High Expression of ACOT2 Predicts Worse Overall Survival and Abnormal Lipid Metabolism: A Potential Target for Acute Myeloid Leukemia.

Acyl-CoA thioesterase (ACOT) plays a considerable role in lipid metabolism, which is closely related to the occurrence and development of cancer, nevertheless, its role has not been fully elucidated in acute myeloid leukemia (AML). To explore the role of ACOT2 in AML and to provide a potential therapeutic target for AML, the expression pattern of ACOT was investigated based on the TNMplot, Gene Expression Profiling Interactive Analysis (GEPIA), and Cancer Cell Line Encyclopedia (CCLE) database, and diagnostic value, prognostic value, and clinical phenotype of ACOT were explored based on data from The Cancer Genome Atlas (TCGA). Functional annotation and enrichment analysis of the common targets between ACOT2 coexpressed and AML-related genes were further performed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) analyses. The protein-protein interaction (PPI) network of ACOT2 coexpressed genes and functional ACOT2-related metabolites association network were constructed based on GeneMANIA and Human Metabolome Database. Among ACOTs, ACOT2 was highly expressed in AML compared to normal control subjects according to TNMplot, GEPIA, and CCLE database, which was significantly associated with poor overall survival (OS) in AML (P=0.003). Moreover, ACOT2 exhibited excellent diagnostic efficiency for AML (AUC: 1.000) and related to French-American-British (FAB) classification and cytogenetics. GO, KEGG, and GSEA analyses of 71 common targets between ACOT2 coexpressed and AML-related genes revealed that ACOT2 is closely related to ACOT1, ACOT4, enoyl-acyl carrier protein reductase, mitochondrial (MECR), puromycin-sensitive aminopeptidase (NPEPPS), SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), and long-chain fatty acid-CoA ligase 1 (ACSL1) in PPI network, and plays a significant role in lipid metabolism, that is, involved in fatty acid elongation and biosynthesis of unsaturated fatty acids. Collectively, the increase of ACOT2 may be an important characteristic of worse OS and abnormal lipid metabolism, suggesting that ACOT2 may become a potential therapeutic target for AML.

Acyl-Coa Thioesterases: A Rheostat That Controls Activated Fatty Acids Modulates Dengue Virus Serotype 2 Replication.

During infection with dengue viruses (DENVs), the lipid landscape within host cells is significantly altered to assemble membrane platforms that support viral replication and particle assembly. Fatty acyl-CoAs are key intermediates in the biosynthesis of complex lipids that form these membranes. They also function as key signaling lipids in the cell. Here, we carried out loss of function studies on acyl-CoA thioesterases (ACOTs), a family of enzymes that hydrolyze fatty acyl-CoAs to free fatty acids and coenzyme A, to understand their influence on the lifecycle of DENVs. The loss of function of the type I ACOTs 1 (cytoplasmic) and 2 (mitochondrial) together significantly increased DENV serotype 2 (DENV2) viral replication and infectious particle release. However, isolated knockdown of mitochondrial ACOT2 significantly decreased DENV2 protein translation, genome replication, and infectious virus release. Furthermore, loss of ACOT7 function, a mitochondrial type II ACOT, similarly suppressed DENV2. As ACOT1 and ACOT2 are splice variants, these data suggest that functional differences and substrate specificities due to the location (cytosol and mitochondria, respectively) of these proteins may account for the differences in DENV2 infection phenotype. Additionally, loss of mitochondrial ACOT2 and ACOT7 expression also altered the expression of several ACOTs located in multiple organelle compartments within the cell, highlighting a complex relationship between ACOTs in the DENV2 virus lifecycle.

Thioesterase superfamily member 1 undergoes stimulus-coupled conformational reorganization to regulate metabolism in mice.

In brown adipose tissue, thermogenesis is suppressed by thioesterase superfamily member 1 (Them1), a long chain fatty acyl-CoA thioesterase. Them1 is highly upregulated by cold ambient temperature, where it reduces fatty acid availability and limits thermogenesis. Here, we show that Them1 regulates metabolism by undergoing conformational changes in response to ß-adrenergic stimulation that alter Them1 intracellular distribution. Them1 forms metabolically active puncta near lipid droplets and mitochondria. Upon stimulation, Them1 is phosphorylated at the N-terminus, inhibiting puncta formation and activity and resulting in a diffuse intracellular localization. We show by correlative light and electron microscopy that Them1 puncta are biomolecular condensates that are inhibited by phosphorylation. Thus, Them1 forms intracellular biomolecular condensates that limit fatty acid oxidation and suppress thermogenesis. During a period of energy demand, the condensates are disrupted by phosphorylation to allow for maximal thermogenesis. The stimulus-coupled reorganization of Them1 provides fine-tuning of thermogenesis and energy expenditure.

Cloning of the gene encoding acyl-CoA thioesterase 11 and its functional characterization in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) under heat stress.

Members of the ACOT (acyl-CoA thioesterase) family hydrolyze fatty acyl-CoA to form free fatty acids (FFAs) and coenzyme A (CoA). These enzymes play important roles in fatty acid metabolism. Here, we report the cloning and functional analysis of acot11ß in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂). The open reading frame of acot11ß was found to be 594 bp in length, encoding 198 amino acids. We determined the transcript levels of acot11ß in ten tissues of hybrid yellow catfish by qRT-PCR and found that it was highly expressed in the liver, so we chose the liver for further analysis. We determined the transcript levels of acot11ß in hybrid yellow catfish under heat stress conditions, and analyzed the changes in serum biochemical parameters, liver biochemical parameters, and transcript levels of lipid metabolism-related genes. Healthy yellow catfish were subjected to heat stress at 35 °C for 96 h, and the experimental results were compared with those from fish in a control group (28 °C). The levels of glucose (GLU), total cholesterol (TC), and triglyceride (TG) in serum were significantly increased in the heat-stressed group compared with the control group (P < 0.05). Acute heat stress led to decreased liver glycogen contents, but significantly increased TC and TG contents in the liver (P < 0.05). The transcript levels of acot11ß, acc, and fas were significantly reduced, while that of pparα was significantly increased in hybrid yellow catfish exposed to heat stress (P < 0.05). Our results indicate that acot11ß plays an important role in regulating lipid metabolism in hybrid yellow catfish, and this metabolic process is greatly affected by temperature. These results may be useful for developing effective strategies to prevent or reduce metabolic disorders of yellow catfish caused by high temperature.

Allosteric regulation of thioesterase superfamily member 1 by lipid sensor domain binding fatty acids and lysophosphatidylcholine.

Nonshivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold ambient temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally up-regulated in brown adipose tissue upon exposure to the cold and suppresses thermogenesis in order to conserve energy reserves. It hydrolyzes long-chain fatty acyl-CoAs that are derived from lipid droplets, preventing their use as fuel for thermogenesis. In addition to its enzymatic domains, Them1 contains a C-terminal StAR-related lipid transfer (START) domain with unknown ligand or function. By complementary biophysical approaches, we show that the START domain binds to long-chain fatty acids, products of Them1's enzymatic reaction, as well as lysophosphatidylcholine (LPC), lipids shown to activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the START domain and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the START domain functions to localize Them1 near lipid droplets. These findings define the role of the START domain as a lipid sensor that allosterically regulates Them1 activity and spatially localizes it in proximity to the lipid droplet.

Interaction between ACOT7 and LncRNA NMRAL2P via Methylation Regulates Gastric Cancer Progression.

PURPOSE: Gastric cancer (GC) has a very poor prognosis when diagnosed at a late stage. Acyl-CoA thioesterase 7 (ACOT7) is a major isoform of the acyl coenzyme family that catalyzes the hydrolysis of fatty acyl-CoAs into unesterified free fatty acid and coenzyme A. The purpose of this study was to investigate the expression levels of ACOT7 in GC and mechanisms related therewith. MATERIALS AND METHODS: Screening of systematic biology studies revealed ACOT7 as a key gene in GC, as well as involvement of the long non-coding RNA NMRAL2P in ACOT7 expression. In this study, GC tissues and adjacent tissue samples were obtained from 10 GC patients at the Department of Gastrointestinal Surgery. GES1 and SGC-7901 cells were collected and treated to silence ACOT7 and overexpress NMRAL2P. The expressions of ACOT7 and NMRAL2P were detected by real-time quantitative PCR and Western blot. Additionally, cell proliferation, apoptosis, migration, and invasion were examined. RESULTS: ACOT7 was upregulated in gastric tumor tissues and GC cell lines. ACOT7 gene silencing induced a less malignant phenotype and was closely correlated to reduced cell proliferation and migration, altered cell cycle, and increased apoptosis. Furthermore, NMRAL2P was downregulated in tumor tissues and GC cell lines. NMRAL2P overexpression induced a more malignant phenotype and significantly inhibited the expression of ACOT7. Importantly, NMRAL2P indirectly methylated ACOT7 by binding to DNMT3b, thereby suppressing ACOT7 expression. CONCLUSION: NMRAL2P activation suppresses ACOT7 expression in GC. Thus, ACOT7 could be a promising target for the treatment of GC.

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications.

Plants use fatty acids to synthesize acyl lipids for many different cellular, physiological, and defensive roles. These roles include the synthesis of essential membrane, storage, or surface lipids, as well as the production of various fatty acid-derived metabolites used for signaling or defense. Fatty acids are activated for metabolic processing via a thioester linkage to either coenzyme A or acyl carrier protein. Acyl synthetases metabolically activate fatty acids to their thioester forms, and acyl thioesterases deactivate fatty acyl thioesters to free fatty acids by hydrolysis. These two enzyme classes therefore play critical roles in lipid metabolism. This review highlights the surprisingly complex and varying roles of fatty acyl synthetases in plant lipid metabolism, including roles in the intracellular trafficking of fatty acids. This review also surveys the many specialized fatty acyl thioesterases characterized to date in plants, which produce a great diversity of fatty acid products in a tissue-specific manner. While some acyl thioesterases produce fatty acids that clearly play roles in plant-insect or plant-microbial interactions, most plant acyl thioesterases have yet to be fully characterized both in terms of their substrate specificities and their functions. The biotechnological applications of plant acyl thioesterases and synthetases are also discussed, as there is significant interest in these enzymes as catalysts for the sustainable production of fatty acids and their derivatives for industrial uses.

Production of adipic acid by short- and long-chain fatty acid acyl-CoA oxidase engineered in yeast Candida tropicalis.

In this study, to produce adipic acid, mutant strains of Candida tropicalis KCTC 7212 deficient of AOX genes encoding acyl-CoA oxidases which are important in the ß-oxidation pathway were constructed. Production of adipic acid in the mutants from the most favorable substrate C12 methyl laurate was significantly increased. The highest level of production of adipic acid was obtained in the C. tropicalis ΔAOX4::AOX5 mutant of 339.8 mg L-1 which was about 5.4-fold higher level compared to the parent strain. The C. tropicalis ΔAOX4::AOX5 mutant was subjected to fed-batch fermentation at optimized conditions of agitation rate of 1000 rpm, pH 5.0 and methyl laurate of 3% (w/v), giving the maximum level of adipic acid of 12.1 g L-1 and production rate of 0.1 g L-1 h-1.

Multiple mitochondrial thioesterases have distinct tissue and substrate specificity and CoA regulation, suggesting unique functional roles.

Acyl-CoA thioesterases (Acots) hydrolyze fatty acyl-CoA esters. Acots in the mitochondrial matrix are poised to mitigate ß-oxidation overload and maintain CoA availability. Several Acots associate with mitochondria, but whether they all localize to the matrix, are redundant, or have different roles is unresolved. Here, we compared the suborganellar localization, activity, expression, and regulation among mitochondrial Acots (Acot2, -7, -9, and -13) in mitochondria from multiple mouse tissues and from a model of Acot2 depletion. Acot7, -9, and -13 localized to the matrix, joining Acot2 that was previously shown to localize there. Mitochondria from heart, skeletal muscle, brown adipose tissue, and kidney robustly expressed Acot2, -9, and -13; Acot9 levels were substantially higher in brown adipose tissue and kidney mitochondria, as was activity for C4:0-CoA, a unique Acot9 substrate. In all tissues, Acot2 accounted for about half of the thioesterase activity for C14:0-CoA and C16:0-CoA. In contrast, liver mitochondria from fed and fasted mice expressed little Acot activity, which was confined to long-chain CoAs and due mainly to Acot7 and Acot13 activities. Matrix Acots occupied different functional niches, based on substrate specificity (Acot9 versus Acot2 and -13) and strong CoA inhibition (Acot7, -9, and -13, but not Acot2). Interpreted in the context of ß-oxidation, CoA inhibition would prevent Acot-mediated suppression of ß-oxidation, while providing a release valve when CoA is limiting. In contrast, CoA-insensitive Acot2 could provide a constitutive siphon for long-chain fatty acyl-CoAs. These results reveal how the family of matrix Acots can mitigate ß-oxidation overload and prevent CoA limitation.

Expression level of ACOT7 influences the prognosis in acute myeloid leukemia patients.

BACKGROUND: ACOT plays an important role in lipid metabolism and recent studies found that ACOT participates in some kinds of tumorigenesis. However, both the role of ACOT and its significance have not been revealed in AML. Therefore, we conduct this study in order to investigate the association between AML and ACOT, and hopefully contributed to the management of AML. METHODS: One hundred and fifty-six AML patients were enrolled in our study whose data were derived from the Cancer Genome Atlas database. There were 85 patients who received only chemotherapy and other 71 patients underwent allo-HSCT. RESULTS: Patients in high ACOT7 group had a significant lower EFS and OS, while patients in high versus low expression levels of other types of ACOT showed no significant difference on the outcome. High level of ACOT7 related with poor outcome in both chemotherapy-only group and HSCT group. CONCLUSIONS: High expression level of ACOT7 indicates unfavorable outcome in AML patients. Allo-HSCT could not overcome the unfavorable effect of ACOT7 in these patients.

Convergent genomic signatures of flight loss in birds suggest a switch of main fuel.

Flight loss in birds is as characteristic of the class Aves as flight itself. Although morphological and physiological differences are recognized in flight-degenerate bird species, their contributions to recurrent flight degeneration events across modern birds and underlying genetic mechanisms remain unclear. Here, in an analysis of 295 million nucleotides from 48 bird genomes, we identify two convergent sites causing amino acid changes in ATGLSer321Gly and ACOT7Ala197Val in flight-degenerate birds, which to our knowledge have not previously been implicated in loss of flight. Functional assays suggest that Ser321Gly reduces lipid hydrolytic ability of ATGL, and Ala197Val enhances acyl-CoA hydrolytic activity of ACOT7. Modeling simulations suggest a switch of main energy sources from lipids to carbohydrates in flight-degenerate birds. Our results thus suggest that physiological convergence plays an important role in flight degeneration, and anatomical convergence often invoked may not.

Loss of ACOT7 potentiates seizures and metabolic dysfunction.

Neurons uniquely antagonize fatty acid utilization by hydrolyzing the activated form of fatty acids, long chain acyl-CoAs, via the enzyme acyl-CoA thioesterase 7, Acot7. The loss of Acot7 results in increased fatty acid utilization in neurons and exaggerated stimulus-evoked behavior such as an increased startle response. To understand the contribution of Acot7 to seizure susceptibility, we generated Acot7 knockout (KO) mice and assayed their response to kainate-induced seizures. Acot7 KO mice exhibited potentiated behavioral and molecular indices of seizure severity following kainic acid administration, suggesting that fatty acid metabolism in neurons can be a critical regulator of neuronal activity. These data are consistent with the presentation of seizures in a human with genomic deletion of ACOT7 demonstrating the conservation of function across species. To further understand the metabolic complications arising from a deletion in Acot7, we subjected Acot7 KO mice to a high-fat diet. While the loss of Acot7 did not result in metabolic complications following a normal chow diet, a high-fat diet induced greater body weight gain, adiposity, and glucose intolerance in Acot7 KO mice. These data demonstrate that Acot7, a fatty acid metabolic enzyme highly enriched in neurons, regulates both brain-specific metabolic processes related to seizure susceptibility and the whole body response to dietary lipid.

The role of the acyl-CoA thioesterase "YciA" in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli.

Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease involving motor neuron death, paralysis and, ultimately, respiratory failure. Motor neuron dysfunction leads to target skeletal muscle atrophy involving dysregulation of downstream cell survival, growth and metabolic signaling. Decreased Akt activity is linked to muscle atrophy in ALS and is associated with increased atrophy gene expression. Unfortunately, the regulating mechanism of Akt activity in atrophic muscle remains unclear. Recent research indicates a role of carboxyl-terminal modulator protein (CTMP) in Akt-signaling related neurologic dysfunction and skeletal muscle metabolism. CTMP is known to bind and reduce Akt phosphorylation and activation. We hypothesized that CTMP expression might progressively increase in ALS skeletal muscle as the disease progresses, downregulating Akt activity. We found that CTMP protein expression significantly increased in hindlimb skeletal muscle in the mSOD1G93A mouse model of ALS in late stages of the disease (P < 0.05), which negatively correlated with Akt phosphorylation over this period (R2 = -0.77). Co-immunoprecipitation of Akt revealed CTMP binding in pre-symptomatic and end-stage skeletal muscle, suggesting a possible direct role in reduced Akt signaling during disease progression. Inflammatory TNFα and downstream cellular degradation process markers for autophagy, lysosome production, and atrophy significantly increased in a pattern corresponding to increased CTMP expression and reduced Akt phosphorylation. In an in vitro model of skeletal muscle atrophy, differentiated C2C12 cells exhibited reduced Akt activity and decreased FOXO1 phosphorylation, a process known to promote transcription of atrophy genes in skeletal muscle. These results corresponded with  increased  Atrogin-1 expression  compared to healthy control cells  (P < 0.05). Transfection with CTMP siRNA significantly increased Akt phosphorylation in atrophic C2C12 cells, corresponding to significantly decreased CTMP expression. In conclusion, this is the first study to provide evidence for a link between elevated CTMP expression, downregulated Akt phosphorylation and muscle atrophy in ALS and clearly demonstrates a direct influence of CTMP on Akt phosphorylation in an in vitro muscle cell atrophy model.

Orlistat Displays Antitumor Activity and Enhances the Efficacy of Paclitaxel in Human Hepatoma Hep3B Cells.

Orlistat has been proved to be an effective fatty acid synthase inhibitor that is able to inhibit the proliferation and induce apoptosis in many cancer cell types. However, the anticancer effects of orlistat on hepatocellular carcinoma are undefined. We found that orlistat inhibited cell growth and induced G0/G1 cell cycle arrest with increased cyclin D, cyclin E, and p21 expression in human hepatoma Hep3B cells. Furthermore, protein expression of cyclin A, cyclin B, Cdk1, Cdk2, and Cdk4 was reduced by orlistat. This study investigated the role of lipid metabolism on orlistat-induced human hepatoma Hep3B cell death. The decrease in the expression of key enzymes in fatty acid metabolism, including FASN, ACOT8, PPT1, FABP1, CPT1 and CPT2, was observed after orlistat treatment. We also demonstrated that peroxisomal activity was involved in the orlistat-induced Hep3B cell death. In this study, we established an in vitro model to investigate the effect of orlistat on lipid accumulation. We found that orlistat significantly inhibited the cellular lipid content when administered in fatty acid overload conditions in Hep3B cells. Combination treatment of orlistat and paclitaxel was able to induce a synergistic effect on growth inhibition and cell apoptosis in Hep3B cells. Our data suggested that orlistat displays antitumor activity and enhances the efficacy of paclitaxel in Hep3B cells.

Up-Regulated MicroRNA-27b Promotes Adipocyte Differentiation via Induction of Acyl-CoA Thioesterase 2 Expression.

Nonalcoholic fatty liver disease (NAFLD) is characterized by a spectrum of liver pathologies, from simple steatosis to steatohepatitis. Recent studies have increasingly noted the aberrant expression of microRNAs closely related to NAFLD pathologies. We have previously shown the presence of increased levels of microRNA-27b (miR-27b) in patients with NAFLD. In this study, we investigated the role of miR-27b in NAFLD by examining the impact of up-regulated miR-27b on the differentiation of preadipocytes into mature adipocytes. We found that miR-27b-3p remarkably enhances the adipocyte differentiation of 3T3-L1 cells associated with lipid accumulation and intracellular triglyceride contents. Furthermore, we have demonstrated not only that miR-27b-3p induces acyl-CoA thioesterase 2 (ACOT2) expression in 3T3-L1 cells, but also that the knockdown of ACOT2 suppresses lipid accumulation and adipocyte differentiation in both the presence and absence of miR-27b-3p treatment. Our data strongly suggest that the miR-27b-ACOT2 axis is an important pathway in adipocyte differentiation and may play a role in the pathogenesis of NAFLD.

Increasing Acyl CoA thioesterase activity alters phospholipid profile without effect on insulin action in skeletal muscle of rats.

Increased lipid metabolism in muscle is associated with insulin resistance and therefore, many strategies have been employed to alter fatty acid metabolism and study the impact on insulin action. Metabolism of fatty acid requires activation to fatty acyl CoA by Acyl CoA synthases (ACSL) and fatty acyl CoA can be hydrolysed by Acyl CoA thioesterases (Acot). Thioesterase activity is low in muscle, so we overexpressed Acot7 in muscle of chow and high-fat diet (HFD) rats and investigated effects on insulin action. Acot7 overexpression modified specific phosphatidylcholine and phosphatidylethanolamine species in tibialis muscle of chow rats to levels similar to those observed in control HFD muscle. The changes in phospholipid species did not alter glucose uptake in tibialis muscle under hyperinsulinaemic/euglycaemic clamped conditions. Acot7 overexpression in white extensor digitorum longus (EDL) muscle increased complete fatty acid oxidation ex-vivo but was not associated with any changes in glucose uptake in-vivo, however overexpression of Acot7 in red EDL reduced insulin-stimulated glucose uptake in-vivo which correlated with increased incomplete fatty acid oxidation ex-vivo. In summary, although overexpression of Acot7 in muscle altered some aspects of lipid profile and metabolism in muscle, this had no major effect on insulin-stimulated glucose uptake.

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and ß-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 ß-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/ß-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities.

Regulation of fatty acid trafficking in liver by thioesterase superfamily member 1.

Thioesterase superfamily member 1 (Them1) is an acyl-CoA thioesterase that is highly expressed in brown adipose tissue, where it functions to suppress energy expenditure. Lower Them1 expression levels in the liver are upregulated in response to high-fat feeding. Them1-/- mice are resistant to diet-induced obesity, hepatic steatosis, and glucose intolerance, but the contribution of Them1 in liver is unclear. To examine its liver-specific functions, we created conditional transgenic mice, which, when bred to Them1-/- mice and activated, expressed Them1 exclusively in the liver. Mice with liver-specific Them1 expression exhibited no changes in energy expenditure. Rates of fatty acid oxidation were increased, whereas hepatic VLDL triglyceride secretion rates were decreased by hepatic Them1 expression. When fed a high-fat diet, Them1 expression in liver promoted excess steatosis in the setting of reduced rates of fatty acid oxidation and preserved glycerolipid synthesis. Liver-specific Them1 expression did not influence glucose tolerance or insulin sensitivity, but did promote hepatic gluconeogenesis in high-fat-fed animals. This was attributable to the generation of excess fatty acids, which activated PPARα and promoted expression of gluconeogenic genes. These findings reveal a regulatory role for Them1 in hepatocellular fatty acid trafficking.

Elevating 4-hydroxycoumarin production through alleviating thioesterase-mediated salicoyl-CoA degradation.

Acyl-CoAs are essential intermediates in the biosynthetic pathways of a number of industrially and pharmaceutically important molecules. When these pathways are reconstituted in a heterologous microbial host for metabolic engineering purposes, the acyl-CoAs may be subject to undesirable hydrolysis by the host's native thioesterases, resulting in a waste of cellular energy and decreased intermediate availability, thus impairing bioconversion efficiency. 4-hydroxycoumarin (4HC) is a direct synthetic precursor to the commonly used oral anticoagulants (e.g. warfarin) and rodenticides. In our previous study, we have established an artificial pathway for 4HC biosynthesis in Escherichia coli, which involves the thioester intermediate salicoyl-CoA. Here, we utilized the 4HC pathway as a demonstration to examine the negative effect of salicoyl-CoA degradaton, identify and inactivate the responsible thioesterase, and eventually improve the 4HC production. We screened a total of 16 E. coli thioesterases and tested their hydrolytic activity towards salicoyl-CoA in vitro. Among all the tested candidate enzymes, YdiI was found to be the dominant contributor to the salicoyl-CoA degradation in E. coli. Remarkably, the ydiI knockout strain carrying the 4HC pathway exhibited an up to 300% increase in 4HC production. An optimized 4HC pathway construct introduced in the ydiI knockout strain led to the accumulation of 935mg/L of 4HC in shake flasks, which is about 1.5 folds higher than the wild-type strain. This study demonstrates a systematic strategy to alleviate the undesirable hydrolysis of thioester intermediates, allowing production enhancement for other biosynthetic pathways with similar issues.