Microbial metabolism of alkylbenzene sulphonates. Bacterial metabolism of undecylbenzene-p-sulphonate and dodecylbenzene-p-sulphonate

1. A study was made of the biodegradation of alkylbenzene sulphonate homologues, one of the major components of commercially marketed detergents. A Bacillus species was elected for growth on alkylbenzene sulphonate homologues as the sole source of carbon and sulphur. 2. The results from both whole-cell and cell-free systems indicated that the alkyl, aryl and sulphonate moieties of alkylbenzene sulphonate homologues were all further metabolized by the Bacillus species. 3. The alkyl side chain, after a presumed initial oxidation of the terminal methyl group, was subsequently oxidized by a beta-oxidation pathway. Three enzymes of the beta-oxidation pathway, i.e. acyl-CoA synthetase, acyl-CoA dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase, were identified in cell-free extracts of the detergent-grown Bacillus species. The substrate specificity of acyl-CoA synthetase indicated activity towards several alkylbenzene sulphonate homologues. 4. The sulphonate moiety was released as sulphite by a desulphonating enzyme. Some kinetic properties of this enzyme were determined. The sulphite was subsequently metabolized to either sulphate or adenosine 5'-sulphatophosphate. Two enzymes involved in sulphite metabolism, i.e. sulphite-cytochrome c reductase and adenosine 5'-sulphatophosphate-cytochrome c reductase were detected in cell-free extracts of undecylbenzene-p-sulphonate-grown Bacillus species. 5. The combined results of continuous sampling programmes monitored by both t.l.c. and sulphite appearance in the growth medium indicated that desulphonation of the aromatic moiety was the likely first step in the overall biodegradation of several alkylbenzene sulphonate homologues. 6. The presence of p-hydroxyphenylpropionate, p-hydroxybenzoate and 3,4-dihydroxybenzoate in cells after growth on several alkylbenzene sulphonate homologues containing an odd number of carbon atoms in the side chain was confirmed by g.l.c. and t.l.c. analysis. Cells grown on several homologues containing an even number of carbon atoms in the side chain were shown to contain p-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate. 7. The aromatic nucleus obtained from undecylbenzene-p-sulphonate was further metabolized by an oxidation sequence involving an ;ortho-cleavage' route. 8. An overall metabolic pathway for the biodegradation of various alkylbenzene sulphonate homologues by this Bacillus species is proposed.     

Microbial metabolism of alkylbenzene sulphonates. Variance in enzyme complement of a Bacillus in response to growth substrate

Growth of a Bacillus species at the expense of an alkylbenzene sulphonate (ABS) synthetic detergent homologue (1-phenylundecane-p-sulphonate, 11-ABS) containing an odd number of carbon atoms in the alkyl side chain induced an enzyme complement able to biodegrade 11-ABS by alkyl side-chain oxidation, and ortho cleavage aromatic-ring oxidation reactions. Growth of the Bacillus at the expense of an ABS homologue containing an even number of carbon atoms in the alkyl side chain (1-phenyldodecane-p-sulphonate, 12-ABS) induced an enzyme complement able to biodegrade 12-ABS by alkyl side-chain oxidation, and meta cleavage aromatic-ring oxidation reactions.The results of a number of different growth and enzyme induction experiments confirm that both 11-ABS and 12-ABS are initially biodegraded by an identical complement of enzymes catalysing the alkyl-side-chain oxidation reactions, but that the subsequent metabolism of the aromatic moieties remaining after the removal of the alkyl side chain from 11-ABS and 12-ABS occurs by two separate pathways requiring the de novo induction of different substrate-specific enzyme complements. The detection of the predicted changes in enzyme complement subsequent to changes in the growth substrate of the Bacillus provide confirmation of the biodegradation pathways operating in the microorganism.     

Microbial metabolism of alkylbenzene sulphonates Enzyme system of a Bacillus species responsible for Β-oxidation of the alkyl side chain of alkylbenzene sulphonates

A Bacillus species was isolated from sewage capable of utilising alkylbenzene sulphonates (ABS) as the sole source of carbon and sulphur. The enzymic mechanism involved in alkyl-side-chain biodegradation of various ABS detergent isomers by the Bacillus species was demonstrated to involve the classical-Β-oxidation equence characteristic of long-chain fatty acid oxidation, by appropriate enzyme inductions. The combined results from both enzyme induction studies and molecular separation of induced enzymes by gel-filtration indicated a single set of enzymes to be responsible for the Β-oxidation of both ABS isomers and long-chain fatty acid isomers in the Bacillus species. The substrate specificity of partially purified enzymes after growth on appropriate substrates confirmed the feasibility of a single Β-oxidation pathway in this microorganism capable of catalising the oxidation of a wide range of different synthetic and naturally occurring chemicals and biochemicals containing alkyl side chains. This work was supported at Newcastle by grants from the Science Research Council and The Royal Society.     

Induction of β-oxidation enzymes and microbody proliferation in Aspergillus nidulans

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced β-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the β-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The β-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans. Received: 8 March 1996 / Accepted: 5 August 1996     

Cladosporium carrionii and Hormoconis resinae (C. resinae): Cell Wall and Melanin Studies

Two phaeoid strains of the fungus Cladosporium carrionii (SR3 from a xerophyte species and PP8201 from a patient), and one strain of Hormoconis resinae (Cladosporium resinae), isolated from oil-impregnated soil, were analyzed for their cell wall composition by colorimetric methods, X-ray diffraction, infrared spectroscopy, and solid-state ¹³C-nuclear magnetic resonance. Results suggested that the cell walls were composed mainly of hexoses (34%–47%) as β-1,3-glucan (some galactose and mannose were also present) and melanin, chitin being absent. Electron microscopic observations suggested that melanin was found not only in the cell wall but also in intracellular bodies resembling melanosomes.     

Peroxisomal β-oxidation activities and γ-decalactone production by the yeast Yarrowia lipolytica

γ-Decalactone is a peachy aroma compound resulting from the peroxisomal β-oxidation of ricinoleic acid by yeasts. The expression levels of acyl-CoA oxidase (gene deletion) and 3-ketoacyl-CoA thiolase activities (gene amplification on replicative plasmids) were modified in the yeast Yarrowia lipolytica. The effects of these modifications on β-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall β-oxidation activity. The disruption of one of the acyl-CoA oxidase genes resulted in an enhanced activity. The enhancement led to an increase of overall β-oxidation activity but reduced the γ-decalactone production rates. This seemed to indicate a non-rate-limiting role for β-oxidation in the biotransformation of ricinoleic acid to γ-decalactone by the yeast Yarrowia lipolytica. All strains produced and then consumed γ-decalactone. We checked the ability of the different strains to consume γ-decalactone in a medium containing the lactone as sole carbon source. The consumption of the strain overexpressing acyl-CoA oxidase activity was higher than that of the wild-type strain. We␣concluded that peroxisomal β-oxidation is certainly involved in γ-decalactone catabolism by the yeast Y.␣lipolytica. The observed production rates probably depend on an equilibrium between production and consumption of the lactone. Received: 13 June 1997 / Received revision: 2 October 1997 / Accepted: 14 October 1997     

Substrate inhibition and affinities of the glyoxysomal β-oxidation of sunflower cotyledons

During the glyoxysomal β-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125–130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal β-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the β-oxidation of acyl-CoAs of various chain lengths showed that the β-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 μM. At substrate concentrations >20 μM, long-chain acyl-CoAs were β-oxidized more slowly than short-chain acyl-CoAs because the β-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5–10 μM substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the β-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the β-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (≤5 μM) of a long-chain acyl-CoA exerted an inhibitory effect on the β-oxidation rate of butyryl-CoA. Reversibility of the inhibition was observed as well as metabolization of the inhibiting long-chain acyl-CoA. Regarding the activities of the individual β-oxidation enzymes towards their C₄ substrates in the presence of a long-chain acyl-CoA, the MFP activity exhibited strong inhibition. This inhibition appears not to be due to the detergent-like physical properties of long-chain acyl-CoAs. The results of the studies, which are consistent with the observation that short-chain intermediates accumulate transiently during complete degradation of a long-chain acyl-CoA, suggest that the substrate concentration-dependent chain-length specificity of the β-oxidation and a metabolic regulation at the level of MFP are factors determining this transient accumulation. Received: 2 February 1999 / Accepted: 14 April 1999     

Production of β-glucosidase byCladosporium resinae

Summary Cladosporium resinae QM 7998 produced high activities of extracellular and constitutive -glucosidase when grown on a variety of sugars or cellulose. Starch and ribose induced enzyme synthesis several fold.Cladosporium resinae could utilize agricultural waste residues for growth and -glucosidase production. The initial pH of the medium had a marked effect on enzyme prowduction and optimum pH was between 4.0 and 5.0 depending on the assay method. Mixed culturing ofC. resinae with yeasts, viz.Saccharomyces cerevisiae andCandida utilis, increased the -glucosidase production while that with other fungi decreased the enzyme yield. The- glucosidase preparation fromC. resinae significantly increased the saccharification of rice and wheat straw (untreated or delignified) withTrichoderma reesei QM 9414 cellulase preparation.

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Résumé Cladosporium resinae QM 7998 produit des concentrations élevées de -glucosidase tant extracellulaire que constitutive lorsqu'elle croît sur une variété de sucres ou sur la cellulose. On a trouvé que l'amidon et le ribose augmentent de plusieurs fois la quantité d'enzyme synthétisée.Cladosporium resinae peut utiliser des résidus agricoles pour sa croissance et pour la production de -glucosidase. Le pH initial du milieu exerce un effet marqué sur la production d'enzyme et le pH optimum est compris entre 4.0 et 5.0 selon les conditions de l'essai. La croissance mixte deCladosporium resinae avec diverses levures, notammentSaccharomyces cerevisiae etCandida utilis, augmente la production de -glucosidase tandis que celle avec d'autres moisissures diminue le rendement en enzyme. La -glucosidase deCladosporium resinae augmente de manière significative la saccharification des pailles de riz et de froment (non-traitées ou délignifiées) traités par la cellulase deTrichoderma reesei QM 9414.

     

Glyoxysomal β-oxidation of long-chain fatty acids: completeness of degradation

The ability of glyoxysomes from sunflower (Helianthusannuus L.) cotyledons to completely degrade long-chain fatty acids into their constituent acetyl units and the time courses of the appearance of acyl-CoA intermediates during β-oxidation have been studied using ¹⁴C-labelled substrates at non-saturating concentrations (1.3 to 1.8 μmol · l⁻¹). [¹⁴C]Acetyl-CoA was formed from [18-¹⁴C]oleate metabolized at a yield of up to 80%, and from [U-¹⁴C]palmitate and [U-¹⁴C]linoleate to an extent indicating that a maximum of 80% and 30%, respectively, of the substrate β-oxidized had been degraded beyond the C₄-CoA intermediate level. To obtain the latter values, an acetyl-CoA-removing system was required during β-oxidation. Constant re-oxidation of the NADH formed during the β-oxidation did not replace the effect of acetyl-CoA removal. Neither the completeness of the linoleate β-oxidation nor the rate of reaction were influenced by NADPH. Medium- and short-chain acyl-CoA intermediates were predominantly detected during β-oxidation of the long-chain substrates employed. The degradation of these intermediates appeared to be stimulated mainly in the presence of an acetyl-CoA-removing system. The time courses of the appearance of intermediates corresponded to a precursor-product relationship between intermediates of decreasing chain lengths. Received: 12 December 1997 / Accepted: 26 January 1998     

Complete degradation of xenobiotic surfactants by consortia of aerobic microorganisms

Linear alkylbenzene sulphonates are primarily attacked via a hydroxylation of the alkyl chain from the methyl group followed by -oxidation. The alkyl chain is metabolized by pure cultures to give sulphophenyl carboxylates which accumulate in the medium. In mixed culture, other microorganisms are capable of degrading sulphophenyl carboxylates. Formation of ethylene glycol monosulphates as major products of alkyl ethoxy sulphates demonstrates that the ether bonds are cleaved. The bacteria involved in growing on the alkyl chain are unable to utilize the hydrophilic moiety. This hydrophilic moiety, in turn, is degraded by other microorganisms.The degradation of alkylphenol ethoxylates and highly branched alcohol ethoxylates proceeds by shortening the polyoxyethylene chain leaving the hydrophobic part of the molecule. The biodegradation of linear alcohol ethoxylates and ethoxylated fatty amines is initiated by a central cleavage or -oxidation. Subsequent oxidation of the alkyl chains results in the production of polyethylene glycols and secondary ethoxylated amines. Both polar moieties are metabolized by other microorganisms. Degradation of alkyltrimethylammonium salts and alkylamines is initiated by a cleavage of the C _alkyl -N bond. The central fission leads to the formation of alkanals which are readily converted by -oxidation. The alkyl chain-utilizing bacteria are not able to degrade the methylamines. The methylamines, in turn, are subject to biodegradation by methylotrophs.The limited metabolic capacities of pure cultures of microorganisms utilizing surfactants point to the requirement of consortia to degrade surfactants completely. Complete degradation of surfactants is accomplished by mixed cultures of microorganisms constructed on the basis of synergistic and commensalistic relationships. However, degradation of a surfactant by one member of a commensalistic consortium may lead to the production of toxic or non-toxic metabolites. Waste water treatment without the build up of such metabolites can be achieved in plants operated with sludge retention times that are suitable for maintaining all microorganisms of the consortium. In contrast, in natural ecosystems the introduction of a surfactant may result in a transient formation of a metabolite.     

Alternate microbial strategies for the metabolism of a 3-methyl branched alkanoic acid

The branched alkanoic acid, 3-methylvaleric acid (3-MVA), was used to test the effect of a β-methyl branch on short chain alkanoic acid biodegradability by various bacteria. Most of the bacteria tested were able to usen-valeric acid as sole carbon source but were unable to assimilate 3-MVA. Three bacterial strains capable of growth on 3-MVA are described here because they exemplify metabolism of the branched compound via different strategies.Pseudomonas citronellolis used a β-methyl activation sequence involving CO₂ fixation, analogous to that seen in the isovalerate pathway. AMycobacterium sp. used an α-oxidation sequence to convert 3-MVA to 2-methyl-butyrate, which was then assimilated via part of the isoleucine pathway. AnArthrobacter sp. metabolized 3-MVA via ω-oxidation to produce 3-methylglutarate that was degraded through the 3-hydroxy-3-methylglutarate pathway.     

IBR3, a novel peroxisomal acyl-CoA dehydrogenase-like protein required for indole-3-butyric acid response

Indole-3-butyric acid (IBA) is an endogenous auxin that acts in Arabidopsis primarily via its conversion to the principal auxin indole-3-acetic acid (IAA). Genetic and biochemical evidence indicates that this conversion is similar to peroxisomal fatty acid β-oxidation, but the specific enzymes catalyzing IBA β-oxidation have not been identified. We identified an IBA-response mutant (ibr3) with decreased responses to the inhibitory effects of IBA on root elongation or the stimulatory effects of IBA on lateral root formation. However, ibr3 mutants respond normally to other forms of auxin, including IAA. The mutant seedlings germinate and develop normally, even in the absence of sucrose, suggesting that fatty acid β-oxidation is unaffected. Additionally, double mutants between ibr3 and acx3, which is defective in an acyl-CoA oxidase acting in fatty acid β-oxidation, have enhanced IBA resistance, consistent with a distinct role for IBR3. Positional cloning revealed that IBR3 encodes a putative acyl-CoA dehydrogenase with a consensus peroxisomal targeting signal. Based on the singular defect of this mutant in responding to IBA, we propose that IBR3 may act directly in the oxidation of IBA to IAA. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Aspergillus nidulans contains six possible fatty acyl-CoA synthetases with FaaB being the major synthetase for fatty acid degradation

Aspergillus nidulans can use a variety of fatty acids as sole carbon and energy sources via its peroxisomal and mitochondrial β-oxidation pathways. Prior to channelling the fatty acids into β-oxidation, they need to be activated to their acyl-CoA derivates. Analysis of the genome sequence identified a number of possible fatty acyl-CoA synthetases (FatA, FatB, FatC, FatD, FaaA and FaaB). FaaB was found to be the major long-chain synthetase for fatty acid degradation. FaaB was shown to localise to the peroxisomes, and the corresponding gene was induced in the presence of short and long chain fatty acids. Deletion of the faaB gene leads to a reduced/abolished growth on a variety of fatty acids. However, at least one additional fatty acyl-CoA synthetase with a preference for short chain fatty acids and a potential mitochondrial candidate (AN4659.3) has been identified via genome analysis.     

β-Oxidation of fatty acids in algae: Localization of thiolase and acyl-CoA oxidizing enzymes in three different organisms

In the algae Mougeotia, Bumilleriopsis and Eremosphaera, recently shown to possess the enzymes hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and enoyl-CoA hydratase (EC 4.2.1.17), the presence of thiolase (EC 2.3.1.9) and acyl-CoA-oxidizing enzymes can also be demonstrated, indicating that -oxidation of fatty acids is possible in these organisms. The compartmentation of enzymes is different in the various algae. In Mougeotia, both thiolase and the acyl-CoA-oxidizing enzyme are located exclusively in the peroxisomes. The latter enzyme was found to be an oxidase using molecular oxygen as an electron acceptor. On the other hand, in Bumilleriopsis all enzymes of the fatty-acid -oxidation pathway tested are constituents only of the mitochondria, and acyl-CoA is oxidized by a dehydrogenase incapable of reducing oxygen. Finally, in Eremosphaera thiolase and acyl-CoA-oxidizing enzymes were found in the peroxisomes as well as in the mitochondria. In the peroxisomes, oxidation of acyl-CoA is catalyzed by an oxidase, whereas the corresponding enzyme in the mitochondria is a dehydrogenase. The acyl-CoA oxidases/dehydrogenases of the three algae differ not only by their capability for oxidation of acyl-CoA of different chain lengths but also with regard to their K_m values and substrate specificities. Indications were obtained that the oxygen is reduced to water rather than to H₂O₂ by the algal acyl-CoA oxidases. When cells of Eremosphaera were cultured with hypolipodemic substances in the growth medium the activities of the peroxisomal enzymes, but not those of the mitochondrial enzymes of the fatty-acid -oxidation pathway, were increased by a factor of two to three.Abbreviations DPIP 2,6-dichlorophenol indophenol - INT p-iodonitrotetrazolium violet - MEHP monoethylhexylphthalate     

Microbial degradation of secondaryn-alkyl sulfates and secondary alkanols

Secondaryn-alkyl sulfates (and secondary alkanols) prove to be degraded aerobically by aPseudomonas species (Ps. B-2) to carbon dioxide, water and sulfate. The proposed mechanism for the biodegradation proceeds via the alcohol → ketone → hydroxyketone → dione → aldehyde + acid. Consequently, two fatty acids are formed which are subsequently degraded by β-oxidation. This pathway requires the presence of molecular oxygen for the hydroxylation of the ketone. Attempts to isolate bacteria growing on secondary alcohols anaerobically were unsuccessful.     

Engineering polyhydroxyalkanoate content and monomer composition in the oleaginous yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> by modifying the ß-oxidation multifunctional protein

Recombinant strains of the oleaginous yeast Yarrowia lipolytica expressing the PHA synthase gene (PhaC) from Pseudomonas aeruginosa in the peroxisome were found able to produce polyhydroxyalkanoates (PHA). PHA production yield, but not the monomer composition, was dependent on POX genotype (POX genes encoding acyl-CoA oxidases) (Haddouche et al. FEMS Yeast Res 10:917–927, 2010). In this study of variants of the Y. lipolytica β-oxidation multifunctional enzyme, with deletions or inactivations of the R-3-hydroxyacyl-CoA dehydrogenase domain, we were able to produce hetero-polymers (functional MFE enzyme) or homo-polymers (with no 3-hydroxyacyl-CoA dehydrogenase activity) of PHA consisting principally of 3-hydroxyacid monomers (>80%) of the same length as the external fatty acid used for growth. The redirection of fatty acid flux towards β-oxidation, by deletion of the neutral lipid synthesis pathway (mutant strain Q4 devoid of the acyltransferases encoded by the LRO1, DGA1, DGA2 and ARE1 genes), in combination with variant expressing only the enoyl-CoA hydratase 2 domain, led to a significant increase in PHA levels, to 7.3% of cell dry weight. Finally, the presence of shorter monomers (up to 20% of the monomers) in a mutant strain lacking the peroxisomal 3-hydroxyacyl-CoA dehydrogenase domain provided evidence for the occurrence of partial mitochondrial β-oxidation in Y. lipolytica.     

Enzyme analyses demonstrate that β-methylbutyric acid is converted to β-hydroxy-β-methylbutyric acid via the leucine catabolic pathway by Galactomyces reessii

Galactomyces reessii accomplishes the enzymatic transformation of β-methylbutyric acid (isovaleric acid) to β-hydroxy-β-methylbutyric acid. The enzymatic basis for this bioconversion was evaluated by analyzing cell-free extracts of G. reessii for enzyme activities commonly associated with leucine catabolism. G. reessii extracts contained activities for acyl-CoA synthetase, acyl-CoA dehydrogenase, and enoyl-CoA hydratase, whereas β-methylbutyric acid hydroxylase, α-ketoisocaproate oxygenase, and acyl-CoA oxidase (with isovaleryl-CoA as substrate) were not observed. Furthermore, β-methylbutyric acid is initially activated to isovaleryl-CoA by acyl-CoA synthetase, dehydrogenated to methylcrotonyl-CoA by acyl-CoA dehydrogenase, hydrated to β-hydroxy-β-methylbutyric acid-CoA by enoyl-CoA hydratase, and hydrolyzed to β-hydroxy-β-methylbutyric acid in G. reessii extracts. Cell-free extracts converted both isovaleryl-CoA and methylcrotonyl-CoA into β-hydroxy-β-methylbutyric acid, thus demonstrating that β-methylbutyric acid is part of the leucine catabolic pathway. The rate of β-methylbutyric acid conversion to β-hydroxy-β-methylbutyric acid with cell-free extract was 0.013 μmol β-hydroxy-β-methylbutyric acid (mg protein)^–1 h^–1, while the conversion rate of leucine was fivefold lower. With whole cells, the highest production rate [0.042 μmol β-hydroxy-β-methylbutyric acid (g cells)^–1 h^–1] was also observed with β-methylbutyric acid. The results indicate that β-methylbutyric acid is transformed to β-hydroxy-β-methylbutyric acid through the leucine catabolic pathway. Received: 18 July 1997 / Accepted: 12 November 1997     

Very-long-chain fatty acid metabolism in adrenoleukodystrophy protein-deficient mice

X-linked adrenoleukodystrophy (X-ALD) is characterized by progressive mental and motor deterioration, with demyelination of the central and peripheral nervous system. Its principal biochemical abnormality is the accumulation of very-long-chain fatty acids (VLCFAs) in tissues and body fluids, caused by the impairment of peroxisomal β-oxidation. The authors have generated a line of mice deficient in ALD protein (ALDP) by gene targeting. ALDP-deficient mice appeared normal clinically, at least up to 12 mo. Western blot analysis showed absence of ALDP in the brain, spinal cord, lung, and kidney. The amounts of C26∶0 increased by 240% in the spinal cord. VLCFA β-oxidation in cultured hepatocytes was reduced to 50% of normal. The authors investigated the roles of ALDP in VLCFA β-oxidation using the ALDP-deficient mice. Very-long-chain acyl-CoA synthetase (VLACS) is functionally deficient in ALD cells. The impairment of VLCFA β-oxidation in the ALDP-deficient fibroblasts was not corrected by overexpression of VLACS only, but was done by co-expression of VLACS and ALDP, suggesting that VLACS requires ALDP to function. VLACS was detected in the peroxisomal and microsomal fractions of the liver from both types of mice. Peroxisomal VLACS was clearly decreased in the ALDP-deficient mouse. Thus, ALDP is involved in the peroxisomal localization of VLACS.