Characterisation of recombinant human fatty aldehyde dehydrogenase: Implications for Sjögren-Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is an NAD⁺-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sjögren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. Microsomal FALDH was expressed in E. coli and purified. Using an in vitro activity assay an optimum pH of ～9.5 and temperature of ～35°C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal, tetradecanal, hexadecanal and octadecanal with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, at 25–30% the rate of aldehyde oxidation. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.     

Fatty aldehyde dehydrogenase: genomic structure,expression and mutation analysis in Sjögren–Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is a microsomal enzyme that catalyzes the oxidation of medium- and long-chain aliphatic aldehydes derived from metabolism of fatty alcohol, phytanic acid, ether glycerolipids and leukotriene B4. The FALDH gene (ALDH3A2) in man and mouse consists of 11 exons and is closely linked to the gene for ALDH3. In both species, alternative splicing results in formation of a second minor protein, FALDHv, that has a unique carboxy-terminal end. The functional significance of this alternate protein is not known. In humans, mutations in the FALDH gene cause Sjögren–Larsson syndrome (SLS), which is characterized by ichthyosis, mental retardation and spasticity. Missense mutations involving 24 amino acid positions in FALDH have been identified. These amino acids are more highly conserved among related class 3 aldehyde dehydrogenase enzymes than expected, suggesting that they are critically important for protein folding, catalysis or stability. Studies of mutations in SLS should prove useful for understanding structure–function correlations in FALDH and other aldehyde dehydrogenase proteins.     

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.     

A recombinant α-dioxygenase from rice to produce fatty aldehydes using <Emphasis Type="Italic">E. coli</Emphasis>

Fatty aldehydes are an important group of fragrance and flavor compounds that are found in different fruits and flowers. A biotechnological synthesis of fatty aldehydes based on Escherichia coli cells expressing an α-dioxygenase (αDOX) from Oryza sativa (rice) is presented. α-Dioxygenases are the initial enzymes of α-oxidation in plants and oxidize long and medium-chain C _n fatty acids to 2-hydroperoxy fatty acids. The latter are converted to C _n − 1 fatty aldehydes by spontaneous decarboxylation. Successful expression of αDOX in E. coli was proven by an in vitro luciferase assay. Using resting cells of this recombinant E. coli strain, conversion of different fatty acids to the respective fatty aldehydes shortened by one carbon atom was demonstrated. The usage of Triton X 100 improves the conversion rate up to 1 g aldehyde per liter per hour. Easy reuse of the cells was demonstrated by performing a second biotransformation without any loss of biocatalytic activity.     

O2 incorporation into a long-chain fatty acid during bacterial luminescence

The bioluminescence-dependent oxidation of a long-chain fatty aldehyde catalyzed by luciferase from Photobacterium phosphoreum has been studied in ¹⁸O₂ experiments. The results show the incorporation of one atom of molecular oxygen into the product, the corresponding fatty acid. This incorporation is not the result of exchange of ¹⁸O₂ with the aldehyde prior to oxidation to the acid, thereby indicating that the bacterial luciferase catalyzes an aldehyde monooxygenase reaction which is coupled with bioluminescence.     

A microsomal fatty acid synthetase coupled to acyl-CoA reductase in Euglena gracilis

Microsomal particles from dark-grown Euglena gracilis incorporated malonyl-CoA into fatty acids and fatty alcohols in the presence of acetyl-CoA, NADH, NADPH, and ATP with an optimum pH of 8.0. Schmidt degradation of the individual fatty acids derived from [l,3-¹⁴C]malonyl-CoA showed that the microsomal fatty acid synthesis was a de novo type. Detailed analysis of the products formed in the absence of various cofactors showed that the role of ATP was specifically in the formation of fatty alcohols and that fatty acid reduction specifically required NADH.The major aliphatic chains synthesized by the microsomes were C₁₆, C₁₈, and C₁₄ in both the acyl portions and alcohols. Although relative concentrations of acetyl-CoA and malonyl-CoA influenced the chain length distribution of products, C₁₆remained the major product in both the alcohol and the acid fractions. Effects of NADPH and NADH concentrations on malonyl-CoA incorporation suggested that the two reductive steps involved in the microsomal fatty acid synthesis have different pyridine nucleotide specificity. The apparent K_m for malonyl-CoA was 4.2 × 10^?4m. Based on the experimental results a mechanism is suggested by which carbon is channeled into wax esters under conditions of nutritional abundance in dark-grown E. gracilis.     

Characterisation of recombinant human fatty aldehyde dehydrogenase: implications for Sjögren-Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is an NAD+-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sj?gren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. Microsomal FALDH was expressed in E. coli and purified. Using an in vitro activity assay an optimum pH of approximately 9.5 and temperature of approximately 35 degrees C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal, tetradecanal, hexadecanal and octadecanal with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, at 25-30% the rate of aldehyde oxidation. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.     

Codon-Optimized NADH Oxidase Gene Expression and Gene Fusion with Glycerol Dehydrogenase for Bienzyme System with Cofactor Regeneration

NADH oxidases (NOXs) play an important role in maintaining balance of NAD⁺/NADH by catalyzing cofactors regeneration. The expression of nox gene from Lactobacillus brevis in Escherichia coli BL21 (BL21 (DE3)) was studied. Two strategies, the high AT-content in the region adjacent to the initiation codon and codon usage of the whole gene sequence consistent with the host, obtained the NOX activity of 59.9 U/mg and 73.3 U/mg (crude enzyme), with enhanced expression level of 2.0 and 2.5-folds, respectively. Purified NOX activity was 213.8 U/mg. Gene fusion of glycerol dehydrogenase (GDH) and NOX formed bifuctional multi-enzymes for bioconversion of glycerol coupled with coenzyme regeneration. Kinetic parameters of the GDH-NOX for each substrate, glycerol and NADH, were calculated as V _{max(Glycerol)} 20 μM/min, K _m(Glycerol) 19.4 mM, V _{max (NADH)} 12.5 μM/min and K _{m (NADH)} 51.3 μM, respectively, which indicated the potential application of GDH-NOX for quick glycerol analysis and dioxyacetone biosynthesis.     

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H₂O₂) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA ^D210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsA ^D210Y mutation into a wild-type strain resulted in lower H₂O₂ concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H₂O₂ concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H₂O₂ concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H₂O₂ concentrations. Among the ROS generated by fatty-acid β-oxidation, H₂O₂ critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.     

Cis-unsaturated fatty acids modulate the function of the cell-surface cAMP receptor of Dictyostelium discoideum

The binding of cAMP to the chemotactic cAMP receptor in intact Dictyostelium discoideum cells and isolated membranes is strongly inhibited by unsaturated fatty acids. In isolated membranes, cis-unsaturated fatty acids decreased the number of accessible cAMP binding sites, without significantly altering their affinity. Most potent were C₁₈ and C₂₀ cis-poly unsaturated fatty acids, like arachidonic acid, linoleic acid and linolenic acid. Trans-unsaturated fatty acid was less potent than its cis isomer, while saturated fatty acids did not affect the binding of cAMP to receptors at all. Oxidation reactions were not important for the effect of unsaturated fatty acids. When membranes were preincubated with millimolar concentrations of Ca²⁺, the effect of unsaturated fatty acids was strongly diminished. Mg²⁺ was ineffective. Ca²⁺, if presented after the incubation of membranes with unsaturated fatty acids, did not reverse the inhibitory effect. The specificity of the fatty acid effect, and the interference with Ca²⁺, but not Mg²⁺, suggest that the properties of the cAMP receptor are changed as a result of alterations in the lipid bilayer structure of the membrane.     

Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: Release of the products as free fatty acids

In marine bacteria and some thraustochytrids (marine stramenopiles) long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are produced de novo by PUFA synthases. These large, multi-domain enzymes carry out the multitude of individual reactions required for conversion of malonyl-CoA to the final LC-PUFA products. Here we report on the release of fatty acids from the PUFA synthase found in Schizochytrium, a thraustochytrid that has been developed as a commercial source for DHA-enriched biomass and oil. Data from in vitro activity assays indicate that the PUFAs are released from the enzyme as free fatty acids (FFAs). Addition of ATP and Mg²⁺ to in vitro assays facilitates appearance of radiolabel from ¹⁴C-malonyl-CoA in a triacylglycerol fraction, suggesting the involvement of acyl-CoA synthetases (ACS). Furthermore, addition of triascin C, an inhibitor of ACSs, to the assays blocks this conversion. When the Schizochytrium PUFA synthase is expressed in Escherichia coli, the products of the enzyme accumulate as FFAs, suggesting that the thioesterase activity required for fatty acid release is an integral part of the PUFA synthase.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and α-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n ? 6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n ? 3 fatty acids (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n ? 6 fatty acids (linoleic acid and arachidonic acid), the total n ? 3 fatty acyl content was reduced in all the phospholipids examined. In n ? 3 and n ? 6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n ? 9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appear to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca²⁺]_i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n ? 3 and n ? 6 PUFA but not in n ? 9 monounsaturated fatty acid modified cells. In Ca²⁺ free medium, OKT3-induced transient increase in [Ca²⁺]_i, representing Ca²⁺ release from the inositol 1,4,5-triphosphate-sensitive Ca²⁺ stores, were similar in control and UFA modified cells. Using Mn²⁺ entry as an index of plasma membrane Ca²⁺ permeability, the rate of fura-2 fluorescence quenching as a result of Mn²⁺ influx stimulated by OKT3 in n ? 9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n ? 3 and n ? 6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca²⁺ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and n ? 9 monounsaturated fatty acids appears to be important for the maintenance of a functional Ca²⁺ influx mechanism.     

Fatty aldehyde and fatty alcohol metabolism: Review and importance for epidermal structure and function

Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren–Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Oleaginous yeast Meyerozyma guilliermondii shows fermentative metabolism of sugars in the biosynthesis of ethanol and converts raw glycerol and cheese whey permeate into polyunsaturated fatty acids

We studied the biotechnological potential of the recently isolated yeast Meyerozyma guilliermondii BI281A to produce polyunsaturated fatty acids and ethanol, comparing products yields using glucose, raw glycerol from biodiesel synthesis, or whey permeate as substrates. The yeast metabolism was evaluated for different C/N ratios (100:1 and 50:1). Results found that M. guilliermondii BI281A was able to assimilate all tested substrates, and the most efficient conversion obtained was observed using raw glycerol as carbon source (C/N ratio 50:1), concerning biomass formation (5.67 g·L⁻¹) and lipid production (1.04 g·L⁻¹), representing 18% of dry cell weight. Bioreactors experiments under pH and aeration-controlled conditions were conducted. Obtained fatty acids were composed of ~67% of unsaturated fatty acids, distributed as palmitoleic acid (C_16:1, 9.4%), oleic acid (C_18:1, 47.2%), linoleic acid (C_{18:2 n−6}, 9.6%), and linolenic acid (C_{18:3 n−3}, 1.3%). Showing fermentative metabolism, which is unusual for oleaginous yeasts, M. guilliermondii produced 13.7 g·L⁻¹ of ethanol (yields of 0.27) when growing on glucose medium. These results suggest the promising use of this uncommonly studied yeast to produce unsaturated fatty acids and ethanol using cheap agro-industrial residues as substrates in bioprocess.     

Dual subcellular localization in the endoplasmic reticulum and peroxisomes and a vital role in protecting against oxidative stress of fatty aldehyde dehydrogenase are achieved by alternative splicing

Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.     

Ultrastructure,composition of neutral lipids and their fatty acids of Candida tropicalis strain D-2 mutants resistant to the polyene antibiotic nystatin

This report deals with data on the cell ultrastructure of Candida tropicals strain D-2 mutants resistant to the polyene antibiotic, nystatin, and with an analysis of the fractional composition of neutral lipids and their fatty acids. The ultrastructural organization of the mutant cells is characterized by thickening of the cell wall and formation of invaginations into the cytoplasm, the appearance of new formations, large vacuoles, and reduction of the system of mitochondrial cristae. Lipids of nys^r mutants differ from those of the nys^s variant in having a decreased content of steroids and some fractions of neutral lipids. Certain nys^r mutants manifest difference in the relative amounts of saturated and unsaturated fatty acids (C_16:0, C_16:1, C_18:0, C_18:1).     

Biodegradation of C5-C8 fatty acids and production of aroma volatiles by Myroides sp. ZB35 isolated from activated sludge

In the effluents of a biologically treated wastewater from a heavy oil-refining plant, C₅-C₈ fatty acids including pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, and 2-methylbutanoic acid are often detected. As these residual fatty acids can cause further air and water pollution, a new Myroides isolate ZB35 from activated sludge was explored to degrade these C₅-C₈ fatty acids in this study. It was found that the biodegradation process involved a lag phase that became prolonged with increasing acyl chain length when the fatty acids were individually fed to this strain. However, when fed as a mixture, the ones with longer acyl chains were found to become more quickly assimilated. The branched 2-methylbutanoic acid was always the last one to be depleted among the five fatty acids under both conditions. Metabolite analysis revealed one possible origin of short chain fatty acids in the biologically treated wastewater. Aroma volatiles including 2-methylbutyl isovalerate, isoamyl 2-methylbutanoate, isoamyl isovalerate, and 2-methylbutyl 2-methylbutanoate were subsequently identified from ZB35 extracts, linking the source of the fruity odor to these esters excreted by Myroides species. To our best knowledge, this is the first finding of these aroma esters in bacteria. From a biotechnological viewpoint, this study has revealed the potential of Myroides species as a promising source of aroma esters attractive for food and fragrance industries.     

Relationship between the pentose-phosphate pathway and the de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cultures

This study focuses on the activity of the pentose-phosphate pathway and its relationship to de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cell cultures derived from 1-week old rat brain. The proportion of glucose that was metabolized along the pentose-phosphate pathway was estimated by measuring ¹⁴CO₂ production from [1-¹⁴C]-, [2-¹⁴C]- and [6-¹⁴C]glucose, the utilization of glucose and the production of lactate. Incorporation of ¹⁴C from [¹⁴C]glucose and from [3-¹⁴C]acetoacetate into lipids was analysed. The pentose- phosphate pathway produced much more CO₂ from glucose than the Krebs cycle, although it accounted for only a small part of the consumption of glucose (< 3%). The higher ¹⁴CO₂ production from [2-¹⁴C]glucose than from [6-¹⁴C]glucose indicated that recycling of the products of the pentose-phosphate pathway takes place in these cells.Gradual inhibition of the pathway with increasing concentrations of 6-aminonicotinamide resulted in a parallel inhibition of the conversion of acetoacetate and of glucose into fatty acids and into cholesterol. Glycolysis was also strongly inhibited in the presence of 6-aminonicotinamide whereas the activity of the Krebs cycle was not affected.These results suggest that de novo synthesis of fatty acids and cholesterol by oligodendrocytes of neonatal rats is closely geared to the activity of the pentose-phosphate pathway in these cells.     

Expansion of the ω‐oxidation system AlkBGTL of Pseudomonas putida GPo1 with AlkJ and AlkH results in exclusive mono‐esterified dicarboxylic acid production in E. coli

The AlkBGTL proteins coded on the alk operon from Pseudomonas putida GPo1 can selectively ω‐oxidize ethyl esters of C6 to C10 fatty acids in whole‐cell conversions with Escherichia coli. The major product in these conversions is the ω‐alcohol. However, AlkB also has the capacity to overoxidize the substrate to the ω‐aldehyde and ω‐acid. In this study, we show that alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH are able to oxidize ω‐alcohols and ω‐aldehydes of esterified fatty acids respectively. Resting E. coli expressing AlkBGTHJL enabled exclusive mono‐ethyl azelate production from ethyl nonanoate, with an initial specific activity of 61 U g_cdw^?1. Within 2 h, this strain produced 3.53 mM mono‐ethyl azelate, with a yield of 0.68 mol mol^?1. This strain also produced mono‐ethyl dicarboxylic acids from ethyl esters of C6 to C10 fatty acids and mono‐methyl azelate from methyl nonanoate. Adding ethyl nonanoate dissolved in carrier solvent bis‐(2‐ethylhexyl) phthalate enabled an increase in product titres to 15.55 mM in two‐liquid phase conversions. These findings indicate that E. coli expressing AlkBGTHJL is an effective producer of mono‐esterified dicarboxylic acids from fatty acid esters.