Acyl Donor Research Articles

Development of the esterase PestE for amide bond synthesis under aqueous conditions: Enzyme cascades for converting waste PET into tamibarotene.

A growing number of hydrolase enzymes show promiscuous acyltransferase activity, even under aqueous conditions. Here we report, for the first time, the ability of Pyrobaculum calidifontis VA1 esterase (PestE) to catalyse the formation of a wide range of amides in buffer, where the acyl donor forms a significant structural component in the amide product. The reactions occur under mild conditions and can achieve conversions up to 97% in 6 h for formation of N-benzylfuranamide as the model reaction. We demonstrate PestE's potential in enzyme cascades to make amides from waste PET plastic and the conversion of the terephthalic acid product to tamibarotene, a drug with activity against acute leukemia. Rational mutagenesis led to identification of PestE variants F33L_F289A and F33L. F33L_F289A increased conversion of N-benzylfuranamide by 1.2-fold, and F33L gave a 4-fold increase in conversion to tamibarotene.

Development of the esterase PestE for amide bond synthesis under aqueous conditions: Enzyme cascades for converting waste PET into tamibarotene.

A growing number of hydrolase enzymes show promiscuous acyltransferase activity, even under aqueous conditions. Here we report, for the first time, the ability of Pyrobaculum calidifontis VA1 esterase (PestE) to catalyse the formation of a wide range of amides in buffer, where the acyl donor forms a significant structural component in the amide product. The reactions occur under mild conditions and can achieve conversions up to 97% in 6 h for formation of N‐benzylfuranamide as the model reaction. We demonstrate PestE’s potential in enzyme cascades to make amides from waste PET plastic and the conversion of the terephthalic acid product to tamibarotene, a drug with activity against acute leukemia. Rational mutagenesis led to identification of PestE variants F33L_F289A and F33L. F33L_F289A increased conversion of N‐benzylfuranamide by 1.2‐fold, and F33L gave a 4‐fold increase in conversion to tamibarotene.

Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling

Streptomyces mobaraenesis transglutaminase (smTG) can be used for site-specific labeling of proteins with chemical groups. Here, we explored the use of modified smTG for the biosynthesis of nanobody-fluorophore conjugates (NFC). smTG catalyzes the conjugation of acyl donors containing glutamine with lysine-containing acceptors, which can lead to non-specific cross-linking. To achieve precise site-specific labeling, we employed molecular docking and virtual mutagenesis to redesign the enzyme's substrate specificity towards the peptide GGGGQR, a non-preferred acyl donor for smTG. Starting with a thermostable and highly active smTG variant (TGm2), we identified that single mutations G250H and Y278E significantly enhanced activity against GGGGQR, increasing it by 41 % and 1.13-fold, respectively. Notably, the Y278E mutation dramatically shifted the enzyme's substrate preference, with the activity ratio against GGGGQR versus the standard substrate CBZ-Gln-Gly rising from 0.05 to 0.93. In case studies, we used nanobodies 1C12 and 7D12 as labeling targets, catalyzing their conjugation with a synthetic fluorophore via smTG variants. Nanobodies fused with GGGGQR were successfully site-specifically labeled by TGm2-Y278E, in contrast to non-specific labeling observed with other variants. These results suggest that engineering smTG for site-specific labeling is a promising approach for the biosynthesis of antibody-drug conjugates.

Functions of Coenzyme A and Acyl-CoA in Post-Translational Modification and Human Disease

Coenzyme A (CoA) is synthesized from pantothenate, L-cysteine and adenosine triphosphate (ATP), and plays a vital role in diverse physiological processes. Protein acylation is a common post-translational modification (PTM) that modifies protein structure, function and interactions. It occurs via the transfer of acyl groups from acyl-CoAs to various amino acids by acyltransferase. The characteristics and effects of acylation vary according to the origin, structure, and location of the acyl group. Acetyl-CoA, formyl-CoA, lactoyl-CoA, and malonyl-CoA are typical acyl group donors. The major acyl donor, acyl-CoA, enables modifications that impart distinct biological functions to both histone and non-histone proteins. These modifications are crucial for regulating gene expression, organizing chromatin, managing metabolism, and modulating the immune response. Moreover, CoA and acyl-CoA play significant roles in the development and progression of neurodegenerative diseases, cancer, cardiovascular diseases, and other health conditions. The goal of this review was to systematically describe the types of commonly utilized acyl-CoAs, their functions in protein PTM, and their roles in the progression of human diseases.

The partitioning of fatty acids between membrane and storage lipids controls ER membrane expansion.

The biogenesis of membrane-bound organelles involves the synthesis, remodelling and degradation of their constituent phospholipids. How these pathways regulate organelle size, remains still poorly understood. Here we demonstrate that a lipid degradation pathway inhibits the expansion of the endoplasmic reticulum (ER) membrane. Phospholipid diacylglycerol acyltransferases (PDATs) use endogenous phospholipids as fatty acyl donors to generate triglyceride stored in lipid droplets. The significance of this non-canonical triglyceride biosynthetic pathway has remained elusive. We find that the activity of the yeast PDAT Lro1 is regulated by a membrane- proximal domain facing the luminal side of the ER bilayer. To reveal the biological roles of PDATs, we engineered an Lro1 variant with derepressed activity. We show that active Lro1 mediates the retraction of ER membrane expansion driven by phospholipid synthesis. Furthermore, the subcellular distribution and membrane turnover activity of Lro1 are controlled by diacylglycerol, produced by the activity of Pah1, a conserved member of the lipin family. Collectively, our findings reveal a lipid metabolic network that regulates endoplasmic reticulum biogenesis by converting phospholipids into storage lipids.

Enzymatic acylation of cyanidin-3-O-glucoside with aromatic and aliphatic acid methyl ester: Structure–stability relationships of acylated derivatives

Anthocyanins are water-soluble pigments, but they tend to be unstable in aqueous solutions. Modification of their molecular structure offers a viable approach to alter their intrinsic properties and enhance stability. Aromatic and aliphatic acid methyl esters were used as acyl donors in the enzymatic acylation of cyanidin-3-O-glucoside (C3G), and their analysis was conducted using ultraperformance liquid chromatography–mass spectrometry (UPLC-MS). The highest conversion rate achieved was 96.41 % for cyanidin-3-O-(6″-feruloyl) glucoside. Comparative evaluations of stability revealed that aromatic acyl group–conjugated C3G exhibited superior stability enhancement compared with aliphatic acyl group derivatives. The stability of aliphatic C3G decreased with increasing carbon chain length. The molecular geometries of different anthocyanins were optimized, and energy level calculations using density functional theory (DFT) identified their sites with antioxidant activities. Computational calculations aligned with the in vitro antioxidant assay results. This study provided theoretical support for stabilizing anthocyanins and broadened the application of acylated anthocyanins as food colorants and nutrient supplements.

A Type of Stable Amides Behaves as Acyl Transfer Reagents upon Visible-Light Irradiation through Self-Aromatization.

Organic molecules with light-modifiable reactivity are important in many fields because they can serve as the "switch" for light to trigger chemical processes. Herein, we disclose a new type of stable non-twisted amides, the reactivity of which can be turned on by light as acyl transfer reagents. Upon photo-activation, these amides react with various nucleophiles including amines, phenols, hydroxide, thiols, boronic acids, and alkynes either under metal-free or metal-catalysis conditions. This reactivity hinges on the design and synthesis of a photo-activatable reagent (7-nitro-5,6-dihydrophenanthridine), which undergoes self-aromatization enabled by an internal oxidant under light. This masked acyl donor group is anticipated to be useful in scenarios where light is preferred to trigger a chemical process.

Synthesis of pinolenic acid‐enriched triacylglycerol from pine nut oil via a two‐step consecutive enzyme reaction: Comparison of acyl donors

AbstractPinolenic acid (PLA) is a plant‐origin Δ5‐unsaturated polymethylene‐interrupted fatty acid that provides several beneficial health effects to the human body. A two‐step lipase‐catalyzed reaction was carried out to synthesize PLA‐enriched triacylglycerol (TAG) with pine nut oil (PNO) in the present study. In the first step, PLA was efficiently enriched from an initial value of 15–43 mol% in ethyl ester of the reaction mixture after Lipozyme 435‐catalyzed ethanolysis with PNO. In the second step, PLA‐enriched TAG was efficiently synthesized with the fatty acid form via Lipozyme 435‐catalyzed esterification at all temperatures when both ethyl ester form and fatty acid form were compared as acyl donors. The effect of vacuum on the synthesis of PLA‐enriched TAG with glycerol and PLA‐enriched fatty acid was studied. The optimum temperature and vacuum were 60°C, and 50 mmHg, respectively. A maximum TAG conversion of approximately 95% was achieved after 12 h under the optimum conditions.

Electronic Control of Polyproline II Helix Stability via the Identity of Acyl Capping Groups: the Pivaloyl Group Particularly Promotes PPII.

The type II polyproline helix (PPII) is a fundamental secondary structure of proteins, important in globular proteins, in intrinsically disordered proteins, and at protein-protein interfaces. PPII is stabilized in part by n→π* interactions between consecutive carbonyls, via electron delocalization between an electron-donor carbonyl lone pair (n) and an electron-acceptor carbonyl (π*) on the subsequent residue. We previously demonstrated that changes to the electronic properties of the acyl donor can predictably modulate the strength of n→π* interactions, with data from model compounds, in solution in chloroform, in the solid state, and computationally. Herein, we examined whether the electronic properties of acyl capping groups could modulate the stability of PPII in peptides in water. In X-PPGY-NH2 peptides (X=10 acyl capping groups), the effect of acyl group identity on PPII was quantified by circular dichroism and NMR spectroscopy. Electron-rich acyl groups promoted PPII relative to the standard acetyl (Ac-) group, with the pivaloyl and iso-butyryl groups most significantly increasing PPII. In contrast, acyl derivatives with electron-withdrawing substituents and the formyl group relatively disfavored PPII. Similar results, though lesser in magnitude, were also observed in X-APPGY-NH2 peptides, indicating that the capping group can impact PPII conformation at both proline and non-proline residues. The pivaloyl group was particularly favorable in promoting PPII. The effects of acyl capping groups were further analyzed in X-DfpPGY-NH2 and X-ADfpPGY-NH2 peptides, Dfp=4,4-difluoroproline. Data on these peptides indicated that acyl groups induced order Piv- > Ac- > For-. These results suggest that greater consideration should be given to the identity of acyl capping groups in inducing structure in peptides.

Vinyl 3-(Dimethylamino)propanoate as an Irreversible Acyl Donor Reagent in a Chromatography-free Lipase-Catalyzed Kinetic Resolution of sec-Alcohols.

The reported chemoenzymatic strategy involves the employment of vinyl 3-(dimethylamino)propanoate as an irreversible acyl donor in a chromatography-free lipase-catalyzed kinetic resolution (KR) of racemic sec-alcohols. This biotransformation is achieved in a sequential manner using CAL-B to affect the kinetic resolution, followed by a simple acidic extractive work-up furnishing both KR products with excellent enantioselectivity (E>200; up to 98 % ee). The elaborated method eliminates a single-use silica gel chromatographic separation and significantly reduces organic solvent consumption to foster a more environmentally friendly chemical industry.

Efficient enzymatic synthesis of d-α-tocopherol acetate by Carica papaya lipase-catalyzed acetylation of d-α-tocopherol in a solvent-free system

Enzymatic synthesis of d-α-tocopherol acetate is a promising approach for cost-effective production of liposoluble food and pharmaceutical antioxidant. In this study, we developed an efficient and environmentally friendly process that employed plant-derived Carica papaya lipase (CPL) as biocatalyst for the synthesis of d-α-tocopherol acetate in a solvent-free system. The optimum conditions were: 400 mg/mL d-α-tocopherol, 125 mg/mL CPL (44 U, aw = 0.328), with acetic anhydride as acyl donor, at 60°C and 200 rpm for 30 h. Interestingly, yields of 90.7%, 77.7%, 60.3% and 48.1% of d-α-tocopherol acetate were achieved after the “naturally immobilized” CPL recycled for 4 times, indicating a novel enzyme immobilization mechanism. Moreover, the synthetic product was purified from scale-up reaction, and confirmed by FTIR and GC-MS analysis, providing a total of 97.5% purity (91.1% d-α-tocopherol acetate plus 6.4% d-α-tocopherol). Overall, this work provided an efficient alternative method for green production of d-α-tocopherol acetate probably being applied in food and pharmaceutical industries, and this could also enhance the comprehensive utilization of Carica papaya.

Improvements in the Production of Isosorbide Monomethacrylate Using a Biobased Catalyst and Liquid-Liquid Extraction Isolation for Modifications of Oil-Based Resins.

The improved production of a polar curable monomer, isosorbide monomethacrylate (MISD), with methacrylic anhydride (MAAH) as an acyl donor, was performed. A sustainable and cheap catalyst, potassium acetate (CH3COOK), was used for a solvent-free synthesis, requiring only the equimolar amount of reagents (no excess). The production included the quantitative separation of the secondary product, methacrylic acid (MAA), preventing the reaction batch from the purification process (neutralization of MAA), and gaining a usable reagent. The synthesis resulted in a sufficient yield of MISD (61.8%) obtained by the liquid-liquid extraction process (LLE), which is a significant improvement in the process, avoiding the flash chromatography step in the isolation of MISD. The purity of synthesized and isolated MISD via the LLE was confirmed by 1H NMR, MS, and FTIR analyses. The thermal analyses, namely, DSC and TGA, were used to characterize the curability and thermal stability of MISD. The activation energy of MISD's curing was calculated (E a = 94.6 kJ/mol) along with the heat-resistant index (T s = 136.8). The polar character of isosorbide monomethacrylate was investigated in a mixture with epoxidized acrylated soybean oil (EASO). It was found that MISD is entirely soluble in EASO and can modify the rheological behavior and surface energy of EASO-based resins. The apparent viscosity of EASO at 30 °C (ηapp = 3413 mPa·s) decreased with the 50% content of MISD significantly (ηapp = 500 mPa·s), and the free surface energy value of EASO (γS = 42.2 mJ/m2) also increased with the 50% content of MISD (γS = 48.7 mJ/m2). The produced MISD can be successfully used as a diluent and the polarity modifier of curable oil-based resins.

Kinetic Resolution of Cyclic Tertiary Alcohols with Lipase A from Candida Antarctica

AbstractEnzyme‐catalyzed acylative kinetic resolution (KR) and dynamic kinetic resolution (DKR) of racemic primary and secondary alcohols have been widely reported to produce esters with high enantiomeric purity. In contrast, similar KRs of tertiary alcohols have been reported for only a limited range of substrates and require prolonged reaction times of several days. To gain deeper insight into the substrate scope and increase the process efficiency, we examined the reaction conditions using commercially available immobilized lipase A from Candida antarctica and found that the addition of the heterogeneous, inorganic base sodium carbonate significantly increased the reaction rate while maintaining high enantioselectivity. The use of vinyl hexanoate as the acyl donor provided esters that were stable during chromatography purification. The optimized reaction conditions were then successfully applied to a range of cyclic tertiary alcohols containing tetralin, dihydroindene, chromane, and thiochromane skeletons having, in part, a substituent on the aromatic ring. In this study on the structure–reactivity relationship of enzymatic KR‐type reactions, we achieved >30 % conversion for various tertiary alcohols in 24 h at 25 °C, producing optically active esters with 88–99 % ee.

Systematic characterization of gene families and functional analysis of PvRAS3 and PvRAS4 involved in rosmarinic acid biosynthesis in Prunella vulgaris.

Prunella vulgaris is an important material for Chinese medicines with rosmarinic acid (RA) as its index component. Based on the chromosome-level genome assembly we obtained recently, 51 RA biosynthesis-related genes were identified. Sequence feature, gene expression pattern and phylogenetic relationship analyses showed that 17 of them could be involved in RA biosynthesis. In vitro enzymatic assay showed that PvRAS3 catalyzed the condensation of p-coumaroyl-CoA and caffeoyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was higher than caffeoyl-CoA. PvRAS4 catalyzed the condensation of p-coumaroyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was lower than PvRAS3. UPLC and LC-MS/MS analyses showed the existence of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid in P. vulgaris. Generation and analysis of pvras3 homozygous mutants showed significant decrease of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid and significant increase of DHPL and pHPL. It suggests that PvRAS3 is the main enzyme catalyzing the condensation of acyl donors and acceptors during RA biosynthesis. The role of PvRAS4 appears minor. The results provide significant information for quality control of P. vulgaris medicinal materials.

Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3β-tigloyloxytropane

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3β-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3β-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3β-tigloyloxytropane synthase (TS), catalyzes 3β-tropanol and tigloyl-CoA to form 3β-tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3β-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3β-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3β-tigloyloxytropane from tiglic acid and 3β-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3β-tigloyloxytropane.

Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta

BackgroundEicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs.ResultIn this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool).ConclusionsThis set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

Integrated Omics Analysis Uncovers the Culprit behind Exacerbated Atopic Dermatitis in a Diet-Induced Obesity Model.

Atopic dermatitis (AD), a chronic inflammatory skin disease, is exacerbated by obesity, yet the precise linking mechanism remains elusive. This study aimed to elucidate how obesity amplifies AD symptoms. We studied skin samples from three mouse groups: sham control, AD, and high-fat (HF) + AD. The HF + AD mice exhibited more severe AD symptoms than the AD or sham control mice. Skin lipidome analysis revealed noteworthy changes in arachidonic acid (AA) metabolism, including increased expression of pla2g4, a key enzyme in AA generation. Genes for phospholipid transport (Scarb1) and acyltransferase utilizing AA as the acyl donor (Agpat3) were upregulated in HF + AD skin. Associations were observed between AA-containing phospholipids and skin lipids containing AA and its metabolites. Furthermore, imbalanced phospholipid metabolism was identified in the HF + AD mice, marked by excessive activation of the AA and phosphatidic acid (PA)-mediated pathway. This imbalance featured increased expression of Plcb1, Plcg1, and Dgk involved in PA generation, along with a decrease in genes converting PA into diglycerol (DG) and CDP-DG (Lpin1 and cds1). This investigation revealed imbalanced phospholipid metabolism in the skin of HF + AD mice, contributing to the heightened inflammatory response observed in HF + AD, shedding light on potential mechanisms linking obesity to the exacerbation of AD symptoms.

Genome-wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid.

Genome-wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD-family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl-CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP-sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate-starvation stress. GGES is a member of a well-supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant-specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

Heterologous phospholipid:diacylglycerol acyltransferase channels eicosapentaenoic acid to triacylglycerol in Phaeodactylum tricornutum

Phaeodactylum tricornutum is a well-studied oleaginous model diatom with high triacylglycerol (TAG) and omega-3 eicosapentaenoic acid (EPA) contents, however, only a small portion of EPA is transferred from the polar lipids to TAG through phospholipid:diacylglycerol acyltransferase (PDAT). In this study, a Nannochloropsis oceanica PDAT (NoPDAT) was heterologously expressed in P. tricornutum to increase the EPA content in TAG. NoPDAT heterologous expression slightly disturbed the growth of the transgenic lines, but enhanced the TAG content by 16–18 %. Considering the localization of NoPDAT in P. tricornutum after heterologous expression together with our lipidomic results, NoPDAT is more likely to utilize phosphatidylcholine and phosphatidylethanolamine on the chloroplast endoplasmic reticulum membrane as the acyl donors. Especially, NoPDAT transferred the EPA acyl moiety from the sn-2 position of the two above-mentioned phospholipids to the sn-3 position of TAG, resulting in the increase of two major EPA-containing TAG molecules (16:1/16:0/20:5 and 16:1/16:1/20:5). Correspondingly, two major 16:0-containing TAG molecules (16,0/16:1/16:1 and 16:0/16:0/16:1) decreased in the transgenic strains. In conclusion, our attempt to express NoPDAT in P. tricornutum increases the EPA content in TAG by approximately one-fold, and demonstrates the versatility of diatom lipid metabolism and acyl editing.

Enzymatic kinetic resolution of an intermediate alcohol of the drug Tegoprazan using lipase from Thermomyces lanuginosus

An enzymatic approach for the synthesis of intermediate alcohol of the drug tegoprazan in 99% ee at 20 °C in hexane has been developed utilizing commercially available immobilized Thermomyces lanuginosus lipase (TL lipase) as a biocatalyst and vinyl acetate as a acyl donor. This method involves the enantioselective esterification of (RS)-5,7-difluorochroman-4-ol with vinyl acetate using TL Lipase. This enzymatic approach provides the (S)-chiral alcohol in 44% yield with 99% enantiomeric excess (ee) and the (R)-acetate in 43% yield and >99% ee. It is a sustainable approach for the synthesis of enantiopure tegoprazan intermediate, contributing to the advancement of green chemistry. The recovered TL lipase was reused in 13 subsequent runs without loss of activity.