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Wax esters play critical roles in biological systems, serving functions from energy storage to chemical signaling. Their diversity is attributed to variations in alcohol and acyl chains, including their length, branching, and the stereochemistry of double bonds. Traditional analysis by mass spectrometry with collisional activations (CID, HCD) offers insights into acyl chain lengths and unsaturation level. Still, it falls short in pinpointing more nuanced structural features like the position of double bonds. As a solution, this study explores the application of 213-nm ultraviolet photodissociation (UVPD) for the detailed structural analysis of wax esters. It is shown that lithium adducts provide unique fragments as a result of Norrish and Norrish-Yang reactions at the ester moieties and photoinduced cleavages of double bonds. The product ions are useful for determining chain lengths and localizing double bonds. UVPD spectra of various wax esters are presented systematically, and the effect of activation time is discussed. The applicability of tandem mass spectrometry with UVPD is demonstrated for wax esters from natural sources. The UHPLC analysis of jojoba oil proves the compatibility of MS2 UVPD with the chromatography time scale, and a direct infusion is used to analyze wax esters from vernix caseosa. Data shows the potential of UVPD and its combination with CID or HCD in advancing our understanding of wax ester structures. [ABSTRACT FROM AUTHOR]

Jojoba (Simmondsia chinensis Schneider), the sole species in the Simmondsiaceae family, holds significant economic value. Understanding the mechanism of seed germination and post-germination is essential for jojoba breeding programs. For the first time, this study employed matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to comprehensively detect and image endogenous lipids in jojoba seeds during germination and post-germination. Meanwhile, a new method of lithium salt-doped 2-mercaptobenzothiazole (2-MBT) matrix was adopted to detect the wax ester in jojoba seeds. The spatial lipidomic analysis of MALDI-MS revealed the dynamics of lipid alterations during three stages of jojoba seed (stage I, quiescent seed; stage II, germination; stage III, post-germination). MALDI-MSI results demonstrated heterogeneous distributions of differential lipids and main wax esters (WEs) in unique tissue regions of the jojoba seed at different stages. For instance, at stages II and III, three phosphatidylcholines (PCs) (PC(36:2), PC(36:4), and PC(35:3)) are mainly localized in the cotyledons, while the other two PCs (PC(34:1) and PC(36:1)) were distributed in both cotyledons and embryonic axis. In contrast, three phosphatidylethanolamines (PE(36:1), PE(42:4), and PE(42:9)) are primarily distributed near the embryonic axis and phosphatidylserine (PS(32:0)) was found exclusively in the embryonic axis area at three stages. In addition, three phosphatidic acids (PA(40:7), PA(46:2), and PA(42:1)) displayed highly accumulated in the middle of the cotyledons at stages II and III. Besides, WEs are continuously degraded mainly in the cotyledons during germination and post-germination processes. This study offers new insights into lipid alterations from a spatial lipidomics perspective and can contribute to our understanding of the role of these specific lipids during jojoba seed germination and post-germination. These findings will deepen our understanding of the metabolic processes involved in jojoba seed germination and seedling development and have underlying implications for crop improvement and seed quality control. [Display omitted] • MALDI-MS imaging of differentially expressed lipids and wax esters in dormant and germinated jojoba seeds for the first time. • Lithium salt-doped 2-mercaptobenzothiazole matrix was developed for the MALDI imaging of wax esters in jojoba seeds. • MS based spatial-omics enables us to better understand the mechanism of jojoba seed germination. [ABSTRACT FROM AUTHOR]

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Current research on wax‐based oleogels indicates wax esters to be the key component in many natural waxes. This necessitates understanding the properties of pure wax esters to unravel the gelling mechanism in wax‐based oleogels. Therefore, available literature data on pure wax esters is summarized and critically reviewed. The detailed analysis of the pre‐existing data on crystallographic (SAXS) and thermal properties, facilitates the interpretation of subsequently performed experiments: Specific wax esters with different carbon numbers and symmetries were studied as such and in oleogels formed in combination with medium chained triglyceride oil at inclusion levels of 10% (w/w). They were characterized regarding their thermal (differential scanning calorimetry [DSC]) and viscoelastic (oscillatory rheology) behavior. It is found that all observations concerning pure wax esters behave systematically, linking molecular makeup, crystal structure, and behavior. The experimental study of oleogels structured by four different binary mixtures of wax esters revealed that substantial chain length differences induce separate crystallization (CN30 + 36 and CN30 + 42). Mixtures of wax esters with only limited chain length difference (≤ 2 carbon atoms) reconfirmed earlier speculations on mixing behavior and crystal structure. Applying mixtures of wax esters only differing in their position of the ester bond (CN36 [14_22] + CN36 [22_14]) indicated ideal mixing behavior in the solid phase of the gels. Surprisingly, the data revealed that additional thermal events occur at specific mixing ratios, predominantly at 1:1 (w/w). Their supposed relation to compound formation certainly needs further confirmation. Rheological analysis confirmed that sequential crystallization results in highest firmness values for the systems studied. [ABSTRACT FROM AUTHOR]

Abstract Background Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings, and cosmetics. They can be produced chemically from fossil fuel or plant-derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, tightly controlled and tailor-made WE synthesis in plants would be a sustainable, beneficial, and valuable commodity. Here, we report the expression of ten combinations of WE producing transgenes in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and composition in the transgenic plants. Results The transgenes consisted of different combinations of a FATTY ACYL-COA/ACP REDUCTASE (FAR) and two WAX SYNTHASES/ACYL-COA:DIACYLGLYCEROL O-ACYLTRANSFERASES (WSD), namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access distinct alcohol and acyl substrate pools within A. thaliana cells. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis. Conclusions A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE that are mainly derived from mid- and long-chain saturated and monounsaturated substrates.

Persister cells constitute a bacterial subpopulation able to survive to high concentrations of antibiotics. This phenotype is temporary and reversible, and thus could be involved in the recurrence of infections and emergence of antibiotic resistance. To better understand how persister cells survive to such high antibiotic concentration, we examined changes in their lipid composition. We thus compared the lipidome of Acinetobacter baumannii ATCC 19606T persister cells formed under ciprofloxacin treatment with the lipidome of control cells grown without antibiotic. Using matrix assisted laser desorption ionisation-Fourier transform ion cyclotron resonance mass spectrometry, we observed a higher abundance of short chains and secondary chains without hydroxylation for lipid A in persister cells. Using liquid chromatography-tandem mass spectrometry, we found that persister cells produced particular phosphatidylglycerols, as LPAGPE and PAGPE, but also lipids with particular acyl chains containing additional hydroxyl group or uncommon di-unsaturation on C18 and C16 acyl chains. In order to determine the impact of these multiple lipidome modifications on membrane fluidity, fluorescence anisotropy assays were performed. They showed an increase of rigidity for the membrane of persister cells, inducing likely a decrease membrane permeability to protect cells during dormancy. Finally, we highlighted that A. baumannii persister cells also produced particular wax esters, composed of two fatty acids and a fatty diol. These uncommon storage lipids are key metabolites allowing a rapid bacterial regrow when antibiotic pressure disappears. These overall changes in persister lipidome may constitute new therapeutic targets to combat these particular dormant cells. [Display omitted] • PAGPE, LPAGPE as discriminant lipids in persister cells • Presence of di-unsaturated and hydroxyl fatty acyl chains in lipids of persister cells • Decrease of membrane fluidity of persister cells • Special WE presenting one diol with two acyl chains in persister cells [ABSTRACT FROM AUTHOR]

Meibomian glands (MGs) and their holocrine secretion—meibum—play crucial roles in the physiology of the eye, providing protection from environmental factors and desiccation, among other functions. Importantly, aging was implicated in the deterioration of the morphology and functions of MGs, and the quantity and quality of meibum they produce, leading to a loss of its protective properties, while the meibum of young individuals and experimental animals provide ample protection to the eye. Currently, the molecular mechanisms of meibum biosynthesis (termed meibogenesis) are not fully understood. To characterize the physiological changes in developing and maturing MGs, we studied the lipidomes and transcriptomes of mouse MGs ranging from newborns to adults. The results revealed a gradual increase in the critical genes of meibogenesis (such as Elovl3, Elovl4, Awat2, and Soat1, among others) that positively correlated with the biosynthesis of their respective lipid products. The MG transcriptomes of young and adult mice were also analyzed using single-cell RNA sequencing. These experiments revealed the existence of multiple unique populations of MG cells (meibocytes, epithelial cells, and others) with specific combinations of genes that encode meibogenesis-related proteins, and identified clusters and subclusters of cells that were tentatively classified as meibocytes at different stages of differentiation/maturation, or their progenitor cells. A hypothesis was formulated that these cells may produce different types of lipids, and contribute differentially to the Meibomian lipidome. [ABSTRACT FROM AUTHOR]

Odontocetes possess specialized fat bodies in and around the mandibles for sound reception which have complex topographical distributions of unique endogenous lipids (triacylglycerols and wax esters [WE]). Although there is diversity across species in the fatty acid (FA) and fatty alcohol (FAlc) components of WE, little is understood about the composition and placement of the intact molecules, which will likely impact acoustic function. We aimed to determine the composition and distribution of intact waxes in the jaw fats from five species representative of three odontocete families: delphinids, kogiids, and ziphiids. Total lipid content was similar in all groups, but the WE content of that lipid (21.3%–53.3% of total lipid) and the identity of intact WE molecules showed a high degree of variation, especially in the short‐chain fatty acid components. In contrast, the FAlc elements were surprisingly well conserved. There were 26 intact WE that were common to all species but the delphinids had 12 additional WE with short‐chain fatty acids (i‐5:0 specifically) not found in the other animals examined here. Our study suggests that this highly specialized tissue has evolved several different biochemical pathways, and that there may be multiple strategies for building acoustic fat bodies. [ABSTRACT FROM AUTHOR]

In this work, the synthesis of oleyl oleate wax ester using Brønsted acidic ionic liquid catalysts was carried out. Confirmation of oleyl oleate molecular structure has been performed using FTIR, NMR, and ESI-MS spectroscopies. The ability of ionic liquid catalysts for catalyzing the esterification reaction of oleic acid and oleyl alcohol to produce oleyl oleate was optimized. The ionic liquid catalyst ([NMP][CH3SO3]) was found to be the best catalyst for the esterification reaction of oleic acid and oleyl alcohol compared with the other acidic ionic liquids studied. The optimal reaction conditions were determined at a reaction time of 8 h; oleic acid to oleyl alcohol mole ratio of 1:1; ([NMP][CH3SO3]) with 9.9 wt.%; and reaction temperature of 90 °C. Under these conditions, the percentage yield of oleyl oleate wax ester was 86%.

The factors affecting lipid and fatty acid composition of copepod Calanus sinicus in the Yellow Sea (YS) and the East China Sea (ECS) were examined in this study. In spring, there were significant differences between these two regions for both environmental conditions and food availability. Such regional difference significantly influenced the lipid and fatty acid profiles of C. sinicus. Our results show that C. sinicus has a higher lipids content in ECS, especially for wax ester and triglyceride lipids, indicating a more active and efficient predation. According to BIO-ENV analysis, the variation of lipids profiles may be influenced majorly by water temperature. Moreover, the fatty acids (FAs) profiles of C. sinicus were also different between YS and ECS, especially in the four major contributors, C22:1ω11, eicosapentaenoic acid (EPA), docosahexenoic acid (DHA), and C20:1ω9. The considerable amounts of self-biosynthesized FAs of herbivorous copepod (C22:1ω11 and C20:1ω9) and low DHA/EPA ratio may indicate that C. sinicus in ECS feed mainly on phytoplankton comparing to those in YS. The fatty acid profiles of C. sinicus were affected by the differences in food availability. [ABSTRACT FROM AUTHOR]

Background: Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings, and cosmetics. They can be produced chemically from fossil fuel or plant-derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, tightly controlled and tailor-made WE synthesis in plants would be a sustainable, beneficial, and valuable commodity. Here, we report the expression of ten combinations of WE producing transgenes in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and composition in the transgenic plants. Results: The transgenes consisted of different combinations of a FATTY ACYL-COA/ACP REDUCTASE (FAR) and two WAX SYNTHASES/ACYL-COA:DIACYLGLYCEROL O-ACYLTRANSFERASES (WSD), namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access distinct alcohol and acyl substrate pools within A. thaliana cells. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis. Conclusions: A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE that are mainly derived from mid- and long-chain saturated and monounsaturated substrates. [ABSTRACT FROM AUTHOR]

Abstract Background Global demand for energy is on the rise at a time when limited natural resources are fast depleting. To address this issue, microalgal biofuels are being recommended as a renewable and eco-friendly substitute for fossil fuels. Euglena gracilis is one such candidate that has received special interest due to their ability to synthesize wax esters that serve as precursors for production of drop-in jet fuel. However, to realize economic viability and achieve industrial-scale production, development of novel methods to characterize algal cells, evaluate its culture conditions, and construct appropriate genetically modified strains is necessary. Here, we report a Raman microspectroscopy-based method to visualize important metabolites such as paramylon and ester during wax ester fermentation in single Euglena gracilis cells in a label-free manner. Results We measured Raman spectra to obtain intracellular biomolecular information in Euglena under anaerobic condition. First, by univariate approach, we identified Raman markers corresponding to paramylon/esters and constructed their time-lapse chemical images. However, univariate analysis is severely limited in its ability to obtain detailed information as several molecules can contribute to a Raman band. Therefore, we further employed multivariate curve resolution analysis to obtain chain length-specific information and their abundance images of the produced esters. Accumulated esters in Euglena were particularly identified to be myristyl myristate (C28), a wax ester candidate suitable to prepare drop-in jet fuel. Interestingly, we found accumulation of two different forms of myristyl myristate for the first time in Euglena through our exploratory multivariate analysis. Conclusions We succeeded in visualizing molecular-specific information in Euglena during wax ester fermentation by Raman microspectroscopy. It is obvious from our results that simple univariate approach is insufficient and that multivariate curve resolution analysis is crucial to extract hidden information from Raman spectra. Even though we have not measured any mutants in this study, our approach is directly applicable to other systems and is expected to deepen the knowledge on lipid metabolism in microalgae, which eventually leads to new strategies that will help to enhance biofuel production efficiency in the future.

Wax synthase (WS) catalyzes the last step in wax ester biosynthesis in green plants. Two unrelated sub-families of WS, including the bifunctional acyltransferase and plant-like WS have been reported, but the latter is largely uncharacterized in microalgae. Here, we functionally characterized a putative plant-like WS (CzWS1) from the emerging model green microalga Chromochloris zofingiensis. Our results showed that plant-like WS evolved under different selection constraints in plants and microalgae, with positive selection likely contributing to functional divergence. Unlike jojoba with high amounts of wax ester in seeds and a highly active WS enzyme, C. zofingiensis has no detectable wax ester but a high abundance of WS transcripts. Co-expression analysis showed that C. zofingiensis WS has different expression correlation with lipid biosynthetic genes from jojoba, and may have a divergent function. In vitro characterization indicated that CzWS1 had diacylglycerol acyltransferase activity along with WS activity, and overexpression of CzWS1 in yeast and Chlamydomonas reinhardtii affected triacylglycerol accumulation. Moreover, biochemical and bioinformatic analyses revealed the relevance of the C-terminal region of CzWS1 in enzyme function. Taken together, our results indicated a functional divergence of plant-like WS in plants and microalgae, and the importance of its C-terminal region in specialization of enzyme function. [ABSTRACT FROM AUTHOR]

Microbial storage compounds, such as wax esters (WE), are potential high‐value lipids for the production of specialty chemicals and medicines. Their synthesis, however, is strictly regulated and competes with cell growth, which leads to trade‐offs between biomass and product formation. Here, we use metabolic engineering and synergistic substrate cofeeding to partition the metabolism of Acinetobacter baylyi ADP1 into two distinct modules, each dedicated to cell growth and WE biosynthesis, respectively. We first blocked the glyoxylate shunt and upregulated the WE synthesis pathway to direct the acetate substrate exclusively for WE synthesis, then we controlled the supply of gluconate so it could be used exclusively for cell growth and maintenance. We show that the two modules are functionally independent from each other, allowing efficient lipid accumulation while maintaining active cell growth. Our strategy resulted in 7.2‐ and 4.2‐fold improvements in WE content and productivity, respectively, and the product titer was enhanced by 8.3‐fold over the wild type strain. Notably, during a 24‐h cultivation, a yield of 18% C‐WE/C‐total‐substrates was achieved, being the highest reported for WE biosynthesis. This study provides a simple, yet powerful, means of controlling cellular operations and overcoming some of the fundamental challenges in microbial storage lipid production. [ABSTRACT FROM AUTHOR]

In the present work, a new, highly efficient and simple strategy has been developed for the synthesis of long chain esters from fatty acids and fatty alcohols as phase change materials. Equivalent amounts of the selected starting compounds were taken to the esterification reaction at 110 °C in a solventless medium. In order to catalyze the esterification reaction, non-hygroscopic triphenylphosphine-sulfur trioxide adduct was used (0.83 mmol%) which is an easily accessible compound. The relevant reaction was completed in a very short time (2 h) and under optimized esterification conditions, excellent conversion were reached. The targeted mono ester compounds (15 examples) were obtained in good to excellent yields even after a simple crystallization step (72-99%). Additionally, a catalyst reuse investigation and study covering the scale-up production of stearyl stearate was also carried out. The triphenylphosphine-sulfur trioxide catalyzed solvent free process can compete with existing processes and proved to be a cheaper, practical and environmentally-friendly method for the esterification of fatty acids and alcohols. [ABSTRACT FROM AUTHOR]

Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal for the production. An example of synthesis strongly dependent on external conditions is the production of storage lipids, which typically requires a high carbon/nitrogen ratio. This requirement also limits the use of abundant nitrogen-rich materials, such as industrial protein by-products, as substrates for lipid production. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered A. baylyi ADP1 by deleting the gene aceA encoding for isocitrate lyase and overexpressing fatty acyl-CoA reductase Acr1 in the wax ester production pathway to allow redirection of carbon towards WEs. This strategy led to 3-fold improvement in yield (0.075 ​g/g glucose) and 3.15-fold improvement in titer (1.82 ​g/L) and productivity (0.038 ​g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract, and baker’s yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.

Abstract Background Fatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different properties are needed for the construction of tailored synthetic pathways for the production of various compounds. Results Fatty aldehyde production in Acinetobacter baylyi ADP1 was increased by the overexpression of three different FARs: a native A. baylyi FAR Acr1, a cyanobacterial Aar, and a putative, previously uncharacterized dehydrogenase (Ramo) from Nevskia ramosa. The fatty aldehyde production was followed in real-time inside the cells with a luminescence-based tool, and the highest aldehyde production was achieved with Aar. The fate of the overproduced fatty aldehydes was studied by measuring the production of wax esters by a native downstream pathway of A. baylyi, for which fatty aldehyde is a specific intermediate. The wax ester production was improved with the overexpression of Acr1 or Ramo compared to the wild type A. baylyi by more than two-fold, whereas the expression of Aar led to only subtle wax ester production. The overexpression of FARs did not affect the length of the acyl chains of the wax esters. Conclusions The fatty aldehyde production, as well as the wax ester production of A. baylyi, was improved with the overexpression of a key enzyme in the pathway. The wax ester titer (0.45 g/l) achieved with the overexpression of Acr1 is the highest reported without hydrocarbon supplementation to the culture. The contrasting behavior of the different reductases highlight the significance of in vivo characterization of enzymes and emphasizes the possibilities provided by the diversity of FARs for pathway and product modulation.

Abstract Background Photosynthetic oleaginous microalgae are promising feedstocks for biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) represent rich sources for engineering microalgal lipid production. The principal activity of DGATs has been defined as a single-function enzyme catalyzing the esterification of diacylglycerol with acyl-CoA. Results A dual-function PtWS/DGAT associated with diatom Phaeodactylum tricornutum is discovered in the current study. Distinctive to documented microalgal DGAT types, PtWS/DGAT exhibits activities of both a wax ester synthase (WS) and a DGAT. WS/DGATs are broadly distributed in microalgae, with different topology and phylogeny from those of DGAT1s, DGAT2s, and DGAT3s. In vitro and in vivo assays revealed that PtWS/DGAT, functioning as either a WS or a DGAT, exhibited a preference on saturated FA substrate. Endogenous overexpression of PtWS/DGAT demonstrated that the DGAT activity was dominant, whereas the WS activity was condition dependent and relatively minor. Compared with the wild type (WT), overexpression of PtWS/DGAT in the diatom resulted in increased levels of total lipids (TL) and triacylglycerol (TAG) regardless of nitrogen availability. The stability and scalability of the introduced traits were further investigated at a 10-L photobioreactor, where the mutant growth resembled WT, with moderately increased productivity of TL and TAG. Furthermore, the production of wax esters increased considerably (from undetectable levels to 2.83%) under nitrogen-deplete conditions. Conclusions PtWS/DGAT is a bifunctional enzyme and may serve as a promising target for the engineering of microalga-based oils and waxes for future industrial use.

The biosynthesis of lipid-based biofuels is an important aspect of developing sustainable alternatives to conventional oils derived from fossil fuel reserves. Many biosynthetic approaches to biodiesel fuels and oils involve fatty acid derivatives as a precursor, and thioesterases have been employed in various strategies to increase fatty acid pools. Thioesterases liberate fatty acids from fatty acyl-coenzyme A or fatty acyl-acyl carrier protein substrates. The role played by thioesterases has not been extensively studied in model bacteria that accumulate elevated levels of biological oils based on fatty acid precursors. In this report, two primary thioesterases from the wax ester accumulating bacterium Marinobacter aquaeolei VT8 were heterologously expressed, isolated and characterized. These genes were further analyzed at the transcriptional level in the native bacterium during wax ester accumulation, and their genes were disrupted to determine the effect these changes had on wax ester levels. Combined, these results indicate that these two thioesterases do not play an integral role in wax ester accumulation in this natural lipid-accumulating model bacterium. [ABSTRACT FROM AUTHOR]

Plant lipids, mainly stored in seeds and other plant parts, are not only a crucial resource for food and fodder but are also a promising alternative to fossil oils as a chemical industry feedstock. Oil crop cultivation and processing are always important parts of agriculture worldwide. Vegetable oils containing polyunsaturated fatty acids, very long chain fatty acids, conjugated fatty acids, hydroxy fatty acids and wax esters, have outstanding nutritional, lubricating, surfactant, and artificial-fibre-synthesis properties, amongst others. Enhancing the production of such specific lipid components is of economic interest. There has been a considerable amount of information reported about plant lipid biosynthesis, including identification of the pathway map of carbon flux, key enzymes (and the coding genes), and substrate affinities. Plant lipid biosynthesis engineering to produce special oil compounds has become feasible, although until now, only limited progress has been made in the laboratory. It is relatively easy to achieve the experimental objectives, for example, accumulating novel lipid compounds in given plant tissues facilitated by genetic modification. Applying such technologies to agricultural production is difficult, and the challenge is to make engineered crops economically attractive, which is impeded by only moderate success. To achieve this goal, more complicated and systematic strategies should be developed and discussed based on the relevant results currently available. [ABSTRACT FROM AUTHOR]

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long‐chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl‐CoA:diacylglycerol O‐acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid‐ and long‐chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta. [ABSTRACT FROM AUTHOR]

Long and very long chain fatty alcohols are produced from their corresponding acyl-CoAs through the activity of fatty acyl reductases (FARs). Fatty alcohols are important components of the cuticle that protects aerial plant organs, and they are metabolic intermediates in the synthesis of the wax esters in the hull of sunflower (Helianthus annuus) seeds. Genes encoding 4 different FARs (named Ha FAR2, Ha FAR3, Ha FAR4 and Ha FAR5) were identified using BLAST, and studies showed that four of the genes were expressed in seed hulls. In this study, the structure and location of sunflower FAR proteins were determined. They were also expressed exogenously in Saccharomyces cerevisiae to evaluate their substrate specificity based on the fatty alcohols synthesized by the transformed yeasts. Three of the four enzymes tested showed activity in yeast. Ha FAR3 produced C18, C20 and C22 saturated alcohols, whereas Ha FAR4 and Ha FAR5 produced C24 and C26 saturated alcohols. The involvement of these genes in the synthesis of sunflower seed wax esters was addressed by considering the results obtained. • FARs (fatty acyl reductases) are involved in the synthesis of fatty alcohols in sunflower seed hull. • 4 of 8 different identified FAR genes were expressed in the hull of developing sunflower seeds. • The activity of these enzymes was evaluated by heterologous expression in yeast. • Studies of expression in tobacco leaves suggested that the Ha FARs were mostly localized in the cytosol. [ABSTRACT FROM AUTHOR]

Docosahexaenoic acid (DHA) is known to have numerous health benefits and immense dietary value. There is a pressing need to have a deeper understanding of DHA metabolism. Acyl CoA: Diacylglycerol Acyltransferase (DGAT) is an important enzyme of lipid anabolism and an essential piece of the puzzle. Aurantiochytrium limacinum , a primary producer of DHA, is a good model for studying DHA metabolism. Thus, we aimed to investigate important lipid metabolic genes from A. limacinum. We cloned four putative DGATs (DGAT2a, DGAT2b, DGAT2c, and DGAT2d) from A. limacinum and performed detailed in vivo and in vitro characterization. Functional characterization showed that not all the studied genes exhibited DGAT activity. DGAT2a and DGAT2d conferred DGAT activity whereas DGAT2b showed wax synthase (WS) activity and DGAT2c showed dual function of both WS and DGAT. Based on their identified function, DGAT2b and DGAT2c were renamed as AlWS and AlWS/DGAT respectively. DGAT2a was found to exhibit a preference for DHA as a substrate. DGAT2d was found to have robust activity and emerged as a promising candidate for genetic engineering aimed at increasing oil yield. The study enriches our knowledge of lipid biosynthetic enzymes in A. limacinum , which can be utilized to design suitable application strategies. [ABSTRACT FROM AUTHOR]

Abstract Background Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT). This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms. To date, knowledge about this class of biologically and biotechnologically important enzymes is mainly from biochemical characterization of WS/DGATs from Arabidopsis, jojoba and some bacteria that can synthesize both TAGs and WEs intracellularly, whereas little is known about WS/DGATs from eukaryotic microorganisms. Results Here, we report the identification and characterization of two bifunctional WS/DGAT enzymes (designated TrWSD4 and TrWSD5) from the marine protist Thraustochytrium roseum. Both TrWSD4 and TrWSD5 comprise a WS-like acyl-CoA acyltransferase domain and the recombinant proteins purified from Escherichia coli Rosetta (DE3) have substantial WS and lower DGAT activity. They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs and fatty alcohols ranging from C10 to C18. TrWSD4 displays WS activity with the lowest K m value of 0.14 μM and the highest k cat/K m value of 1.46 × 105 M−1 s−1 for lauroyl-CoA (C12:0) in the presence of 100 μM hexadecanol, while TrWSD5 exhibits WS activity with the lowest K m value of 0.96 μM and the highest k cat/K m value of 9.83 × 104 M−1 s−1 for decanoyl-CoA (C10:0) under the same reaction condition. Both WS/DGAT enzymes have the highest WS activity at 37 and 47 °C, and WS activity was greatly decreased when temperature exceeds 47 °C. TrWSD4 and TrWSD5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mM. The potential of T. roseum WS/DGATs to establish novel process for biotechnological production of WEs was demonstrated by heterologous expression in recombinant yeast. Expression of either TrWSD4 or TrWSD5 in Saccharomyces cerevisiae quadruple mutant H1246, which is devoid of storage lipids, resulted in the accumulation of WEs, but not any detectable TAGs, indicating a predominant WS activity in yeast. Conclusions This study demonstrates both in vitro WS and DGAT activity of two T. roseum WS/DGATs, which were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs and fatty alcohols as substrates for WS activity but displaying substrate preference for medium-chain acyl-CoAs. In vivo characterization shows that these two WS/DGATs predominantly function as wax synthase and presents the feasibility for production of WEs by heterologous hosts.

Heterotrophic protists play pivotal roles in aquatic ecosystems by transferring matter and energy, including lipids, from primary producers to higher trophic predators. Using Oxyrrhis marina as a model organism, changes to the non-saponifiable protist lipids were investigated under satiation and starvation conditions. During active feeding on the alga Cryptomonas sp., the O. marina hexane soluble non-saponifiable fraction lipid profile reflected its food source with the observed presence of long chain mono-unsaturated fatty alcohols up to C25:1. Evidence of trophic upgrading in O. marina was observed with long chain mono-unsaturated fatty alcohol accumulation of up to C35:1. To the best of our knowledge, this is the first evidence that heterotrophic dinoflagellates are capable of producing ester derived alcohols and that dinoflagellates like O. marina are capable of synthesizing fatty alcohols up to C35. Additionally, we show evidence of trophic upgrading of lipids. During a 20-day resource deprivation, the lipid profile remained constant. During starvation, the mobilization of wax esters as energy stores was observed with long chain fatty alcohols mobilized first. Changes in lipid class profile and utilization of wax esters in O. marina provides insight into the types of lipids available for energy demand, the transfer of lipids through the base of marine food webs, and the catabolic response induced by resource deprivation.

Heterotrophic protists play pivotal roles in aquatic ecosystems by transferring matter and energy, including lipids, from primary producers to higher trophic predators. Using Oxyrrhis marina as a model organism, changes to the non-saponifiable protist lipids were investigated under satiation and starvation conditions. During active feeding on the alga Cryptomonas sp., the O. marina hexane soluble non-saponifiable fraction lipid profile reflected its food source with the observed presence of long chain mono-unsaturated fatty alcohols up to C25:1. Evidence of trophic upgrading in O. marina was observed with long chain mono-unsaturated fatty alcohol accumulation of up to C35:1. To the best of our knowledge, this is the first evidence that heterotrophic dinoflagellates are capable of producing ester derived alcohols and that dinoflagellates like O. marina are capable of synthesizing fatty alcohols up to C35. Additionally, we show evidence of trophic upgrading of lipids. During a 20-day resource deprivation, the lipid profile remained constant. During starvation, the mobilization of wax esters as energy stores was observed with long chain fatty alcohols mobilized first. Changes in lipid class profile and utilization of wax esters in O. marina provides insight into the types of lipids available for energy demand, the transfer of lipids through the base of marine food webs, and the catabolic response induced by resource deprivation. [ABSTRACT FROM AUTHOR]

Lignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in the industry. Here, we investigated the properties of lignin‐related aromatic compounds (LRAs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in an LRA‐utilizing bacterial host Acinetobacter baylyi ADP1. The biosynthesis products, wax esters, and alkanes are relevant compounds for the chemical and fuel industries. Here, wax esters were produced by a native pathway of ADP1, whereas alkanes were produced by a synthetic pathway introduced to the host. Using individual LRAs as substrates, the growth and product formation were monitored with internal biosensors and off‐line analytics. Of the tested LRAs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with yield and titer of 37 mg/gcoumarate and 202 mg/L, whereas alkanes were produced with a yield of 62.3 µg /gcoumarate and titer of 152 µg/L. This study demonstrates the microbial preference for certain LRAs and highlights the potential of A. baylyi ADP1 as a host for LRA upgrading to value‐added products. [ABSTRACT FROM AUTHOR]

Jojoba is one of the main two known plant source of natural liquid wax ester for use in various applications, including cosmetics, pharmaceuticals, and biofuel. Due to the lack of transcriptomic and genomic data on lipid biosynthesis and accumulation, molecular marker breeding has been used to improve jojoba oil production and quality. In the current study, the transcriptome of developing jojoba seeds was investigated using the Illunina NovaSeq 6000 system, 100 × 106 paired end reads, an average length of 100 bp, and a sequence depth of 12 Gb per sample. A total of 176,106 unigenes were detected with an average contig length of 201 bp. Gene Ontology (GO) showed that the detected unigenes were distributed in the three GO groups biological processes (BP, 5.53%), cellular component (CC, 6.06%), and molecular functions (MF, 5.88%) and distributed in 67 functional groups. The lipid biosynthesis pathway was established based on the expression of lipid biosynthesis genes, fatty acid (FA) biosynthesis, FA desaturation, FA elongation, fatty alcohol biosynthesis, triacylglycerol (TAG) biosynthesis, phospholipid metabolism, wax ester biosynthesis, and lipid transfer and storage genes. The detection of these categories of genes confirms the presence of an efficient lipid biosynthesis and accumulation system in developing jojoba seeds. The results of this study will significantly enhance the current understanding of wax ester biology in jojoba seeds and open new routes for the improvement of jojoba oil production and quality through biotechnology applications.

Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes. [ABSTRACT FROM AUTHOR]

Structural and rheological properties of oleogels consisting of 0.5–25 wt% rice bran wax (RBX) in rice bran oil (RBO) were explored. RBX was an efficient, thermoreversible oleogelator capable of structuring RBO at concentrations as low as 0.5 wt% RBX. A qualitative temperature-composition phase diagram showed that oleogels containing higher concentrations of RBX were expectedly the most resistant to melting. In oleogels at higher RBX concentrations, polarized light microscopy revealed the presence of a network of interlinked, long aspect ratio wax crystal needles up to 50 μm long. Upon heating, RBX crystals did not undergo any structural transition, based on the constant short spacings at ~ 4.16 and ~ 3.73 Å, indicative of an orthorhombic subcell, and d 001 long spacing at 74–76 Å that persisted until RBX fusion. This long spacing was ascribed to the presence of wax esters consisting of long-chain saturated fatty acids (C24 and C22) esterified to C28 – C34 saturated fatty alcohols. During cooling from 90 to 20 °C, the increase in oleogel viscosity resulting from the RBX liquid-solid phase transition was corroborated by DSC-based crystallization onset and enthalpy data. Similarly, elastic moduli and hardness both rose with increasing RBX concentration. This study, which demonstrated that RBX can structure RBO with distinct concentration-dependent properties, serves as the foundation for the development of oleogel-based approaches to saturated and trans fats replacement in processed foods. [ABSTRACT FROM AUTHOR]

Cetyl palmitate was synthetized by esterification the cetyl alcohol with palmitic acid in a solvent-free system. Reactions were performed in batch mode using commercial lipase Lipozyme RM IM as catalyst. Stirring speed (180, 480 and 720 rpm), temperature (60, 70, 80 and 87 °C), amount of catalyst (0.5, 1.0 and 1.5% wt% related to the mass of substrates) and alcohol:acid molar ratio (0.5:1, 1:1 and 2:1) were evaluated and best reaction conditions obtained were 480 rpm, 70 °C, 1.0% of enzyme and 1:1 M ratio. A novel kinetic model based on random-sequential bi-bi mechanism was proposed and showed a good agreement with experimental data. Furthermore, this model was proved to be suitable for kinetics predictions with a small uncertainty. The catalyst was recycled and showed good stability since reaction conversion decreased only 6.8% after 15 reuses. [ABSTRACT FROM AUTHOR]

Lipids secreted from the meibomian gland (meibum) form the superficial layer of the tear film and prevent water evaporation from the ocular surface and infection. Here, we identified the fatty acid (FA) elongases responsible for the synthesis of very long-chain FAs (VLCFAs) that constitute the meibum lipids. Elongation of VLCFAs (ELOVL)1 is primarily responsible for the production of saturated VLCFAs, whereas ELOVL1, ELOVL3, and ELOVL4 redundantly participate in the synthesis of monounsaturated VLCFAs. Gene disruption of Elovl1 in mice shortened acyl moieties in the 2 major meibum lipids: cholesteryl esters and wax esters. These changes were associated with dry eye phenotypes, including increases in eye-blink frequency and water evaporation from the ocular surface at younger ages. Aged Elovl1 mutant mice developed corneal opacity with vascular invasion, accompanied by epidermalization of the cornea. Thus, in addition to the well-known VLC ceramides (acylceramides) in the epidermis, VLC meibum lipids are barrier-forming lipids. [ABSTRACT FROM AUTHOR]

Wax esters (WEs) synthesized by Euglena gracilis are potential sources for alternative fuels because of their high productivity, recent success in mass cultivation, and low energy consumption in extraction. In this study, deoxygenation of Euglena WE and conversion to hydrocarbons in a catalytic cracking process under a hydrogen-free atmosphere was investigated using a residue fluid catalytic cracking equilibrium catalyst with enhanced hydrogen-transfer activity. The deoxygenation of Euglena WE proceeded more rapidly with higher H 2 O selectivity than that of saturated triglycerides. This is because initial β-elimination of WEs produces saturated fatty acids and higher olefins; the higher olefins rapidly release hydrogen species during cracking, cyclization and aromatization, and the hydrogen species accelerate hydrodeoxygenation of the saturated fatty acids. Furthermore, the cracking of Euglena WE produced large amounts of paraffins and olefins instead of aromatics. Therefore, Euglena WE was confirmed to be a preferable feedstock for the catalytic cracking process for hydrocarbon fuel production. [ABSTRACT FROM AUTHOR]

The Odontocetes (toothed whales) possess two types of specialized fat and, therefore, represent an interesting group when considering the evolution and function of adipose tissue. All whales have a layer of superficial blubber, which insulates and streamlines, provides buoyancy and acts as an energy reserve. Some toothed whales deposit large amounts of wax esters, rather than triacylglycerols, in blubber, which is unusual. Waxes have very different physical and physiological properties, which may impact blubber function. The cranial acoustic fat depots serve to focus sound during echolocation and hearing. The acoustic fats have unique morphologies; however, they are even more specialized biochemically because they are composed of a mix of endogenous waxes and triacylglycerols with unusual branched elements (derived from amino acids) that are not present in other mammals. Both waxes and branched elements alter how sound travels through a fat body; they are arranged in a 3D topographical pattern to focus sound. Furthermore, the specific branched-chain acid/alcohol synthesis mechanisms and products vary phylogenetically (e.g. dolphins synthesize lipids from leucine whereas beaked whales use valine). I propose that these specialized lipids evolved first in the head: wax synthesis first emerged to serve an acoustic function in toothed whales, with branched-chain synthesis adding additional acoustic focusing power, and some species secondarily retained wax synthesis pathways for blubber. Further research is necessary to elucidate specific molecular mechanisms controlling the synthesis and deposition of wax esters and branched-chain fatty acids, as well as their spatial deposition within tissues and within adipocytes. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate whether wax esters could be produced, and to determine what types of wax esters could be produced in the wild oil species field cress ( Lepidium campestre L.). Wax esters produced in plants are perhaps best suited for lubrication applications; however, natural sources of plant-derived wax esters are limited and new resources need to be generated. Moreover, the composition of wax esters needs to be optimized in planta in order to suit various end uses for commercial applications. In this study, three jojoba wax ester synthesis genes under a seed specific promoter were introduced into field cress. The results show that expressing the jojoba genes produced various amounts of wax esters in the seed oil of field cress with C42 and C44 as the most abundant wax ester molecular species. The additional expression of a jojoba fatty acyl-coenzyme A elongase resulted in an increased level of longer chain C46 and C48 wax esters. The cellular distribution of neutral lipids appeared to be disrupted in the transgenic seeds with high wax ester content compared with wild type and wax esters were mainly localized in the seed coat. Collectively, the results indicate that field cress has the potential to be developed as a new industrial oil crop for wax ester production, and an improved understanding of mechanisms for the correct packaging of wax esters in seeds may influence wax ester amounts accumulated in transgenic crops. [ABSTRACT FROM AUTHOR]

Over-summering is a crucial period for Calanus sinicus in the southern Yellow Sea, where it is a key member of the zooplankton community. Lipids play an important role in copepod diapause, which is part of their over-summering strategy. We investigated how different fatty acids and lipid classes, including wax esters, changed during over-summering of C. sinicus during three cruises in June and August 2011 and November 2010, corresponding to the pre-, during and post-diapause periods, respectively. Large amounts of lipids were accumulated, mainly wax esters as previously found in C. finmarchicus during its diapause, and most of the storage lipids were used during over-summering. Wax ester polyunsaturated fatty acids (PUFAs) showed the most variation of the fatty acids (FAs), while the percentage composition of FAs in polar lipids was relatively stable. Selective use of wax ester PUFAs has already been shown to play important roles in the winter diapause of Calanus species in other regions, and our FA results show that this is the case for the Yellow Sea Cold Bottom Water (YSCBW) population that diapauses in summer. [ABSTRACT FROM AUTHOR]

Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes from Marinobacter aquaeolei VT8 and an additional enzyme from Acinetobacter baylyi were heterologously expressed in Escherichia coli and shown to display FAldDH activity. Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572, and the enzyme reported under RefSeq accession no. WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu_3316) from M. aquaeolei VT8. Crystals were independently treated with both the NAD+ cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided. IMPORTANCE This study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids and provides a likely picture of how the fatty aldehyde and NAD+ are bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and comparisons of specificities for the five enzymes that were characterized, correlations to the potential roles played by specific residues within the structure may be drawn. [ABSTRACT FROM AUTHOR]

Studi kinetika reaksi esterifikasi enzimatisasam miristat dan oleil alkohol telah dilakukan. Penelitian ini bertujuan untuk mendapatkan hasil sintesis yang optimal, sehingga meningkatkan konversi dan kecepatan reaksi pembentukan wax ester. Analisis kualitatif wax ester yang terbentuk dilakukan secara TLC dan GC, sedangkan analisis secara kuantitatif dilakukan secara titrasi asam-basa. Dari hasil penelitian diperoleh % konversi wax ester dengan menggunakan enzim tanpa perlakuan sebesar 94,44%. Nilai Km dan Vmax untuk asam miristat sebesar 2,5649 dan 14,3823 mmol x10-5 mmol/menit.mg katalis sedangkan untuk oleil alkohol sebesar 7,0345 dan 29,2292 mmol x10-mmol/menit.mg katalis. Sehingga dapat disimpulkan bahwa kinetika reaksi esterifikasi asam miristat dengan oleil Alkohol yang dikatalisis oleh enzim lipase tanpa perlakuan mengikuti Model Michaelis-Menten sedangkan mekanisme reaksi esterifikasi mengikuti mekanisme Bi Bi Ping-Pong