Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Antifungal Hydroxy Fatty Acids Produced during Sourdough Fermentation: Microbial and Enzymatic Pathways,and Antifungal Activity in Bread

Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C_18:1 fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter⁻¹. Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter⁻¹. L. hammesii accumulated the monohydroxy C_18:1 fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter⁻¹ (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.     

On the Specificity of a Fatty Acid Epoxygenase in Broad Bean (Vicia faba L.)

Seeds of broad bean (Vicia faba L.) contain a hydroperoxide-dependent fatty acid epoxygenase. Hydrogen peroxide served as an effective oxygen donor in the epoxygenase reaction. Fifteen unsaturated fatty acids were incubated with V. faba epoxygenase in the presence of hydrogen peroxide and the epoxy fatty acids produced were identified. Examination of the substrate specificity of the epoxygenase using a series of monounsaturated fatty acids demonstrated that (Z)-fatty acids were rapidly epoxidized into the corresponding cis-epoxy acids, whereas (E)-fatty acids were converted into their trans-epoxides at a very slow rate. In the series of (Z)-monoenoic acids, the double bond position as well as the chain length influenced the rate of epoxidation. The best substrates were found to be palmitoleic, oleic, and myristoleic acids. Steric analysis showed that most of the epoxy acids produced from monounsaturated fatty acids as well as from linoleic and α-linolenic acids had mainly the (R),(S) configuration. Exceptions were C₁₈ acids having the epoxide group located at C-12/13, in which cases the (S),(R) enantiomers dominated. 13(S)-Hydroxy-9(Z),11(E)-octadecadienoic acid incubated with epoxygenase afforded the epoxy alcohol 9(S),10(R)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as the major product. Smaller amounts of the diastereomeric epoxy alcohol 9(R),10(S)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as well as the α,β-epoxy alcohol 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoic acid were also obtained. The soluble fraction of homogenate of V. faba seeds contained an epoxide hydrolase activity that catalyzed the conversion of cis-9,10-epoxyoctadecanoic acid into threo-9,10-dihydroxyoctadecanoic acid.     

Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for In Vivo Application of Cytochrome P-450BM-3 Monooxygenase

The application of whole cells containing cytochrome P-450_BM-3 monooxygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to ω-1, ω-2, and ω-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450_BM-3 were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid uptake system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system of E. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in E. coli W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of E. coli K27, which is a fadD mutant and therefore unable to consume substrates or products via the β-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450_BM-3 monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from P. oleovorans, cytochrome P-450_BM-3 recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids.Cytochrome P-450_BM-3 monooxygenase (CytP450_BM-3) is a soluble NADPH-dependent monooxygenase from Bacillus megaterium ATCC 14581 (13). It is a class II P-450 enzyme that contains flavin adenine dinucleotide, flavin mononucleotide, and a heme moiety (17). Unlike most CytP450 monooxygenases, which consist of a distinct monooxygenase and a reductase, CytP450_BM-3 contains these functionalities in a single polypeptide (3, 15, 18).The enzyme hydroxylates a variety of long-chain aliphatic substrates, such as fatty acids, alkanols, and alkylamides at the ω-1, ω-2, and ω-3 positions (4, 17), and oxidizes unsaturated fatty acids to epoxides in vitro (17, 23) with high enantioselectivity. Oxidation of eicosapentenoic acid (C_20:5) and arachidonic acid (C_20:4) yielded 17(S),18(R)-epoxyeicosatetraenoic acid (94% enantiomeric excess [e.e.]) for the former and a mixture of 18-(R)-hydroxyarachidonic acid (92% e.e.) and 14(S),15(R)-epoxyeicosatrienoic acid at 98% e.e. for the latter substrate (8). Recently, it has been demonstrated that the enzyme also produces α,ω diacids from ω-oxo fatty acids by oxidation of the terminal aldehyde functionality (9). The catalytic constant (k_cat) of CytP450_BM-3 is among the highest found for P-450 monooxygenases, ranging from 15 s⁻¹ for laureate to 75 s⁻¹ for pentadecanoic acid (11). For comparison, a typical microsomal P-450 monooxygenase from human liver (CYP2J2) had a k_cat of 10⁻³ s⁻¹ for arachidonic acid (32), compared to a k_cat of 55 s⁻¹ for CytP450_BM-3 for the same substrate (8).This high catalytic efficiency prompted us to investigate the applicability of CytP450_BM-3 as a biocatalyst for the subterminal hydroxylation of long-chain fatty acids (LCFAs). Since these subterminal hydroxy LCFAs are chiral molecules, their application in the production of enantiopure synthetic building blocks, especially for pharmaceutical agents, could be envisioned. Further, long-chain hydroxy acids find applications as precursors for polymers or cyclic lactones, which are used as components of fragrances and as antibiotics. Although chemical syntheses have been developed for ω-1 hydroxy fatty acids (from C₁₂ to C₁₈) (26, 28, 29) and for ω-2 and ω-3 hydroxyoctadecanoic acids (2), they require expensive functionalized substrates and are in general complicated, multistep processes (26, 28, 29) which cannot be carried out with unmodified fatty acids as inexpensive starting material. In principle, such inexpensive substrates can be oxidized to hydroxy fatty acids by biocatalysts, either in vitro or in vivo. The latter is preferred, since whole cells actively regenerate the NADPH required for fatty acid oxidation with monooxygenases such as CytP450_BM-3. In designing a suitable whole-cell biocatalyst, several additional points had to be considered.First, uptake must be efficient. Second, degradation of substrate or product must be avoided. In fact, biotransformations of fatty acids with whole cells are usually inefficient due to limited uptake of these compounds at neutral pH, and when taken up, they are degraded via β-oxidation. The transport of LCFAs in Escherichia coli is mediated via the fadL and fadD gene products. FadL is the transporter that carries LCFAs across the outer membrane and is absolutely required for LCFA transport (20). FadD, the acyl coenzyme A (CoA) synthetase, is located at the inner side of the cytoplasmic membrane and is required for formation of the acyl coenzyme A thioester, after which the activated fatty acids are channeled into the β-oxidation cycle for fatty acid degradation (21, 22). Thus, we used a FadD mutant, E. coli K27, as a suitable host for the production of subterminal hydroxyalkanoic acids (20). E. coli K27 cannot couple free fatty acids to coenzyme A, thus preventing substrate or product degradation by the host. Such fadD mutants are, however, also impaired in efficient uptake of fatty acids (20). We circumvented this by introducing a fatty acid uptake system from Pseudomonas oleovorans encoded on pGEc47. Finally, we introduced the P-450_BM-3 monooxygenase on pCYP102 into the fadD mutant E. coli. The resulting recombinant, E. coli K27(pCYP102, pGEc47), is a promising tailored biocatalyst for the oxidation of saturated LCFAs to ω-1, ω-2, and ω-3 hydroxy fatty acids.     

The Microbial Community Structure of Drinking Water Biofilms Can Be Affected by Phosphorus Availability

Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 μg of phosphorus liter⁻¹ and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1ω7c and 18:1ω7c and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.     

Absolute configuration of ceriporic acids, the iron redox-silencing metabolites produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe³⁺. Ceriporic acids have an asymmetric centre at carbon-3, but absolute configuration has not been determined. We have isolated a series of ceriporic acids from the cultures of C. subvermispora, and measured their NMR spectra using a chiral shift reagent. In comparison with NMR spectra of (R)-(−)- and (S)-(+)-methylsuccinic acid and those of natural and chemically synthesized racemic mixtures of ceriporic acids, we have determined the absolute configuration of ceriporic acids as (R)-3-tetradecylitaconic acid (ceriporic acid A), (R)-3-hexadecylitaconic acid (ceriporic acid B) and (R,Z)-2-(hexadec-7-enyl)-3-itaconic acid (ceriporic acid C). We herein discuss their stereoselective biosynthetic pathway and the structural diversity of fungal secondary metabolites.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase

A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD⁺-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg^–1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg^–1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg^–1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 mol g^–1 (cell dry weight) min^–1. In the presence of formate, the intracellular [NADH]/[NAD⁺] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.     

Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C_18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C_14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.     

Mechanism and stereospecificity of alpha-hydroxylation of lignoceric acid in rat brain.

Cerebronic acid (2-hydroxytetracosanoic acid) is the major fatty acid component of cerebrosides and sulfatides in mammalian brain. Our previous communication demonstrated the synthesis of cerebronic acid from lignoceric acid (tetracosanoic acid) by a rat brain preparation in the presence of molecular oxygen and a reduced pyridine nucleotide (Hoshi, M., and Kishimoto, Y. (1973) J. Biol. Chem., 248, 4123–4130). The present'studies on the conversion of (RS)-[2-³H]-, (RS)-[3-³H]-, (R)-[2-³H]-, and (S)-[2-³H]lignoceric acids to cerebronic acid by rat brain preparations establish that the pro-R hydrogen at the α-carbon of lignoceric acid is replaced by a hydroxyl group with overall retention of configuration.     

Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-²H]18:2n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. α-Linolenic acid and 20:2n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.     

Stereo- and Regioselective Hydroxylation of α-Ionone by Streptomyces Strains

A total of 215 Streptomyces strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15 Streptomyces strains, S. fradiae Tü 27, S. arenae Tü 495, S. griseus ATCC 13273, S. violaceoniger Tü 38, and S. antibioticus Tü 4 and Tü 46 converted α-ionone to 3-hydroxy-α-ionone with significantly higher hydroxylation activity compared to that of β-ionone. Hydroxylation of racemic α-ionone [(6R)-(−)/(6S)-(+)] resulted in the exclusive formation of only the two enantiomers (3R,6R)- and (3S,6S)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the Streptomyces strains tested proceeds with both high regio- and stereoselectivity.     

Genetic loci associated with circulating levels of very long-chain saturated fatty acids

Very long-chain saturated fatty acids (VLSFAs) are saturated fatty acids with 20 or more carbons. In contrast to the more abundant saturated fatty acids, such as palmitic acid, there is growing evidence that circulating VLSFAs may have beneficial biological properties. Whether genetic factors influence circulating levels of VLSFAs is not known. We investigated the association of common genetic variation with plasma phospholipid/erythrocyte levels of three VLSFAs by performing genome-wide association studies in seven population-based cohorts comprising 10,129 subjects of European ancestry. We observed associations of circulating VLSFA concentrations with common variants in two genes, serine palmitoyl-transferase long-chain base subunit 3 (SPTLC3), a gene involved in the rate-limiting step of de novo sphingolipid synthesis, and ceramide synthase 4 (CERS4). The SPTLC3 variant at rs680379 was associated with higher arachidic acid (20:0 , P = 5.81 × 10⁻¹³). The CERS4 variant at rs2100944 was associated with higher levels of 20:0 (P = 2.65 × 10⁻⁴⁰) and in analyses that adjusted for 20:0, with lower levels of behenic acid (P = 4.22 × 10⁻²⁶) and lignoceric acid (P = 3.20 × 10⁻²¹). These novel associations suggest an inter-relationship of circulating VLSFAs and sphingolipid synthesis.     

Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters

Deuterated styrene ([²H₈]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [²H₈]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [²H₈]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.     

Purification and characterization of an (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum

An NADP⁺ —dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified. As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps. Its molecular mass was estimated to be 40–43 kDa. The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate. Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate. Except for the latter all substrates were converted enantioselectively to (S)-3-hydroxy acids in the presence of NADPH. 4-Chloro-3-oxobutyrate was reduced to the (R)-3-hydroxy acid. The specific activity of the enzyme was about 1400 mol min^–1 mg^–1 protein for the reduction of 3-oxobutyrate at pH 5.0. The Michaelis constant (K _m) values for 3-oxobutyrate, 3-oxovalerate and 3-oxocaproate were determined to be 0.22, 1.6 and 3.0 mM, respectively. The K _m values for dehydrogenation of (S)-3-hydroxybutyrate, (S)-3-hydroxyvalerate and (S)-3-hydroxycaproate were found to be 2.6, 1.1 and 5.2 mM, respectively. The identity of 43 of the first 45 N-terminal amino acid residues has been determined. So far such enzyme activities have been described in eucaryotes only.Dedicated to Prof. A. Trebst on the occasion of his 65th birthday     

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Cloning and Characterization of Three Fatty Alcohol Oxidase Genes from Candida tropicalis Strain ATCC 20336

Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of ω-hydroxy fatty acids to ω-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and K_m values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes ω-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not ω-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the K_m values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.     

PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi

Recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxy-alkanoate) consisting of medium-chain-length constituents (C₆ to C₁₄) (PHA_MCL), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylase (PhaG), has been identified in Pseudomonas putida (B. H. A. Rehm, N. Krüger, and A. Steinbüchel, J. Biol. Chem. 273:24044–24051, 1998). To establish this PHA-biosynthetic pathway in a non-PHA-accumulating bacterium, we functionally coexpressed phaC1 (encoding PHA synthase 1) from Pseudomonas aeruginosa and phaG (encoding the transacylase) from P. putida in Pseudomonas fragi. The recombinant strains of P. fragi were cultivated on gluconate as the sole carbon source, and PHA accumulation to about 14% of the total cellular dry weight was achieved. The respective polyester was isolated, and GPC analysis revealed a weight average molar mass of about 130,000 g mol⁻¹ and a polydispersity of 2.2. The PHA was composed mainly (60 mol%) of 3-hydroxydecanoate. These data strongly suggested that functional expression of phaC1 and phaG established a new pathway for PHA_MCL biosynthesis from nonrelated carbon sources in P. fragi. When fatty acids were used as the carbon source, no PHA accumulation was observed in PHA synthase-expressing P. fragi, whereas application of the β-oxidation inhibitor acrylic acid mediated PHA_MCL accumulation. The substrate for the PHA synthase PhaC1 is therefore presumably directly provided through the enzymatic activity of the transacylase PhaG by the conversion of (R)-3-hydroxydecanoyl-ACP to (R)-3-hydroxydecanoyl-CoA when the organism is cultivated on gluconate. Here we demonstrate for the first time the establishment of PHA_MCL synthesis from nonrelated carbon sources in a non-PHA-accumulating bacterium, employing fatty acid de novo biosynthesis and the enzymes PhaG (a transacylase) and PhaC1 (a PHA synthase).