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Yeast are a powerful material for the encapsulation of compounds. Usually, yeast used as capsules are inactivated by the encapsulation treatment, which is stressful to cells. However, if kept active, cells can bring their own activity in addition to the encapsulated compound. We have observed previously that lipid-grown Yarrowia lipolytica were more resistant to encapsulation. The objective of the present study was to identify physiological markers involved in this resistance. Cells were cultured in the presence of glucose or methyl-oleate as the sole carbon source and submitted to a γ-dodecalactone stress. This paper focuses on the role of intracellular lipid droplets (LDs) and of the ergosteryl content to protect cells during the lactone treatment. Lipid-grown cells were more resistant to lactone and the presence of LDs before the shock increased significantly the resistance. The ergosteryl esters from the LD pool were hydrolysed to release ergosterol able to strenghten the plasma membrane during the shock. For cells devoid of LDs, membrane ergosterols were esterified concomitantly with LDs growth, resulting in a membrane weakening. By using pox3-mutant strains, which possesse numerous and small-sized LDs, we observed the original behaviour: these mutants showed no increased resistance and their LDs exploded in the cytoplasma during the shock. These results point out the role of LDs in cell resistance to amphiphilic stresses as a storage compartment as well as in ergosterol homeostasis.

Abstract Peroxisomes are organelles that play essential roles in many metabolic processes, but also play roles in innate immunity, signal transduction, aging and cancer. One of the main functions of peroxisomes is the processing of very-long chain fatty acids into metabolites that can be directed to the mitochondria. One key family of enzymes in this process are the peroxisomal acyl-CoA oxidases (ACOX1, ACOX2 and ACOX3), the expression of which has been shown to be dysregulated in some cancers. Very little is however known about the expression of this family of oxidases in non-small cell lung cancer (NSCLC). ACOX2 has however been suggested to be elevated at the mRNA level in over 10% of NSCLC, and in the present study using both standard and bioinformatics approaches we show that expression of ACOX2 is significantly altered in NSCLC. ACOX2 mRNA expression is linked to a number of mutated genes, and associations between ACOX2 expression and tumour mutational burden and immune cell infiltration were explored. Links between ACOX2 expression and candidate therapies for oncogenic driver mutations such as KRAS were also identified. Furthermore, levels of acyl-CoA oxidases and other associated peroxisomal genes were explored to identify further links between the peroxisomal pathway and NSCLC. The results of this biomarker driven study suggest that ACOX2 may have potential clinical utility in the diagnosis, prognosis and stratification of patients into various therapeutically targetable options.

In this study, we report the rejuvenation of aged muscle fibers facilitated by heterochronic parabiosis, underscored by a notable upregulation in peroxisomal gene expression. Our findings highlight the pivotal role of dysregulation in peroxisomal biogenesis and consequential impairment of fatty acid β-oxidation in myoblast differentiation and muscle regeneration. Notably, we demonstrate the novel therapeutic strategy that involves the administration of peroxisome-COS-FITC complexes and nanozymes that mimic peroxisomal function, effectively rejuvenating aged muscle tissues. This innovative approach paves the way for new methods to mitigate or reverse sarcopenia, offering new avenues of the treatment of age-related muscle atrophy and a significant advancement in our understanding for the molecular underpinnings in muscle aging. [Display omitted] • Peroxisome dysfunction linked to sarcopenia and muscle degeneration. • Innovative peroxisomal transplant therapy rejuvenates aged muscles. • Nanozyme technology mimics peroxisomal β-oxidation for muscle health. • Interplay between peroxisomes and mitochondria restores muscle function. [ABSTRACT FROM AUTHOR]

Peroxisomes account for ~35% of total H2O2 generation in mammalian tissues. Peroxisomal ACOX1 (acyl-CoA oxidase 1) is the first and rate-limiting enzyme in fatty acid β-oxidation and a major producer of H2O2 ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5-mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H2O2 production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome-induced oxidative stress, in liver protection, and in suppressing HCC development.

Peroxisomes are organelles that play essential roles in many metabolic processes, but also play roles in innate immunity, signal transduction, aging and cancer. One of the main functions of peroxisomes is the processing of very-long chain fatty acids into metabolites that can be directed to the mitochondria. One key family of enzymes in this process are the peroxisomal acyl-CoA oxidases (ACOX1, ACOX2 and ACOX3), the expression of which has been shown to be dysregulated in some cancers. Very little is however known about the expression of this family of oxidases in non-small cell lung cancer (NSCLC). ACOX2 has however been suggested to be elevated at the mRNA level in over 10% of NSCLC, and in the present study using both standard and bioinformatics approaches we show that expression of ACOX2 is significantly altered in NSCLC. ACOX2 mRNA expression is linked to a number of mutated genes, and associations between ACOX2 expression and tumour mutational burden and immune cell infiltration were explored. Links between ACOX2 expression and candidate therapies for oncogenic driver mutations such as KRAS were also identified. Furthermore, levels of acyl-CoA oxidases and other associated peroxisomal genes were explored to identify further links between the peroxisomal pathway and NSCLC. The results of this biomarker driven study suggest that ACOX2 may have potential clinical utility in the diagnosis, prognosis and stratification of patients into various therapeutically targetable options. [ABSTRACT FROM AUTHOR]

Objective To report a case of peroxisomal acyl⁃CoA oxidase (ACOX1) deficiency, which was caused by an unreported mutation of ACOX1 gene, and review its clinical characteristics. Methods and Results The male child patient suffered from neonatal hypotonia，developmental retardation and epilepsy seizure once since neonatal period. Neurodegeneration appeared at the age of 3 and developed rapidly to difficulty of sitting, standing and walking independently and retrogressive language expression ability. Physical examination showed special face, pyramidal tract sign and cerebellar signs. Cranial MRI revealed symmetric demyelinating lesions in bilateral brain stem and cerebellum along with increased serum very⁃long⁃chain fatty acids (VLCFAs). Whole exome sequencing revealed c. 1589A＞G (p. His530Arg) homozygous mutation in ACOX1 gene. His parents carried the same heterozygous mutation without symptoms and this phenomenon was in accord with co⁃segregation. According to the guidelines of American College of Medical Genetics and Genomics (ACMG), we considered this new variation to be possibly pathogenic variation. Finally, the patient was diagnosed as ACOX1 deficiency. Conclusions As for patients who suffer from hypotonia, epilepsy seizure and growth retardation since neonatal period, they should be altered to ACOX1 deficiency if cranial MRI shows symmetrical abnormal foci in cerebellum and brain stem. Positive family history, increased VLCFAs level, and gene detection would be beneficial to definite diagnosis. doi:Peroxisomes; Acyl⁃CoA oxidase; Genes; Mutation10.3969/j.issn.1672⁃6731.2021.04.009

Peroxisomal acyl-CoA oxidase 1a (ACOX1a) catalyzes the first and rate-limiting step of fatty acid oxidation, the conversion of acyl-CoAs to 2- trans -enoyl-CoAs. The dysfunction of human ACOX1a (hACOX1a) leads to deterioration of the nervous system manifesting in myeloneuropathy, hypotonia and convulsions. Crystal structures of hACOX1a in apo- and cofactor (FAD)-bound forms were solved at 2.00 and 2.09 Å resolution, respectively. hACOX1a exists as a homo-dimer with solvation free energy gain (ΔGo) of −44.7 kcal mol−1. Two FAD molecules bind at the interface of protein monomers completing the active sites. The substrate binding cleft of hACOX1a is wider compared to human mitochondrial very-long chain specific acyl-CoA dehydrogenase. Mutations (p.G178C, p.M278V and p.N237S) reported to cause dysfunctionality of hACOX1a are analyzed on its 3D-structure to understand structure-function related perturbations and explain the associated phenotypes. • Crystal structures of apo- and FAD-bound human peroxisomal ACOX1a are solved. • Human ACOX1a exists as dimer like other homologues. • FAD molecules bind at the interface of ACOX1a monomers completing the active sites. • Mechanistic basis for known pathogenic mutations in human ACOX1a are explained. [ABSTRACT FROM AUTHOR]

The synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D) functions as an agronomic weed control herbicide. High concentrations of 2,4‐D induce plant growth defects, particularly leaf epinasty and stem curvature. Although the 2,4‐D triggered reactive oxygen species (ROS) production, little is known about its signalling. In this study, by using a null mutant in peroxisomal acyl CoA oxidase 1 (acx1‐2), we identified acyl‐coenzyme A oxidase 1 (ACX1) as one of the main sources of ROS production and, in part, also causing the epinastic phenotype following 2,4‐D application. Transcriptomic analyses of wild type (WT) plants after treatment with 2,4‐D revealed a ROS‐related peroxisomal footprint in early plant responses, while other organelles, such as mitochondria and chloroplasts, are involved in later responses. Interestingly, a group of 2,4‐D‐responsive ACX1‐dependent transcripts previously associated with epinasty is related to auxin biosynthesis, metabolism, and signalling. We found that the auxin receptor auxin signalling F‐box 3 (AFB3), a component of Skp, Cullin, F‐box containing complex (SCF) (ASK‐cullin‐F‐box) E3 ubiquitin ligase complexes, which mediates auxin/indole acetic acid (AUX/IAA) degradation by the 26S proteasome, acts downstream of ACX1 and is involved in the epinastic phenotype induced by 2,4‐D. We also found that protein degradation associated with ubiquitin E3‐RING and E3‐SCF‐FBOX in ACX1‐dependent signalling in plant responses to 2,4‐D is significantly regulated over longer treatment periods. Summary Statement: 2,4‐D induced a reactive oxygen species (ROS)‐related peroxisomal footprint in early plant responses. ACX1 is one of the main sources of ROS production and associated epinasty phenotype following 2,4‐D application in plants. ACX1‐dependent signalling in plant responses to 2,4‐D point to protein degradation associated with auxin signalling at the early response and with ubiquitin E3‐RING and E3‐SCF‐FBOX/proteasome as key processes in the development of epinasty over longer treatment periods. [ABSTRACT FROM AUTHOR]

ObjectiveAcyl-CoA oxidase (ACOX1) deficiency is a rare disorder of peroxisomal very-long chain fatty acid oxidation. No reports detailing attempted treatment, longitudinal imaging, or neuropathology exist. We describe the natural history of clinical symptoms and brain imaging in two siblings with ACOX1 deficiency, including the younger sibling's response to allogeneic unrelated donor hematopoietic stem cell transplantation (HSCT).MethodsWe conducted retrospective chart review to obtain clinical history, neuro-imaging, and neuropathology data. ACOX1 genotyping were performed to confirm the disease. In vitro fibroblast and neural stem cell fatty acid oxidation assays were also performed.ResultsBoth patients experienced a fatal neurodegenerative course, with late-stage cerebellar and cerebral gray matter atrophy. Serial brain magnetic resonance imaging in the younger sibling indicated demyelination began in the medulla and progressed rostrally to include the white matter of the cerebellum, pons, midbrain, and eventually subcortical white matter. The successfully engrafted younger sibling had less brain inflammation, cortical atrophy, and neuronal loss on neuro-imaging and neuropathology compared to the untreated older sister. Fibroblasts and stem cells demonstrated deficient very long chain fatty acid oxidation.InterpretationAlthough HSCT did not halt the course of ACOX1 deficiency, it reduced the extent of white matter inflammation in the brain. Demyelination continued because of ongoing neuronal loss, which may be due to inability of transplant to prevent progression of gray matter disease, adverse effects of chronic corticosteroid use to control graft-versus-host disease, or intervention occurring beyond a critical point for therapeutic efficacy.

Objective To report a case of peroxisomal acyl-CoA oxidase (ACOX1) deficiency, which was caused by an unreported mutation of ACOX1 gene, and review its clinical characteristics. Methods and Results The male child patient suffered from neonatal hypotonia, developmental retardation and epilepsy seizure once since neonatal period. Neurodegeneration appeared at the age of 3 and developed rapidly to difficulty of sitting, standing and walking independently and retrogressive language expression ability. Physical examination showed special face, pyramidal tract sign and cerebellar signs. Cranial MRI revealed symmetric demyelinating lesions in bilateral brain stem and cerebellum along with increased serum very-long-chain fatty acids (VLCFAs). Whole exome sequencing revealed c. 1589A > G (p. His530Arg) homozygous mutation in ACOX1 gene. His parents carried the same heterozygous mutation without symptoms and this phenomenon was in accord with co-segregation. According to the guidelines of American College of Medical Genetics and Genomics (ACMG), we considered this new variation to be possibly pathogenic variation. Finally, the patient was diagnosed as ACOX1 deficiency. Conclusions As for patients who suffer from hypotonia, epilepsy seizure and growth retardation since neonatal period, they should be altered to ACOX1 deficiency if cranial MRI shows symmetrical abnormal foci in cerebellum and brain stem. Positive family history, increased VLCFAs level, and gene detection would be beneficial to definite diagnosis. [ABSTRACT FROM AUTHOR]

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems. [ABSTRACT FROM AUTHOR]

Dicarboxylic fatty acids, taken as a nutritional supplement or produced endogenously via omega oxidation of monocarboxylic fatty acids, may have therapeutic potential for rare inborn errors of metabolism as well as common metabolic diseases such as type 2 diabetes. Breakdown of dicarboxylic acids yields acetyl-CoA and succinyl-CoA as products, the latter of which is anaplerotic for the TCA cycle. However, little is known about the metabolic pathways responsible for degradation of dicarboxylic acids. Here, we demonstrated with whole-cell fatty acid oxidation assays that both mitochondria and peroxisomes contribute to dicarboxylic acid degradation. Several mitochondrial acyl-CoA dehydrogenases were tested for activity against dicarboxylyl-CoAs. Medium-chain acyl-CoA dehydrogenase (MCAD) exhibited activity with both six and 12 carbon dicarboxylyl-CoAs, and the capacity for dehydrogenation of these substrates was significantly reduced in MCAD knockout mouse liver. However, when dicarboxylic acids were fed to normal mice, the expression of MCAD did not change, while expression of peroxisomal fatty acid oxidation enzymes was greatly upregulated. In conclusion, mitochondrial fatty acid oxidation, and in particular MCAD, contributes to dicarboxylic acid degradation, but feeding dicarboxylic acids induces only the peroxisomal pathway. • Both mitochondria and peroxisomes chain-shorten dicarboxylic fatty acids (DCAs). • Medium-chain acyl-CoA dehydrogenase is active against dicarboxylyl-CoAs. • Feeding DCAs induces liver expression of peroxisomal, but mitochondrial, enzymes. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

Epigenetic markers, such as histone acetylation and DNA methylation, determine chromatin organization. In eukaryotic cells, metabolites from organelles or the cytosol affect epigenetic modifications. However, the relationships between metabolites and epigenetic modifications are not well understood in plants. We found that peroxisomal acyl-CoA oxidase 4 (ACX4), an enzyme in the fatty acid ß-oxidation pathway, is required for suppressing the silencing of some endogenous loci, as well as Pro35S:NPTII in the ProRD29A:LUC/C24 transgenic line. The acx4 mutation reduces nuclear histone acetylation and increases DNA methylation at the NOS terminator of Pro35S:NPTII and at some endogenous genomic loci, which are also targeted by the demethylation enzyme REPRESSOR OF SILENCING 1 (ROS1). Furthermore, mutations in multifunctional protein 2 (MFP2) and 3-ketoacyl- CoA thiolase-2 (KAT2/PED1/PKT3), two enzymes in the last two steps of the ß-oxidation pathway, lead to similar patterns of DNA hypermethylation as in acx4. Thus, metabolites from fatty acid ß-oxidation in peroxisomes are closely linked to nuclear epigenetic modifications, which may affect diverse cellular processes in plants. [ABSTRACT FROM AUTHOR]

Abstract Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) β–oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1 -deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H 2 O 2 , was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1β and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1 -deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal β–oxidation defect on oxidative stress, inflammation and cellular functions. Highlights • Generation by CRISPR/Cas9 editing of a mouse microglial cell line with ACOX1 deficiency • Accumulation of VLCFA and changes in lipid droplets distribution in mutant cells • Increased catalase activity in Acox1-deficient cells • Increased number of peroxisomes and mitochondria in mutant cells • Expression of key microglial markers is modified in mutant cells [ABSTRACT FROM AUTHOR]

Abstract The biosynthesis of jasmonic acid (JA) in plant peroxisomes requires the action of acyl-CoA oxidase (ACX; EC 1.3.3.6). Multiple isoforms of ACXs have been identified in various annual herbaceous plants, but the genes encoding these enzymes in perennial woody plants are yet to be fully investigated. In this study, an ACX gene named CsACX1 (GeneBank accession: KX650077.1) was isolated from tea plant (Camellia sinensis L.). CsACX1 was predicted to consist of 664 amino acid residues. Transcriptional analysis revealed that CsACX1 can be induced by mechanical wounding, JA application, and infestation by the tea geometrid Ectropis obliqua Prout and the tea green leafhopper Empoasca (Matsumurasca) onukii Matsuda. To further elucidate the function of CsACX1 , it was heterologously expressed in a bacterial system and characterized. Recombinant CsACX1 showed preference for C 12 ∼ C 16 -CoA substrates. The constitutive expression of CsACX1 can rescue wound-related JA biosynthesis in Arabidopsis mutant acx1. CsACX1 was expressed in different organs, predominantly in flowers. Notably, CsACX1 transcripts were detected up-regulated during flower opening, and the JA levels were correlated with CsACX1 expression. All these results enrich our knowledge of the regulatory pathway involved in the JA biosynthesis in tea, and helps further understand the defense mechanism of tea plant against insects. Highlights • A putative ACX gene, CsACX1 , was isolated from tea plants. • Herbivore infestation, wound and JA application could elevate the CsACX1 expression. • Recombinant CsACX1 showed preference for C12- ∼ C16-CoA substrates. • Overexpression of CsACX1 can rescue JA biosynthesis in Arabidopsis mutant acx1. • CsACX1 transcripts were detected up-regulated during flower opening. [ABSTRACT FROM AUTHOR]

A lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A β-oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of β-oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.

Caenorhabditis elegans produces ascaroside pheromones to control its development and behavior. Even minor structural differences in the ascarosides have dramatic consequences for their biological activities. Here, we identify a mechanism that enables C. elegans to dynamically tailor the fatty-acid side chains of the indole-3-carbonyl (IC)-modified ascarosides it has produced. In response to starvation, C. elegans uses the peroxisomal acyl-CoA synthetase ACS-7 to activate the side chains of medium-chain IC-ascarosides for β-oxidation involving the acyl-CoA oxidases ACOX-1.1 and ACOX-3. This pathway rapidly converts a favorable ascaroside pheromone that induces aggregation to an unfavorable one that induces the stress-resistant dauer larval stage. Thus, the pathway allows the worm to respond to changing environmental conditions and alter its chemical message without having to synthesize new ascarosides de novo. We establish a new model for biosynthesis of the IC-ascarosides in which side-chain β-oxidation is critical for controlling the type of IC-ascarosides produced.

Acyl-coA oxidase (ACO) is an important flavoenzyme responsible for the first step of peroxisomal fatty acid β-oxidation. In this study, the roles of Tyr232 and Tyr401 in flavin adenine dinucleotide (FAD) binding and enzyme catalysis of ACO were explored using site-directed mutagenesis. For mutant proteins, different levels of activity loss were observed. Wavelength scanning of Y232 and Y401 mutant proteins indicated that there is no FAD binding in Y401S and Y401G mutant ACO. Structure analysis indicated that the phenolic hydroxyl and benzene ring of the side chain could stabilize FAD binding through hydrogen bonds network and hydrophobic pocket formation. These results indicated that these two tyrosine residues play a critical role in the FAD binding of ACO. [ABSTRACT FROM AUTHOR]

Summary: Lipid degradation processes are important in microalgae because survival and growth of microalgal cells under fluctuating environmental conditions require permanent remodeling or turnover of membrane lipids as well as rapid mobilization of storage lipids. Lipid catabolism comprises two major spatially and temporarily separated steps, namely lipolysis, which releases fatty acids and head groups and is catalyzed by lipases at membranes or lipid droplets, and degradation of fatty acids to acetyl‐CoA, which occurs in peroxisomes through the β‐oxidation pathway in green microalgae, and can sometimes occur in mitochondria in some other algal species. Here we review the current knowledge on the enzymes and regulatory proteins involved in lipolysis and peroxisomal β‐oxidation and highlight gaps in our understanding of lipid degradation pathways in microalgae. Metabolic use of acetyl‐CoA products via glyoxylate cycle and gluconeogenesis is also reviewed. We then present the implication of various cellular processes such as vesicle trafficking, cell cycle and autophagy on lipid turnover. Finally, physiological roles and the manipulation of lipid catabolism for biotechnological applications in microalgae are discussed. [ABSTRACT FROM AUTHOR]

Peroxisomal acyl-CoA oxidases catalyze the first step of beta-oxidation of a variety of substrates broken down in the peroxisome. These include the CoA-esters of very long-chain fatty acids, branched-chain fatty acids and the C27-bile acid intermediates. In rat, three peroxisomal acyl-CoA oxidases with different substrate specificities are known, whereas in humans it is believed that only two peroxisomal acyl-CoA oxidases are expressed under normal circumstances. Only three patients with ACOX2 deficiency, including two siblings, have been identified so far, showing accumulation of the C27-bile acid intermediates. Here, we performed biochemical studies in material from a novel ACOX2-deficient patient with increased levels of C27-bile acids in plasma, a complete loss of ACOX2 protein expression on immunoblot, but normal pristanic acid oxidation activity in fibroblasts. Since pristanoyl-CoA is presumed to be handled by ACOX2 specifically, these findings prompted us to re-investigate the expression of the human peroxisomal acyl-CoA oxidases. We report for the first time expression of ACOX3 in normal human tissues at the mRNA and protein level. Substrate specificity studies were done for ACOX1, 2 and 3 which revealed that ACOX1 is responsible for the oxidation of straight-chain fatty acids with different chain lengths, ACOX2 is the only human acyl-CoA oxidase involved in bile acid biosynthesis, and both ACOX2 and ACOX3 are involved in the degradation of the branched-chain fatty acids. Our studies provide new insights both into ACOX2 deficiency and into the role of the different acyl-CoA oxidases in peroxisomal metabolism. [ABSTRACT FROM AUTHOR]

Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S)-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S)-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S)-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS) based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases. [ABSTRACT FROM AUTHOR]

Single cell oil (SCO) is the lipid accumulated in the cells of oleaginous microorganisms. Cellular lipids can be synthesized in two different pathways: de novo by metabolizing hydrophilic substrates and ex novo by fermenting hydrophobic substrates. The aim of the study was to evaluate the effect of carbon source (glucose and olive oil) in the culture medium on the course of microbial oil accumulation in Y. lipolytica cells. The level of selected gene expression by real time quantitative PCR method was investigated. The significant increase in expression of the POX2 gene encoding acyl-CoA oxidase II, which preferentially oxidizes long-chain acyl-CoAs formed from substrate fatty acids incorporated inside the microbial cell, was observed in medium with olive oil in relation to glucose containing medium. Noteworthily, the presence of lipid carbon substrate did not inhibit the level of ACL gene transcription coding for ATP-citrate lyase, the key enzyme of the lipid de novo accumulation process. The present study indicated that de novo lipid biosynthesis could occur despite the presence of fatty acids in the medium, and the synthesis of storage lipids in the presence of lipid carbon substrates could be carried out with the use of both pathways (de novo and ex novo).

Increased fatty acid β-oxidation is essential for early postgerminative growth in seedlings, but high levels of H2O2 produced by β-oxidation can induce oxidative stress. Whether and how catalase (CAT) functions in fine-tuning H2O2 homeostasis during seedling growth remain unclear. Here, we report that CAT2 functions in early seedling growth. Compared to the wild type, the cat2-1 mutant, with elevated H2O2 levels, exhibited reduced root elongation on sucrose (Suc)-free medium, mimicking soils without exogenous sugar supply. Treatment with the H2O2 scavenger potassium iodide rescued the mutant phenotype of cat2-1. In contrast to the wild type, the cat2-1 mutant was insensitive to the CAT inhibitor 3-amino-1,2,4-triazole in terms of root elongation when grown on Suc-free medium, suggesting that CAT2 modulates early seedling growth by altering H2O2 accumulation. Furthermore, like cat2-1, the acyl-CoA oxidase (ACX) double mutant acx2-1 acx3-6 showed repressed root elongation, suggesting that CAT2 functions in early seedling growth by regulating ACX activity, as this activity was inhibited in cat2-1. Indeed, decreased ACX activity and short root of cat2-1 seedlings grown on Suc-free medium were rescued by overexpressing ACX3. Together, these findings suggest that CAT2 functions in early seedling growth by scavenging H2O2 and stimulating ACX2/3 activity. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of a high fat diet with experimental oil consisting of 60% MUFAs (monounsaturated fatty acids) with a P/S ratio of 5 on fat deposition and lipid metabolism in obese hamsters. Hamsters were randomly assigned to a control group and a diet-induced obesity group for nine weeks. Then an additional eight-week experimental period began, during which obese hamsters were randomly divided into three groups and fed different amounts of the experimental oil mixture in their diets as follows: 5%, 15%, and 20% w/w (OB-M5, OB-M15, and OB-M20 groups, respectively). The results showed that the OB-M15 and OB-M20 groups had significantly lower blood cholesterol and higher insulin levels. Compared to the control group, the three obese groups exhibited higher hepatic fatty acid synthase activity; however, the acyl-CoA oxidase activities were also enhanced. Although dietary fat content differed, there were no differences in energy intake, final body weights, and epididymal fat weights among the four groups. These results suggest that regardless of whether the specimens had a high fat intake or not, dietary fat containing high MUFAs with a high P/S ratio had beneficial effects on maintaining blood lipid profiles and may not result in body fat accumulation in obese hamsters, possibly by promoting lipolytic enzyme activities. [ABSTRACT FROM AUTHOR]

Key message: γ-Decalactone accumulation in peach mesocarp was highly correlated with ACX enzyme activity and natural PpACX1 content. Adding the purified recombinant PpACX1 induced γ-decalactone biosynthesis in cultured mesocarp discs in vitro. Abstract: Previous gene expression studies have implied that acyl coenzyme A oxidase (ACX) is related to lactones synthesis, the characteristic aroma compounds of peach. Here, we analysed the correlation between γ-decalactone content and ACX enzyme activity in mesocarp of five different types of fully ripe peach varieties. Furthermore, 'Hu Jing Mi Lu' ('HJ') and 'Feng Hua Yu Lu' ('YL'), which have strong aroma among them, at four ripening stages were selected to study the role of ACX in lactone biosynthesis. The result showed that γ-decalactone was the most abundant lactone compound. γ-Decalactone accumulation was highly correlated with ACX enzyme activity. Mass spectrometry (MS) showed that PpACX1 was the most abundant PpACX protein in fully ripe mesocarp of cv. 'HJ'. To further elucidate the function of the PpACX1 protein, the PpACX1 gene was heterologously expressed in a bacterial system and characterized in vitro. MS identification gave the molecular weight of the recombinant PpACX1 as 94.44 kDa and the coverage rate of the peptide segments was 47.3%. In cultured mesocarp discs in vitro, adding the purified recombinant PpACX1 and C-CoA substrate induced the expected γ-decalactone biosynthesis. Using a sandwich ELISA based on mixed mono- and polyclonal antibodies against recombinant PpACX1, PpACX1 content in mesocarp was found to be highly correlated with γ-decalactone accumulation in mesocarp of five fully ripe varieties and four ripening stages of 'HJ' and 'YL'. This study revealed the vital function of PpACX1 in γ-decalactone biosynthesis in peach fruit. [ABSTRACT FROM AUTHOR]

Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 ( CrACX2), a gene encoding a member of the acyl-CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl-CoAs into trans-2-enoyl-CoA and produced H2O2. This result demonstrated that CrACX2 is a genuine acyl-CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) β-oxidation spiral. A fluorescent protein-tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA β-oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of β-oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant-type FA β-oxidation involving H2O2-producing acyl-CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii. [ABSTRACT FROM AUTHOR]

A chronic high fat diet results in hepatic mitochondrial dysfunction and induction of peroxisomal fatty acid oxidation (FAO); whether specific inhibition of peroxisomal FAO benefits mitochondrial FAO and reactive oxygen species (ROS) metabolism remains unclear. In this study a specific inhibitor for the rate-limiting enzyme involved in peroxisomal FAO, acyl-CoA oxidase-1 (ACOX1) was developed and used for the investigation of peroxisomal FAO inhibition upon mitochondrial FAO and ROS metabolism. Specific inhibition of ACOX1 by 10,12-tricosadiynoic acid increased hepatic mitochondrial FAO via activation of the SIRT1-AMPK (adenosine 5′-monophosphateactivated protein kinase) pathway and proliferator activator receptor α and reduced hydrogen peroxide accumulation in high fat diet-fed rats, which significantly decreased hepatic lipid and ROS contents, reduced body weight gain, and decreased serum triglyceride and insulin levels. Inhibition of ACOX1 is a novel and effective approach for the treatment of high fat dietor obesity-induced metabolic diseases by improving mitochondrial lipid and ROS metabolism. [ABSTRACT FROM AUTHOR]

Background & Aims Acyl-CoA oxidase (ACOX2) is involved in the shortening of C27 cholesterol derivatives to generate C24 bile acids. Inborn errors affecting the rest of peroxisomal enzymes involved in bile acid biosynthesis have been described. Here we aimed at investigating the case of an adolescent boy with persistent hypertransaminasemia of unknown origin and suspected dysfunction in bile acid metabolism. Methods Serum and urine samples were taken from the patient, his sister and parents and underwent HPLC-MS/MS and HPLC-TOF analyses. Coding exons in genes of interest were amplified by high-fidelity PCR and sequenced. Wild-type or mutated (mutACOX2) variants were overexpressed in human hepatoblastoma HepG2 cells to determine ACOX2 enzymatic activity, expression and subcellular location. Results The patient’s serum and urine showed negligible amounts of C24 bile acids, but augmented levels of C27 intermediates, mainly tauroconjugated trihydroxycholestanoic acid (THCA). Genetic analysis of enzymes potentially involved revealed a homozygous missense mutation (c.673C>T; R225W) in ACOX2 . His only sister was also homozygous for this mutation and exhibited similar alterations in bile acid profiles. Both parents were heterozygous and presented normal C24 and C27 bile acid levels. Immunofluorescence studies showed similar protein size and peroxisomal localization for both normal and mutated variants. THCA biotransformation into cholic acid was enhanced in cells overexpressing ACOX2, but not in those overexpressing mutACOX2. Both cell types showed similar sensitivity to oxidative stress caused by C24 bile acids. In contrast, THCA-induced oxidative stress and cell death were reduced by overexpressing ACOX2, but not mutACOX2. Conclusion ACOX2 deficiency, a condition characterized by accumulation of toxic C27 bile acid intermediates, is a novel cause of isolated persistent hypertransaminasemia. Lay summary Elevation of serum transaminases is a biochemical sign of liver damage due to multiplicity of causes (viruses, toxins, autoimmunity, metabolic disorders). In rare cases the origin of this alteration remains unknown. We have identified by the first time in a young patient and his only sister a familiar genetic defect of an enzyme called ACOX2, which participates in the transformation of cholesterol into bile acids as a cause of increased serum transaminases in the absence of any other symptomatology. This treatable condition should be considered in the diagnosis of those patients where the cause of elevated transaminases remains obscure. [ABSTRACT FROM AUTHOR]

Objectives: To explore Candida guilliermondii for the production of long-chain dicarboxylic acids (DCA), we performed metabolic pathway engineering aiming to prevent DCA consumption during β-oxidation, but also to increase its production via the ω-oxidation pathway. Results: We identified the major β- and ω-oxidation pathway genes in C. guilliermondii and performed first steps in the strain improvement. A double pox disruption mutant was created that slowed growth with oleic acid but showed accelerated DCA degradation. Increase in DCA production was achieved by homologous overexpression of a plasmid borne cytochrome P450 monooxygenase gene. Conclusion: C. guilliermondii is a promising biocatalyst for DCA production but further insight into its fatty acid metabolism is necessary. [ABSTRACT FROM AUTHOR]