An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor

Apple flavor is characterized by combinations of ester compounds, which increase markedly during fruit ripening. The final step in ester biosynthesis is catalyzed by alcohol acyl transferases (AATs) that use coenzyme A (CoA) donors together with alcohol acceptors as substrates. The gene MpAAT1, which produces a predicted protein containing features of other plant acyl transferases, was isolated from Malus pumila (cv. Royal Gala). The MpAAT1 gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme can utilize a range of alcohol substrates from short to medium straight chain (C3-C10), branched chain, aromatic and terpene alcohols. The enzyme can also utilize a range of short to medium chain CoAs. The binding of the alcohol substrate is rate limiting compared with the binding of the CoA substrate. Among different alcohol substrates there is more variation in turnover compared with K(m) values. MpAAT1 is capable of producing many esters found in Royal Gala fruit, including hexyl esters, butyl acetate and 2-methylbutyl acetate. Of these, MpAAT1 prefers to produce the hexyl esters of C3, C6 and C8 CoAs. For the acetate esters, however, MpAAT1 preference depends upon substrate concentration. At low concentrations of alcohol substrate the enzyme prefers utilizing the 2-methylbutanol over hexanol and butanol, while at high concentrations of substrate hexanol can be used at a greater rate than 2-methylbutanol and butanol. Such kinetic characteristics of AATs may therefore be another important factor in understanding how the distinct flavor profiles of different fruit are produced during ripening.     

Engineering promiscuity of chloramphenicol acetyltransferase for microbial designer ester biosynthesis

Robust and efficient enzymes are essential modules for metabolic engineering and synthetic biology strategies across biological systems to engineer whole-cell biocatalysts. By condensing an acyl-CoA and an alcohol, alcohol acyltransferases (AATs) can serve as interchangeable metabolic modules for microbial biosynthesis of a diverse class of ester molecules with broad applications as flavors, fragrances, solvents, and drop-in biofuels. However, the current lack of robust and efficient AATs significantly limits their compatibility with heterologous precursor pathways and microbial hosts. Through bioprospecting and rational protein engineering, we identified and engineered promiscuity of chloramphenicol acetyltransferases (CATs) from mesophilic prokaryotes to function as robust and efficient AATs compatible with at least 21 alcohol and 8 acyl-CoA substrates for microbial biosynthesis of linear, branched, saturated, unsaturated and/or aromatic esters. By plugging the best engineered CAT (CATec3 Y20F) into the gram-negative mesophilic bacterium Escherichia coli, we demonstrated that the recombinant strain could effectively convert various alcohols into desirable esters, for instance, achieving a titer of 13.9 g/L isoamyl acetate with 95% conversion by fed-batch fermentation. The recombinant E. coli was also capable of simulating the ester profile of roses with high conversion (>97%) and titer (>1 g/L) from fermentable sugars at 37 °C. Likewise, a recombinant gram-positive, cellulolytic, thermophilic bacterium Clostridium thermocellum harboring CATec3 Y20F could produce many of these esters from recalcitrant cellulosic biomass at elevated temperatures (>50 °C) due to the engineered enzyme's remarkable thermostability. Overall, the engineered CATs can serve as a robust and efficient platform for designer ester biosynthesis from renewable and sustainable feedstocks.     

Functional characterization of enzymes forming volatile esters from strawberry and banana

Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alcohol acyltransferases (AATs), which link alcohols to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene from the cultivated strawberry (Fragaria x ananassa). The potential role of these enzymes in fruit flavor formation was assessed. To this end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alcohol and acyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic analysis of the various members of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequence similarity. In addition, the substrate preference of recombinant enzymes was not necessarily reflected in the representation of esters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significant extent determined by the supply of precursors. To study the in planta activity of an alcohol acyltransferase and to assess the potential for metabolic engineering of ester production, we generated transgenic petunia (Petunia hybrida) plants overexpressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detected in transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alcohol to explants of transgenic lines resulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alcohol substrates is an important parameter to consider when engineering volatile ester formation in plants.     

Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.     

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

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

Microbial formation of esters

Small aliphatic esters are important natural flavor and fragrance compounds and have numerous uses as solvents and as chemical intermediates. Besides the chemical or lipase-catalyzed formation of esters from alcohols and organic acids, small volatile esters are made by several biochemical routes in microbes. This short review will cover the biosynthesis of esters from acyl-CoA and alcohol condensation, from oxidation of hemiacetals formed from aldehydes and alcohols, and from the insertion of oxygen adjacent to the carbonyl group in a straight chain or cyclic ketone by Baeyer–Villiger monooxygenases. The physiological role of the ester-forming reactions can allow degradation of ketones for use as a carbon source and may play a role in detoxification of aldehydes or recycling cofactors. The enzymes catalyzing each of these processes have been isolated and characterized, and a number of genes encoding the proteins from various microbes have been cloned and functionally expressed. The use of these ester-forming organisms or recombinant organisms expressing the appropriate genes as biocatalysts in biotechnology to make specific esters and chiral lactones has been studied in recent years.     

Modulation of Triglyceride and Cholesterol Ester Synthesis Impairs Assembly of Infectious Hepatitis C Virus

In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.     

Physiological characterization of lipid accumulation and in vivo ester formation in Gordonia sp. KTR9

Previous work has demonstrated the feasibility of in vivo biodiesel synthesis in Escherichia coli, however, ethyl ester formation was dependent on an external fatty acid feedstock. In contrast to E. coli, actinomycetes may be ideal organisms for direct biodiesel synthesis because of their capacity to synthesize high levels of triacylglcerides (TAGs). In this study, we investigated the physiology and associated TAG accumulation along with the in vivo ability to catalyze ester formation from exogenous short chain alcohol sources in Gordonia sp. KTR9, a strain that possesses a large number of genes dedicated to fatty acid and lipid biosynthesis. Total lipid fatty acids content increased by 75 % and TAG content increased by 50 % under nitrogen starvation conditions in strain KTR9. Strain KTR9 tolerated the exogenous addition of up to 4 % methanol, 4 % ethanol and 2 % propanol in the media. Increasing alcohol concentrations resulted in a decrease in the degree of saturation of recovered fatty acid alcohol esters and a slight increase in the fatty acid chain length. A linear dose dependency in fatty alcohol ester synthesis was observed in the presence of 0.5–2 % methanol and ethanol compared to control KTR9 strains grown in the absence of alcohols. An inspection of the KTR9 genome revealed the presence of several putative wax ester synthase/acyl-coenzyme A?:?diacylglycerol acyltransferase (WS/DGAT) enzymes, encoded by atf gene homologs, that may catalyze the in vivo synthesis of fatty acid esters from short chain alcohols. Collectively, these results indicate that Gordonia sp. KTR9 may be a suitable actinomycete host strain for in vivo biodiesel synthesis.     

Parameters Affecting Ethyl Ester Production by Saccharomyces cerevisiae during Fermentation

Volatile esters are responsible for the fruity character of fermented beverages and thus constitute a vital group of aromatic compounds in beer and wine. Many fermentation parameters are known to affect volatile ester production. In order to obtain insight into the production of ethyl esters during fermentation, we investigated the influence of several fermentation variables. A higher level of unsaturated fatty acids in the fermentation medium resulted in a general decrease in ethyl ester production. On the other hand, a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, while a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that provision of medium-chain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes a strong increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability has an important role in ethyl ester production. We concluded that, at least in our fermentation conditions and with our yeast strain, the fatty acid precursor level rather than the activity of the biosynthetic enzymes is the major limiting factor for ethyl ester production. The expression level and activity of the fatty acid biosynthetic enzymes therefore appear to be prime targets for flavor modification by alteration of process parameters or through strain selection.     

The fruit ripening-related gene FaAAT2 encodes an acyl transferase involved in strawberry aroma biogenesis

Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour.     

Structural and Affinity Determinants in the Interaction between Alcohol Acyltransferase from F. x ananassa and Several Alcohol Substrates: A Computational Study

Aroma and flavor are important factors of fruit quality and consumer preference. The specific pattern of aroma is generated during ripening by the accumulation of volatiles compounds, which are mainly esters. Alcohol acyltransferase (AAT) (EC 2.3.1.84) catalyzes the esterification reaction of aliphatic and aromatic alcohols and acyl-CoA into esters in fruits and flowers. In Fragaria x ananassa, there are different volatiles compounds that are obtained from different alcohol precursors, where octanol and hexanol are the most abundant during fruit ripening. At present, there is not structural evidence about the mechanism used by the AAT to synthesize esters. Experimental data attribute the kinetic role of this enzyme to 2 amino acidic residues in a highly conserved motif (HXXXD) that is located in the middle of the protein. With the aim to understand the molecular and energetic aspects of volatiles compound production from F. x ananassa, we first studied the binding modes of a series of alcohols, and also different acyl-CoA substrates, in a molecular model of alcohol acyltransferase from Fragaria x ananassa (SAAT) using molecular docking. Afterwards, the dynamical behavior of both substrates, docked within the SAAT binding site, was studied using routine molecular dynamics (MD) simulations. In addition, in order to correlate the experimental and theoretical data obtained in our laboratories, binding free energy calculations were performed; which previous results suggested that octanol, followed by hexanol, presented the best affinity for SAAT. Finally, and concerning the SAAT molecular reaction mechanism, it is suggested from molecular dynamics simulations that the reaction mechanism may proceed through the formation of a ternary complex, in where the Histidine residue at the HXXXD motif deprotonates the alcohol substrates. Then, a nucleophilic attack occurs from alcohol charged oxygen atom to the carbon atom at carbonyl group of the acyl CoA. This mechanism is in agreement with previous results, obtained in our group, in alcohol acyltransferase from Vasconcellea pubescens (VpAAT1).     

蜡酯合成途径及关键酶的研究进展

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

Rapid ester biosynthesis screening reveals a high activity alcohol‐O‐acyltransferase (AATase) from tomato fruit

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl‐CoA with an alcohol by alcohol‐O‐acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short‐ and medium‐chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf‐S.l). Atf1‐S.l exhibited broad specificity towards acyl‐CoAs with chain length from C₄ to C₁₀ and was specific towards 1‐pentanol. The AATase screen also revealed new acyl‐CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf‐C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester‐based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.     

Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase

Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.     

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

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

Alcohol acyl transferase 1 links two distinct volatile pathways that produce esters and phenylpropenes in apple fruit

Fruit accumulate a diverse set of volatiles including esters and phenylpropenes. Volatile esters are synthesised via fatty acid degradation or from amino acid precursors, with the final step being catalysed by alcohol acyl transferases (AATs). Phenylpropenes are produced as a side branch of the general phenylpropanoid pathway. Major quantitative trait loci (QTLs) on apple (Malus × domestica) linkage group (LG)2 for production of the phenylpropene estragole and volatile esters (including 2‐methylbutyl acetate and hexyl acetate) both co‐located with the MdAAT1 gene. MdAAT1 has previously been shown to be required for volatile ester production in apple (Plant J., 2014, https://doi.org/10.1111/tpj.12518 ), and here we show it is also required to produce p‐hydroxycinnamyl acetates that serve as substrates for a bifunctional chavicol/eugenol synthase (MdoPhR5) in ripe apple fruit. Fruit from transgenic ‘Royal Gala’ MdAAT1 knockdown lines produced significantly reduced phenylpropene levels, whilst manipulation of the phenylpropanoid pathway using MdCHS (chalcone synthase) knockout and MdMYB10 over‐expression lines increased phenylpropene production. Transient expression of MdAAT1, MdoPhR5 and MdoOMT1 (O‐methyltransferase) genes reconstituted the apple pathway to estragole production in tobacco. AATs from ripe strawberry (SAAT1) and tomato (SlAAT1) fruit can also utilise p‐coumaryl and coniferyl alcohols, indicating that ripening‐related AATs are likely to link volatile ester and phenylpropene production in many different fruit.     

Generation of Phenylpropanoid Pathway-Derived Volatiles in Transgenic Plants: Rose Alcohol Acetyltransferase Produces Phenylethyl Acetate and Benzyl Acetate in Petunia Flowers

Esters are important contributors to the aroma of numerous flowers and fruits. Acetate esters such as geranyl acetate, phenylethyl acetate and benzyl acetate are generated as a result of the action of alcohol acetyltransferases (AATs). Numerous homologous AATs from various plants have been characterized using in-vitro assays. To study the function of rose alcohol acetyltransferase (RhAAT) in planta, we generated transgenic petunia plants expressing the rose gene under the control of a CaMV-35S promoter. Although the preferred substrate of RhAAT in vitro is geraniol, in transgenic petunia flowers, it used phenylethyl alcohol and benzyl alcohol to produce the corresponding acetate esters, not generated by control flowers. The level of benzyl alcohol emitted by the flowers of different transgenic lines was ca. three times higher than that of phenylethyl alcohol, which corresponded to the ratio between the respective products, i.e. ca. three times more benzyl acetate than phenylethyl acetate. Feeding of transgenic petunia tissues with geraniol or octanol led to the production of their respective acetates, suggesting the dependence of volatile production on substrate availability.     

Inhibition of hepatic gluconeogenesis and lipogenesis by benzoic acid,p-tert.-butylbenzoic acid,and a structurally related hypolipidemic agent SC-33459

Benzoic acid, p-tert.-butylbenzoic acid, and a structurally related hypolipidemic agent SC-33459 were found to inhibit glucose synthesis by hepatocytes isolated from 48-h fasted rats as well as fatty acid synthesis by hepatocytes isolated from meal-fed rats. Glucose synthesis was less sensitive than fatty acid synthesis. Benzoic acid was the least effective inhibitor of both processes; SC-33459 and p-tert.-butylbenzoic acid were very potent inhibitors with similar efficacy. Glycine prevented the inhibition of fatty acid synthesis caused by benzoic acid, but had no effect on that caused by p-tert.-butylbenzoic acid. Octanoate opposed the inhibitory effects of both benzoic acid and p-tert.-butylbenzoic acid. Oxidation of [1-¹⁴C]oleate to ketone bodies and acid-soluble radioactive products was inhibited by both p-tert.-butylbenzoic acid and SC-33459. Preincubation of hepatocytes with SC-33459 was required for the latter effect, suggesting catabolism of this compound may be involved. SC-33459 is a p-tert.-butylphenyl derivative which should be readily converted to p-tert.-butylbenzoic acid by β oxidation. Both p-tert.-butylbenzoic acid and SC-33459 decreased citrate levels dramatically. All three compounds reduced CoA and acetyl-CoA levels and increased medium-chain acyl-CoA ester levels. p-tert.-Butylbenzoic acid and SC-33459 also increased long-chain acyl-CoA ester levels. The increase in medium-chain acyl-CoA levels presumably reflects benzoyl-CoA formation from benzoic acid and p-tert.-butylbenzoyl-CoA formation from p-tert.-butylbenzoic acid and SC-33459. Inhibition of glucose and fatty acid synthesis by these compounds may be due to effects on specific enzymes or to CoA sequestration.     

Disappearance of short chain acids from the preen gland wax of male mallard ducks during eclipse

Wax esters constitute the major products of the uropygial glands of male mallard ducks (Anas platyrhynchos). Combined gas-liquid chromatography and mass spectrometry of the acyl and alcohol portions of the wax ester revealed dramatic seasonal variation. Of the total of nearly 100 compounds detected in the acyl portion, more than 90 were identified as n-, monomethyl-, dimethyl-, and trimethyl-C6 to C20 acids. The major components were 2-methyl- and 4-methyl C6 acids which constituted nearly 75% of the total acids during most of the year. As the birds went through the post-nuptial molt into eclipse, these short chain acids decreased to only 4% of the total acids. Fatty acids longer than C12 became the major components during the summer months when the birds were in eclipse and by fall the characteristic shorter branched acids reappeared as the dominant components. Fatty alcohols did not undergo such dramatic changes. This is the first report of biochemical changes that occur during eclipse.