiting conditions. The chemical structure is similar to those wax esters produced by jojoba and the sperm whale with mainly a C32-C36 carbon length composed of saturated and unsaturated C16 and C18 fatty acid and fatty alcohol moieties (21). Thus, analysis of microbial wax ester biosynthesis could provide a promising basis for the biotechnological production of low-cost jojoba-like wax esters. The key enzymatic step in wax ester biosynthesis is the final condensation of acyl-CoA and fatty alcohol yielding the wax ester catalyzed by the acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS). The only WS gene described so far has been cloned from jojoba, but this WS was not active in Escherichia coli or yeast (22). Here we report on the identification of a bifunctional WS/DGAT, which is the first description of a bacterial protein mediating WS or DGAT activity. This bifunctional WS/DGAT is a new type of long-chain acyl-CoA acyltransferase, which also might have great importance for Mycobacterium and Arabidopsis thaliana.

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Strains, Plasmids, and Growth Conditions-- The strains used are: A. calcoaceticus ADP1 (ATCC 33305), Escherichia coli XL1-Blue (23), E. coli S17-1 (24), Pseudomonas citronellolis (DSM 50332), Rhodococcus opacus PD630 (DSM 44193), and Mycobacterium smegmatis mc2155 (25). Plasmids used for cloning are pBluescript SK- and KS- (Stratagene, Heidelberg, Germany), pBBR1MCS-2 (26), and the E. coli-Rhodococcus shuttle vector pBBRKmNC903.2 Cells of A. calcoaceticus were cultivated aerobically in LB medium (27) in Erlenmeyer flasks at 30 °C. For the induction of wax ester and TAG formation and for determination of enzyme activities, cells were cultivated 24 h in mineral salts medium (MSM) (28) with 0.1 g liter-1 NH4Cl and with 1% (w/v) sodium gluconate as carbon source. These conditions are referred to as "storage conditions." Cells of E. coli and P. citronellolis were cultivated in LB medium at 37 and 30 °C, respectively. For recombinant wax ester production, P. citronellolis was cultivated under storage conditions for 48 h at 30 °C with 0.3% (w/v) 1-hexadecanol as sole carbon source. For determination of enzyme activities in recombinant R. opacus PD630 cells were cultivated for 24 h under storage conditions at 30 °C with 1% (w/v) gluconate. M. smegmatis mc2155 was cultivated for 48 h under storage conditions at 30 °C with 1% (w/v) glucose. Antibiotics were added at the following concentrations if appropriate: ampicillin, 75 µg ml-1; nalidixic acid, 10 µg ml-1; kanamycin, 50 µg ml-1; tetracycline, 12.5 µg ml-1.

Isolation of Mutants of A. calcoaceticus ADP1 Defective in Storage Lipid Accumulation-- miniTn10Km-induced mutants of an isolated spontaneous nalidixic acid-resistant strain of A. calcoaceticus ADP1 were generated according to Ref. 29. Non-auxotrophic transposon mutants were selected on MSM agar plates containing gluconate, kanamycin, and nalidixic acid. Mutants defective in the accumulation of storage lipids were identified by staining with the lipophilic dye Sudan Black B as described in Ref. 30 and subjected to TLC for further analysis.

Lipid Analysis-- Thin-layer chromatography (TLC) was performed as described (31) using the solvent systems hexane/diethylether/acetic acid (90:15:1, v/v/v) for wax ester analysis or (80:20:1, v/v/v) for TAG analysis. Triolein was used as TAG reference substance and cetylpalmitate as wax ester reference substance. Fatty acid analysis of whole cells and purified TAGs and wax esters was done by gas chromatography (GC) according to Ref. 31.

Genotypic Characterization of the miniTn10Km Insertion Mutant A. calcoaceticus ACM7-- An 8.9-kbp EcoRI fragment conferring kanamycin-resistance to E. coli due to the presence of miniTn10Km was isolated from total DNA of the mutant A. calcoaceticus ACM7. After subcloning into pBluescript SK-, DNA sequence was determined, and data were analyzed with the online program pack Biology WorkBench 3.2 at workbench.sdsc.edu.

Cloning of the WS/DGAT Gene and Heterologous Expression-- The WS/DGAT gene was amplified by tailored PCR employing the oligonucleotides 5'-AAAGAATTCAAGGAGGTATCCACGCTATGCGCCCATTAC-3' (5'-end) introducing a ribosome binding site and 5'-TTTGGATCCAGGGCTAATTTAGCCCTTTAGTT-3' (3'-end) and was cloned into pBluescript KS- collinear to the lacZ promoter, resulting in pKS::wax/dgat, and into pBBR1MCS-2 collinear to the lacZ promoter, resulting in pBBR1MCS-2::wax/dgat. The WS/DGAT homologue from M. smegmatis mc2155 exhibiting the highest similarity to the A. calcoaceticus ADP1 gene (designated as wdh3269 because of its localization on contig 3269) was amplified by tailored PCR using the following oligonucleotides: 5'-AAAGAATTCAAGGAGGTCAGCGTTGAATGAACCGGATGCA-3' (5'-end) introducing a ribosome binding site and 5'-TTTAAGCTTTCAGGCGCCTGTGGCCGTCTCGA-3' (3'-end). The obtained 1403-bp PCR product was cloned into pBluescript SK- collinear to the lacZ promoter, resulting in pSK::wdh3269. For heterologous expression in R. opacus PD630, HindIII-restricted pSK::wdh3269 was fused with HindIII-restricted pBBRKmNC903 resulting in pBBRKmNC903-pSK::wdh3269. Transfer of the fusion plasmid to R. opacus was done according to Ref. 32. For heterologous expression, recombinant E. coli were cultivated for 6 h in the presence of 1 mM IPTG and recombinant P. citronellolis for 6 h and recombinant R. opacus PD630 for 24 h without IPTG, respectively.

Determination of Enzyme Activities-- WS activity was measured in a total volume of 250 µl containing 3.75 mM 1-hexadecanol, 4.63 mg ml-1 bovine serum albumin, 10 mM MgCl2, 4.72 µM [1-14C]palmitoyl-CoA (specific activity 1.961 Bq pmol-1), and 125 mM sodium phosphate buffer (pH 7.4). Hexadecanol and bovine serum albumin were emulsified by ultrasonification. The assays were incubated at 35 °C for 30 min, and the reactions were stopped by extraction with 500 µl of chloroform/methanol (1:1, v/v). After centrifugation, the chloroform phase was withdrawn, evaporated to dryness, and 40 µg of chloroform-dissolved unlabeled reference wax ester (cetylpalmitate) were added. After separation of lipids by TLC and staining of TLC plates with iodine vapor, spots corresponding to waxes were scraped from the plates, and radioactivity was measured by scintillation counting. The DGAT activity assay was identical to the WS assay except that the reaction mixture contained 3.75 mM 1,2-dipalmitoyl-rac-glycerol instead of 1-hexadecanol. Here triolein was used as the unlabeled TAG reference substance. Acyl-CoA specificity of the WS and DGAT reactions was assayed in a total volume of 250 µl containing 0.19 mM [1-14C]hexadecanol (specific activity 1.924 Bq pmol-1) or 0.09 mM oleic [1-14C]diolein (specific activity 2.035 Bq pmol-1), respectively, 4.63 mg ml-1 bovine serum albumin, 10 mM MgCl2, 50 µM acyl-CoA, and 125 mM sodium phosphate buffer (pH 7.4) under the conditions described above.

Data Deposition-- The WS/DGAT nucleotide sequence has been deposited in the GenBankTM data base under GenBank Accession Number AF529086.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Identification of a Gene Locus Involved in Storage Lipid Accumulation-- A. calcoaceticus ADP1 accumulates wax ester and TAG intracellularly during cultivation under so-called storage conditions from gluconate with TAGs and wax esters amounting to up to 1.4 and 6.9% (w/v) of the cellular dry weight, respectively, as estimated by gas chromatographic fatty acid analysis of isolated TAGs and wax esters purified by TLC. We isolated the miniTn10Km-induced mutant A. calcoaceticus ACM7, which was unable to synthesize wax ester and accumulated only trace amounts of TAG as estimated by TLC (Fig. 1B). Genotypical characterization of the mutant revealed insertion of the transposon with a 9-bp direct repeat (5'-CGCTATGCG-3') 4-bp upstream of the ATG start codon (shown in bold face) of a 1377-bp open reading frame, which we designated as wax/dgat (Fig. 1A). Since the coding region of wax/dgat remained intact in A. calcoaceticus ACM7, we generated the knock-out mutant A. calcoaceticus ADP1wax/dgatOmega Km by disrupting wax/dgat by insertion of a Omega Km gene cassette (33) (Fig. 1A). The knock-out mutant also exhibited the wax ester-negative and TAG-leaky phenotype, thus demonstrating that the strong impact on biosynthesis of both storage lipids was really attributed to wax/dgat (Fig. 1B).


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Fig. 1.   Influence of wax/dgat on wax ester and TAG accumulation in A. calcoaceticus ADP1. A, molecular organization of the 6.9-kbp EcoRI fragment harboring wax/dgat. The triangle indicates the miniTn10Km insertion locus in A. calcoaceticus ACM7. The NruI site was used for gene disruption by inserting a Omega Km gene cassette (33). The bar shows the DNA region whose sequence has been deposited in GenBankTM. The remaining part was obtained from preliminary sequence data of the A. calcoaceticus ADP1 genome project accessible online at www.genoscope.fr. Designations of putative genes were based on homologies found in a BLAST search: mreC, rod-shape determining protein; maf, putative inhibitor of septum formation; cysH, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase; thrH, homoserine kinase. B, TLC of whole cell extracts of A. calcoaceticus strains grown under storage conditions. Lanes: TAG, TAG standard; Wax, wax ester standard; 1, A. calcoaceticus ADP1; 2, A. calcoaceticus ACM7; 3, A. calcoaceticus ADP1wax/dgatOmega Km.

wax/dgat Encodes a Bifunctional WS/DGAT-- Wild-type A. calcoaceticus ADP1 exhibited a WS activity of 90.37 pmol (mg min)-1 and a ~10-fold lower DGAT activity (Table I), which corresponded approximately with the amounts of wax esters and TAGs accumulated under storage conditions as estimated by TLC (Fig. 1B). Inactivation of wax/dgat not only caused the loss of the ability for wax ester and TAG biosynthesis; it also abolished WS and DGAT activity in the transposon-induced as well as in the knock-out mutant (Table I).

                              
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Table I
WS and DGAT activities in crude cell extracts of different strains
Cell cultivations and enzyme assays were performed as described "Experimental Procedures." Values are averages of at least three independent experiment.

Heterologous expression of wax/dgat conferred the capability to recombinant E. coli XL1-Blue harboring pKS::wax/dgat to catalyze the acyl-CoA-dependent acylation of fatty alcohol as well as of diacylglycerol (Fig. 2A) at rates similar to those of A. calcoaceticus ADP1 (Table I). These results clearly show that both, WS and DGAT activity, arise from wax/dgat, which therefore codes for a bifunctional WS/DGAT enzyme. Furthermore, the experiments with the knock-out mutant indicate that no other protein exhibiting WS or DGAT activity contributes significantly to wax ester or TAG biosynthesis in A. calcoaceticus ADP1. This was supported by the fact that no other wax/dgat homologue could be identified by a BLAST (34) search in the preliminary A. calcoaceticus ADP1 genome sequence data accessible online at www.genoscope.fr. However, residual trace amounts of TAGs accumulated in the mutants indicate the presence of a minor alternative pathway, which is, however, only active at a very low rate. Furthermore, functional heterologous expression of wax/dgat was not only demonstrated in E. coli S17-1 but also in P. citronellolis (Table I).


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Fig. 2.   Properties of the bifunctional WS/DGAT. A, reactions catalyzed by the bifunctional enzyme. B, multiple sequence alignment of WS/DGAT with some related proteins from M. tuberculosis H37Rv and A. thaliana using the ClustalW program (43). Only the region corresponding to the A. calcoaceticus ADP1 WS/DGAT amino acid residues 75-151 are shown. Residues identical in seven or more sequences are shaded in gray. A putative active site is boxed. M. tuberculosis H37Rv: a, Rv3740c; b, Rv3734c; c, Rv1425; d, Rv3480c; e, Rv2285; A. thaliana: f, At5g53380; g, At5g16350; h, At5g12420; i, At5g22490; j, At1g72110; k, A. calcoaceticus ADP1 WS/DGAT (for accession numbers see Table II). C, hydrophobicity plot (44) of WS/DGAT (window size 9). A putative transmembrane domain predicted by the TMAP program (45) is indicated by the gray bar. The black bar region exhibits some homology to a conserved condensing domain containing a putative active site.

Characteristics of the Bifunctional WS/DGAT-- The bifunctional WS/DGAT comprises 458 amino acids with a theoretical molecular mass of 51.8 kDa. The basic theoretical pI of 9.05 is consistent with those observed for the jojoba WS (22) and other lipid biosynthetic enzymes (35). It is a rather amphiphilic protein, and it possesses one putative predicted membrane-spanning region (Fig. 2C). A broad range of long-chain fatty alcohols could be utilized by the bifunctional enzyme as acyl acceptor in the WS reaction with lower specificities toward longer chain length (Fig. 3A). Whereas the WS reaction accepted a wide range of various chain length acyl-CoA molecules almost equally, the DGAT reaction preferred longer chain length acyl-CoAs (Fig. 3, B and C). Kinetic studies on the bifunctional enzyme by monitoring the formation of radiolabeled reaction products as a function of time revealed no obvious lag phase and approximately constant WS and DGAT reaction rates over a period of 40 min (Fig. 4A). Recording the dependence of enzyme activities on palmitoyl-CoA concentration, hyperbolic saturation curves were obtained indicating that the bifunctional WS/DGAT does not behave as an allosteric enzyme (Fig. 4B). Assuming substrate saturation for 1-hexadecanol and dipalmitin under assay conditions, Lineweaver-Burk plot analysis revealed Km values for palmitoyl-CoA of 15.6 and 21.1 µM and Vmax of 212.8 and 54.3 pmol (mg min)-1 for WS and DGAT reactions, respectively. In direct competition experiments, in which dipalmitin and hexadecanol were provided together at various concentrations, the WS/DGAT exhibited a considerably higher substrate specificity toward 1-hexadecanol in comparison to dipalmitin, which explains the rather low DGAT activity compared with the WS activity (Fig. 5).


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Fig. 3.   Substrate specificities of the bifunctional WS/DGAT. Measurements were done using insoluble fraction of crude extract of E. coli XL1-Blue (pKS::wax/dgat) obtained after a 30-min centrifugation at 35,000 × g. Assay conditions were as described under "Experimental Procedures." Values are averages of two independent experiments. A, fatty alcohol specificity of the WS reaction. B, acyl-CoA specificity of the WS reaction. C, acyl-CoA specificity of the DGAT reaction.


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Fig. 4.   Enzyme kinetics of the bifunctional WS/DGAT. Measurements were done using insoluble fraction of crude extract of E. coli XL1-Blue (pKS::wax/dgat) obtained after a 30-min centrifugation at 35,000 × g. Assay conditions were as described under "Experimental Procedures." Values are averages of two independent experiments. A, time course of wax ester and TAG formation. B, palmitoyl-CoA dependence of WS and DGAT reactions. black-diamond , WS; black-square, DGAT.


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Fig. 5.   Competition between 1-hexadecanol and dipalmitin as substrates for WS/DGAT. Measurements were done using the insoluble fraction of crude extract of E. coli XL1-Blue (pKS::wax/dgat) obtained after a 30-min centrifugation at 35,000 × g. Assay conditions were as described under "Experimental Procedures" but providing 1-hexadecanol and dipalmitin together at various ratios in the same assay with a constant total concentration of 1-hexadecanol and dipalmitin as 3.75 mM. Values are averages of two independent experiments. black-diamond , WS; black-square, DGAT.

Heterologous Wax Ester Production in P. citronellolis-- Heterologous functional expression of the wax/dgat gene in the alkane-degrading bacterium P. citronellolis resulted in an active enzyme, which maintained its bifunctionality (Table I). During cultivation of P. citronellolis (pBBR1MCS-2::wax/dgat) under storage conditions, no accumulation of wax esters could be detected by TLC if 0.5% (w/v) gluconate, 0.3% (w/v) 1-hexadecane, or 0.3% (w/v) palmitate were used as carbon sources (data not shown). However, cultivation on 0.3% (w/v) 1-hexadecanol, which can serve as a direct substrate for the WS, resulted in recombinant production of wax esters (Fig. 6B). No TAG accumulation could be observed under either condition.


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Fig. 6.   Wax ester biosynthesis in M. smegmatis mc2155 and recombinant P. citronellolis. A TLC of whole cell extracts of M. smegmatis mc2155 grown under storage conditions with 1% (w/v) glucose (lane 1), 0.3% (w/v) 1-hexadecanol (lane 2), or 1% (w/v) glucose + 0.3% (w/v) 1-hexadecanol (lane 3). TAG, TAG standard; Wax, wax ester standard. The arrow indicates residual 1-hexadecanol used as carbon source. B, TLC of whole cell extracts of P. citronellolis strains grown under storage conditions with 0.3% (w/v) 1-hexadecanol. Lanes: Wax, wax ester standard; lane 1, P. citronellolis (pBBR1MCS-2); lane 2, P. citronellolis (pBBR1MCS-2::wax/dgat).

WS/DGAT-related Proteins-- The A. calcoaceticus ADP1 WS/DGAT exhibits no sequence similarity to any known acyltransferase including the WS from jojoba (Simmondsia chinensis) (22), the DGAT1 (9-12) and DGAT2 (13-15) gene family and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants (17). Thus, it represents a new type of long-chain acyl-CoA acyltransferase. A BLAST search comprising 189 eubacterial and 18 archaeal finished and unfinished microbial genome sequences (as of November 2002) publicly accessible via the National Center of Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov) revealed that WS/DGAT-related proteins are not widely distributed among prokaryotes. Aside from the Gram-negative bacterium A. calcoaceticus, related proteins were found only in some actinomycetes, and interestingly within all members of the genus Mycobacterium (Table III). Whereas only one gene coding for WS/DGAT occurs in A. calcoaceticus ADP1, numerous genes for related proteins were found in mycobacteria (Table III). These WS/DGAT-related proteins constitute a remarkable large group of conserved proteins in Mycobacterium with an up to now unknown function. M. tuberculosis H37Rv, for instance, possesses 13 genes coding for WS/DGAT-related proteins exhibiting up to 39.7% identity with the A. calcoaceticus enzyme (Table II), i.e. 1.43% of the 912 genes encoding conserved hypothetical proteins and 0.86% of all 1518 genes with unknown function in this strain (36, 37). A BLAST search with 50 eukaryotic genome sequences at NCBI identified in A. thaliana a large group of conserved putative proteins with unknown function exhibiting some similarity to the A. calcoaceticus WS/DGAT (Table II).

                              
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Table II
WS/DGAT-related proteins in M. tuberculosis and A. thaliana

Wax Ester Biosynthesis in M. smegmatis mc2155-- Eight WS/DGAT homologous genes could be identified in the preliminary genome sequence of the non-pathogenic strain M. smegmatis mc2155 (see Table III), which is publicly accessible online via NCBI. The gene with the highest similarity exhibits 41.0% amino acid identity to WS/DGAT (Table II). Recombinant E. coli expressing this gene on plasmid pSK::wdh3269 showed weak WS and DGAT activity, which was slightly but reproducible higher than the vector control (Table I). R. opacus PD630 is a TAG-accumulating actinomycete (3), which itself exhibits WS and DGAT activity, but heterologous expression of wdh3269 clearly elevated both activities in this strain (Table I).

                              
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Table III
Distribution of WS/DGAT-related proteins in bacteria
Data were obtained using BLAST search with microbial genomes at NCBI comprising 189 eubacterial + 18 archaeal finished and unfinished genome sequences (as of November 2002). Identities were calculated for full-length sequences

M. smegmatis mc2155 cultivated under storage conditions with glucose as sole carbon source exhibited both high WS as well as DGAT activity in vitro (Table I), although in vivo only TAGs were intracellularly accumulated (Fig. 6A). However, M. smegmatis mc2155 was also capable of substantial wax ester biosynthesis in vivo when 1-hexadecanol was provided as a sole carbon source or as a cosubstrate (Fig. 6A).

Identification of a Putative Active Site-- The A. calcoaceticus ADP1 WS/DGAT and the related proteins in Mycobacterium and A. thaliana exhibit in their N-terminal region partial similarity to a conserved condensing domain found in many multidomain enzymes synthesizing peptide antibiotics (NCBI Conserved Domain Data Base accession pfam00668). This condensing domain contains an active-site motif (HHXXXDG), whose second histidine residue is strictly conserved and has been demonstrated to be essential for catalytic activity in non-ribosomal peptide bond formation (38). The WS/DGAT and related proteins also contain this putative active site with the motif (133HXXXDG138) being strictly conserved (Fig. 2B). Thus, it is very likely that this site is catalytically participating in the acyl-CoA acyltransferase reactions involved in wax ester and TAG formation (Fig. 2A).

    DISCUSSION
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ABSTRACT
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TAGs and wax esters are rather uncommon storage lipids in bacteria. Large amounts of TAG accumulation has been reported particularly for actinomycetes (2-4), whereas wax esters occur frequently among species of the genus Acinetobacter, which are able to accumulate wax esters of up to 14% of the cellular dry weight depending on the culture conditions (5). The A. calcoaceticus strain ADP1 synthesizes both classes of storage lipids under growth-limiting conditions. In the present study we identified a novel bifunctional enzyme from this strain, which mediates the final reaction steps in the biosynthesis of both lipids simultaneously. This bifunctional WS/DGAT is a new and unique type of long-chain acyltransferase, which is not related to members of the DGAT1 and 2 families or phospholipid:DAG acyltransferases mediating TAG synthesis, or to the jojoba WS, or to any other known gene in the data base.

The residual trace amounts of TAGs, which are still present in the mutants, indicate that there might be an alternative low-rate pathway for TAG synthesis. Multiple alternative TAG biosynthesis pathways have been reported for Saccharomyces cerevisiae, where a DGAT, a phospholipid:DAG acyltransferase, and the DGAT side-activity of a sterol:acyl-CoA acyltransferase contribute cumulatively at various rates to TAG synthesis (15). In the preliminary A. calcoaceticus ADP1 genome sequence; however, no gene similar to those mentioned could be identified by BLAST search that could be a candidate for residual TAG synthesis.

The highly conserved motif HHXXXDG corresponding to amino acids 132-138 of the A. calcoaceticus ADP1 WS/DGAT (Fig. 2B) may be the catalytic site responsible for ester bond formation. In analogy to the mechanism of non-ribosomal peptide bond formation catalyzed by condensing domains, which also contain this putative active site motif (38), a proposed catalytic mechanism for the WS/DGAT could involve abstraction of a proton from the hydroxyl group of the fatty alcohol or DAG, respectively, by the strictly conserved histidine residue His133 acting as base catalyst, which would than enable the nucleophilic attack on the thioester bond of the fatty acyl-CoA molecule (see Fig. 2A).

Beside the genus Acinetobacter, WS/DGAT-related proteins seem to be restricted almost exclusively to mycobacteria among prokaryotes (Table III). Whereas only one WS/DGAT gene is present in A. calcoaceticus ADP1, an extensive group of related proteins occurs in mycobacteria (Table III). Wax ester synthesis and WS activity in vitro have been reported for M. tuberculosis (39), and TAG accumulation and DGAT activity have been shown for M. smegmatis (40, 41). In the present study, we demonstrated in addition in vitro WS activity (Table I) and in vivo wax ester production in M. smegmatis mc2155 (Fig. 6A). Until this study, however, no proteins or genes had been reported to which these activities could be attributed. By heterologous functional expression of the homologue from M. smegmatis mc2155, which exhibited the highest similarity to the A. calcoaceticus ADP1 WS/DGAT (wdh3269), in recombinant E. coli and R. opacus, it was unambiguously demonstrated that wdh3269 also codes for a bifunctional WS/DGAT. However, it is not known yet to what extent wdh3269 contributes to storage lipid accumulation in M. smegmatis mc2155. It is likely that the observed in vitro and in vivo WS and DGAT activities in this strain are the cumulative result of several WS/DGAT homologues. Alternatively to storage lipid synthesis, some of the WS/DGAT homologues could also participate in biosynthesis of other lipids like mycolic acids.

Interestingly, only recently the first evidence for lipid accumulation in M. tuberculosis, in vivo, has been reported (42). By employing a combined staining method, the occurrence of substantial lipophilic intracellular inclusions in mycobacterial cells in sputum samples from patients with clinical tuberculosis was demonstrated, which indicates that lipid accumulation could be an essential factor participating in pathogenesis. Simply the large number of WS/DGAT-related proteins identified in mycobacteria already supposes an important function, and it is likely that a least some of them are involved in lipid accumulation in M. tuberculosis. Thus, detailed investigations on the biological function of these WS/DGAT homologous proteins and their importance for the pathogenesis of harmful mycobacteria seem to be worthwhile. Since they can be found almost exclusively within mycobacteria and are not widely distributed among other prokaryotes or eukaryotes, they could be an ideal therapeutical target for treatment of major global health problems caused by mycobacteria like tuberculosis.

A large group of conserved hypothetical proteins similar to WS/DGAT was also found in A. thaliana (Table II). Members of the DGAT1 and 2 families were already described for this plant (11, 14), thus the presence of WS/DGAT-related proteins could possibly indicate that acyl-CoA-dependent TAG synthesis is mediated in A. thaliana by three different non-related enzyme groups. It would also be conceivable that WS/DGAT homologues are involved in the biosynthesis of epicuticular wax esters.

A strong demand exists for large-scale production of cheap jojoba-like wax esters, which have multiple commercial uses. Jojoba oil is the only alternative natural source of wax esters to sperm whale oil, which is used at a commercial scale, but the high production costs restrict its use currently on cosmetic applications. The jojoba WS could not be functionally expressed in microorganisms like E. coli and S. cerevisiae (22). In contrast, we have demonstrated that the A. calcoaceticus ADP1 WS/DGAT was active in different bacterial hosts. In P. citronellolis, the heterologous expression of WS/DGAT led to production of wax esters if a long-chain fatty alcohol was provided as a carbon source that also delivers fatty acyl-CoA during catabolism by the alkane degradation pathway. By variation of the fatty alcohol used as carbon source it should be possible to vary the composition of the produced wax esters, because the bifunctional enzyme can utilize a broad range of substrates (Fig. 3). This study provides the basis for a potential microbial biotechnological production of cheap jojoba-like wax esters.

    ACKNOWLEDGEMENTS

We thank Toni Voelker and Kathryn D. Lardizabal for helpful discussions.

    FOOTNOTES

* This study was supported by a grant from Monsanto Co. (St. Louis, MO).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF529086.

Dagger To whom correspondence should be addressed. Tel.: 49-251-8339821; Fax: 49-251-8338388; E-mail: steinbu@uni-muenster.de.

Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M210533200

2 R. Kalscheuer, unpublished results.

    ABBREVIATIONS

The abbreviations used are: TAG, triacylglycerol; WS, wax ester synthase; DGAT, acyl-CoA:diacylglycerol acyltransferase; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; DAG, diacylglycerol; MSM, mineral salts medium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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