Acetyl-CoA Derived from Hepatic Peroxisomal ß-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 Activation.

Autophagy is activated by prolonged fasting but cannot overcome the ensuing hepatic lipid overload, resulting in fatty liver. Here, we describe a peroxisome-lysosome metabolic link that restricts autophagic degradation of lipids. Acyl-CoA oxidase 1 (Acox1), the enzyme that catalyzes the first step in peroxisomal ß-oxidation, is enriched in liver and further increases with fasting or high-fat diet (HFD). Liver-specific Acox1 knockout (Acox1-LKO) protected mice against hepatic steatosis caused by starvation or HFD due to induction of autophagic degradation of lipid droplets. Hepatic Acox1 deficiency markedly lowered total cytosolic acetyl-CoA levels, which led to decreased Raptor acetylation and reduced lysosomal localization of mTOR, resulting in impaired activation of mTORC1, a central regulator of autophagy. Dichloroacetic acid treatment elevated acetyl-CoA levels, restored mTORC1 activation, inhibited autophagy, and increased hepatic triglycerides in Acox1-LKO mice. These results identify peroxisome-derived acetyl-CoA as a key metabolic regulator of autophagy that controls hepatic lipid homeostasis.

SH-SY5Y cells undergo changes in peroxisomal metabolism when exposed to decanoic acid.

Medium-chain fatty acids (MCFAs), particularly decanoic acid (C10) and octanoic acid (C8), have garnered attention in recent years for their potential antiepileptic properties. A previous study from our laboratory demonstrated that C10 targets the PPARγ nuclear receptor, increasing the activity of the antioxidant enzyme catalase and thereby possibly modulating peroxisomal content. Here, we examined markers of peroxisomal content and activity in response to C10 and C8 exposure in neuronal-like SH-SY5Y cells. SH-SY5Y were treated with 250 mM C10 or C8 for a period of 6 days. Following this, biochemical markers of peroxisomal content and function were assessed, including acyl-coA oxidase activity, peroxisomal gene expression and peroxisomal VLCFA ß-oxidation. Our findings revealed that C10 treatment augments acyl-CoA oxidase 1 (ACOx1) activity by 129% in comparison to control cells. An exploration into genes related to peroxisomal biosynthesis showed 23% increased expression of PEX11α upon C10 exposure, implying peroxisomal proliferation. Furthermore, it was observed that C10 exposure not only elevated ACOx1 activity but also enhanced peroxisomal ß-oxidation of docosanoic acid (C22). Our findings bolster the premise that C10 functions as a peroxisome proliferator, influencing peroxisomal content and function. Further investigations are required to fully understand the mechanistic details as to how this may be beneficial in epilepsy and the potential implications with regards to peroxisomal disease.

ACOX1 deficiency-induced lipid metabolic disorder facilitates chronic interstitial fibrosis development in renal allografts.

Chronic interstitial fibrosis presents a significant challenge to the long-term survival of transplanted kidneys. Our research has shown that reduced expression of acyl-coenzyme A oxidase 1 (ACOX1), which is the rate-limiting enzyme in the peroxisomal fatty acid ß-oxidation pathway, contributes to the development of fibrosis in renal allografts. ACOX1 deficiency leads to lipid accumulation and excessive oxidation of polyunsaturated fatty acids (PUFAs), which mediate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) reorganization respectively, thus causing fibrosis in renal allografts. Furthermore, activation of Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) signaling induced ACOX1 downregulation in a DNA methyltransferase 1 (DNMT1)-dependent manner. Overconsumption of PUFA resulted in endoplasmic reticulum (ER) stress, which played a vital role in facilitating ECM reorganization. Supplementation with PUFAs contributed to delayed fibrosis in a rat model of renal transplantation. The study provides a novel therapeutic approach that can delay chronic interstitial fibrosis in renal allografts by targeting the disorder of lipid metabolism.

SLC9A5 promotes tumor growth and cell motility via ACOX1-mediated peroxisomal fatty acid oxidation.

Growing evidence suggests a strong association between decreased lipid catabolism and the development of cancer. Solute carrier family 9 member A5 (SLC9A5) plays a regulatory role in colorectal function. However, the specific involvement of SLC9A5 in colorectal cancer (CRC) remains unclear, as well as its potential connection to lipid catabolism. We found that SLC9A5 exhibited significantly higher expression in CRC tumor tissues compared to adjacent paratumor tissues, as confirmed through analysis of the TCGA database and validation on a CRC tissue chip using IHC. Furthermore, in vitro experiments showed that knockdown of SLC9A5 resulted in suppressed cell proliferation, migration, and invasion. Then we performed bioinformatics analysis and found that SLC9A5 was significantly enriched in peroxisomal fatty acid oxidation (FAO) pathway and negatively correlated with its first rate-limiting enzyme acyl-CoA oxidases (ACOX). Interestingly, the expression of ACOX1, as well as FAO process indicated by changes in very long chain fatty acid levels, were enhanced upon SLC9A5 knockdown in CRC cells. Moreover, the attenuated tumor growth, migration, invasion, and increased FAO observed after SLC9A5 knockdown could be reversed by simultaneous knockdown of both SLC9A5 and ACOX1. In summary, these findings reveal the oncogenic role of SLC9A5 in CRC, particularly in relation to ACOX1-mediated peroxidation, and might serve as a promising therapeutic target for inhibiting the progression of colorectal cancer.

Sequencing at lymphoid neoplasm susceptibility loci maps six myeloma risk genes.

Inherited genetic risk factors play a role in multiple myeloma (MM), yet considerable missing heritability exists. Rare risk variants at genome-wide association study (GWAS) loci are a new avenue to explore. Pleiotropy between lymphoid neoplasms (LNs) has been suggested in family history and genetic studies, but no studies have interrogated sequencing for pleiotropic genes or rare risk variants. Sequencing genetically enriched cases can help discover rarer variants. We analyzed exome sequencing in familial or early-onset MM cases to identify rare, functionally relevant variants near GWAS loci for a range of LNs. A total of 149 distinct and significant LN GWAS loci have been published. We identified six recurrent, rare, potentially deleterious variants within 5 kb of significant GWAS single nucleotide polymorphisms in 75 MM cases. Mutations were observed in BTNL2, EOMES, TNFRSF13B, IRF8, ACOXL and TSPAN32. All six genes replicated in an independent set of 255 early-onset MM or familial MM or precursor cases. Expansion of our analyses to the full length of these six genes resulted in a list of 39 rare and deleterious variants, seven of which segregated in MM families. Three genes also had significant rare variant burden in 733 sporadic MM cases compared with 935 control individuals: IRF8 (P = 1.0 × 10-6), EOMES (P = 6.0 × 10-6) and BTNL2 (P = 2.1 × 10-3). Together, our results implicate six genes in MM risk, provide support for genetic pleiotropy between LN subtypes and demonstrate the utility of sequencing genetically enriched cases to identify functionally relevant variants near GWAS loci.

Acyl CoA oxidase: from its expression, structure, folding, and import to its role in human health and disease.

ß-oxidation of fatty acids is an important metabolic pathway and is a shared function between mitochondria and peroxisomes in mammalian cells. On the other hand, peroxisomes are the sole site for the degradation of fatty acids in yeast. The first reaction of this pathway is catalyzed by the enzyme acyl CoA oxidase housed in the matrix of peroxisomes. Studies in various model organisms have reported the conserved function of the protein in fatty acid oxidation. The importance of this enzyme is highlighted by the lethal conditions caused in humans due to its altered function. In this review, we discuss various aspects ranging from gene expression, structure, folding, and import of the protein in both yeast and human cells. Further, we highlight recent findings on the role of the protein in human health and aging, and discuss the identified mutations in the protein associated with debilitating conditions in patients.

Acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 to play roles in larval development of Haemonchus contortus.

Hypobiosis (facultative developmental arrest) is the most important life-cycle adaptation ensuring survival of parasitic nematodes under adverse conditions. Little is known about such survival mechanisms, although ascarosides (ascarylose with fatty acid-derived side chains) have been reported to mediate the formation of dauer larvae in the free-living nematode Caenorhabditis elegans. Here, we investigated the role of a key gene acox-1, in the larval development of Haemonchus contortus, one of the most important parasitic nematodes that employ hypobiosis as a routine survival mechanism. In this parasite, acox-1 encodes three proteins (ACOXs) that all show a fatty acid oxidation activity in vitro and in vivo, and interact with a peroxin PEX-5 in peroxisomes. In particular, a peroxisomal targeting signal type1 (PTS1) sequence is required for ACOX-1 to be recognised by PEX-5. Analyses on developmental transcription and tissue expression show that acox-1 is predominantly expressed in the intestine and hypodermis of H. contortus, particularly in the early larval stages in the environment and the arrested fourth larval stage within host animals. Knockdown of acox-1 and pex-5 in parasitic H. contortus shows that these genes play essential roles in the post-embryonic larval development and likely in the facultative arrest of this species. A comprehensive understanding of these genes and the associated ß-oxidation cycle of fatty acids should provide novel insights into the developmental regulation of parasitic nematodes, and into the discovery of novel interventions for species of socioeconomic importance.

Beneficial effect of ursodeoxycholic acid in patients with acyl-CoA oxidase 2 (ACOX2) deficiency-associated hypertransaminasemia.

BACKGROUND AND AIMS: A variant (p.Arg225Trp) of peroxisomal acyl-CoA oxidase 2 (ACOX2), involved in bile acid (BA) side-chain shortening, has been associated with unexplained persistent hypertransaminasemia and accumulation of C27-BAs, mainly 3α,7α,12α-trihydroxy-5ß-cholestanoic acid (THCA). We aimed to investigate the prevalence of ACOX2 deficiency-associated hypertransaminasemia (ADAH), its response to ursodeoxycholic acid (UDCA), elucidate its pathophysiological mechanism and identify other inborn errors that could cause this alteration. METHODS AND RESULTS: Among 33 patients with unexplained hypertransaminasemia from 11 hospitals and 13 of their relatives, seven individuals with abnormally high C27-BA levels (>50% of total BAs) were identified by high-performance liquid chromatography-mass spectrometry. The p.Arg225Trp variant was found in homozygosity (exon amplification/sequencing) in two patients and three family members. Two additional nonrelated patients were heterozygous carriers of different alleles: c.673C>T (p.Arg225Trp) and c.456_459del (p.Thr154fs). In patients with ADAH, impaired liver expression of ACOX2, but not ACOX3, was found (immunohistochemistry). Treatment with UDCA normalized aminotransferase levels. Incubation of HuH-7 hepatoma cells with THCA, which was efficiently taken up, but not through BA transporters, increased reactive oxygen species production (flow cytometry), endoplasmic reticulum stress biomarkers (GRP78, CHOP, and XBP1-S/XBP1-U ratio), and BAXα expression (reverse transcription followed by quantitative polymerase chain reaction and immunoblot), whereas cell viability was decreased (tetrazolium salt-based cell viability test). THCA-induced cell toxicity was higher than that of major C24-BAs and was not prevented by UDCA. Fourteen predicted ACOX2 variants were generated (site-directed mutagenesis) and expressed in HuH-7 cells. Functional tests to determine their ability to metabolize THCA identified six with the potential to cause ADAH. CONCLUSIONS: Dysfunctional ACOX2 has been found in several patients with unexplained hypertransaminasemia. This condition can be accurately identified by a noninvasive diagnostic strategy based on plasma BA profiling and ACOX2 sequencing. Moreover, UDCA treatment can efficiently attenuate liver damage in these patients.

PPARα agonist WY-14,643 induces the PLA2/COX-2/ACOX1 pathway to enhance peroxisomal lipid metabolism and ameliorate alcoholic fatty liver in mice.

Peroxisome proliferator-activated receptor α (PPARα) regulates fatty acid oxidation (FAO). Usually, very-long chain fatty acids are first activated by acyl-CoA synthetase (ACS) to generate acyl-CoA for oxidation by acyl-CoA oxidase (ACOX) in peroxisomes, and the resultant shorter chain fatty acids will be further oxidized in mitochondria. ACS long-chain family member 4 (ACSL4) preferentially uses arachidonic acid (AA) as substrates to synthesize arachidonoyl-CoA. Arachidonoyl-CoA is usually esterified into phospholipids. When AA is released by phospholipase A2 (PLA2) from phospholipids, it will be used for prostaglandin synthesis by cyclooxygenases (COX). In this study, when PPARα agonist WY-14,643 was mixed in liquid Lieber-DeCarli ethanol or control diets and fed to mice, liver PLA2, COX-2, and ACOX1 were induced but ACSL4 was inhibited, suggesting that AA released by PLA2 from phospholipid will be metabolized to prostaglandin via COX-2 instead of being synthesized into acyl-CoA by ACSL4. However, liver prostaglandin E2 (PGE2), a major component of prostaglandin, was not increased with the induced COX-2 but decreased by WY-14,643. ACOX1 specific inhibitor mixed in the liquid diets restored both the WY-14,643-suppressed liver TG and PGE2, but COX-2 specific inhibitor celecoxib mixed in the liquid diets reversed the WY-14,643-suppressed liver TG but not liver PGE2 contents. These results suggest that induction of PLA2, COX-2 and ACOX1 orchestrates to increase oxidation of AA/PGE2, which constitutes one new mechanism by which PPARα induces peroxisomal FAO and inhibits ethanol-induced liver fat accumulation.

MiR-103-3p promotes hepatic steatosis to aggravate nonalcoholic fatty liver disease by targeting of ACOX1.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma, and alterations in miRNA expression are related to the development of NAFLD. However, the role of miRNAs in regulating the development of NAFLD is still poorly understood. METHODS: We used qRT-PCR to detect the level of miR-103-3p in both cell and mouse models of NAFLD. Biochemical assays, DCF-DA assays, Oil red O staining and HE staining were used to detect the role of miR-103-3p in NAFLD development. Target genes of miR-103-3p were predicted using the TargetScan database and verified by qRT-PCR, western blot and dual-luciferase assays. RESULTS: The expression of miR-103-3p increased in both NAFLD model cells and liver tissues from the NAFLD mouse model. Inhibition of miR-103-3p significantly alleviated the accumulation of lipid droplets in free fatty acid-treated L02 cells and liver tissues from mice with NAFLD. Inhibition of miR-103-3p reduced the contents of H2O2, TG, ALT, and AST and ROS production while increasing the ATP content. Moreover, the miR-103-3p antagomir alleviated liver tissue lesions in mice with NAFLD. Further studies identified ACOX1, a key enzyme for the oxidation and decomposition of fatty acids, as a direct target of miR-103-3p. CONCLUSIONS: These findings identified a negative regulatory mechanism between ACOX1 and miR-103-3p that promotes the pathogenesis of NAFLD and suggested that inhibition of miR-103-3p may be a potential treatment strategy for NAFLD.

Altered expression of ACOX2 in non-small cell lung cancer.

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.

Expression Plasticity of Peroxisomal Acyl-Coenzyme A Oxidase Genes Implies Their Involvement in Redox Regulation in Scallops Exposed to PST-Producing Alexandrium.

Filter-feeding bivalves can accumulate paralytic shellfish toxins (PST) produced by toxic microalgae, which may induce oxidative stress and lipid peroxidation. Peroxisomal acyl-coenzyme A oxidases (ACOXs) are key enzymes functioning in maintaining redox and lipid homeostasis, but their roles in PST response in bivalves are less understood. Herein, a total of six and six ACOXs were identified in the Chlamys farreri and Patinopecten yessoensis genome, respectively, and the expansion of ACOX1s was observed. Gene expression analysis revealed an organ/tissue-specific expression pattern in both scallops, with all ACOXs being predominantly expressed in the two most toxic organs, digestive glands and kidneys. The regulation patterns of scallop ACOXs after exposure to different PST-producing algaes Alexandrium catenella (ACDH) and A. minutum (AM-1) were revealed. After ACDH exposure, more differentially expressed genes (DEGs) were identified in C. farreri digestive glands (three) and kidneys (five) than that in P. yessoensis (two), but the up-regulated DEGs showed similar expression patterns in both species. In C. farreri, three DEGs were found in both digestive glands and kidneys after AM-1 exposure, with two same CfACOX1s being acutely and chronically induced, respectively. Notably, these two CfACOX1s also showed different expression patterns in kidneys between ACDH (acute response) and AM-1 (chronic response) exposure. Moreover, inductive expression of CfACOXs after AM-1 exposure was observed in gills and mantles, and all DEGs in both tissues were up-regulated and their common DEGs exhibited both acute and chronic induction. These results indicate the involvement of scallop ACOXs in PST response, and their plasticity expression patterns between scallop species, among tissues, and between the exposure of different PST analogs.

Peroxisomal ß-oxidation regulates histone acetylation and DNA methylation in Arabidopsis.

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.

Quercetin, Engelitin and Caffeic Acid of Smilax china L. Polyphenols, Stimulate 3T3-L1 Adipocytes to Brown-like Adipocytes Via ß3-AR/AMPK Signaling Pathway.

The aim of the present study was to investigate the browning effects mechanism of Smilax china L. polyphenols (SCLP) and its monomer. In this study, polyphenols (SCLP, engeletin, quercetin and caffeic acid) markedly suppressed lipid accumulation. Polyphenols significantly up-graded the expression of protein kinase A (PKA), adipose triglyceride lipase (ATGL), peroxisome proliferators-activated receptors alpha (PPARα), carnitine palmitoyl transferase (CPT) and acyl-CoA oxidase (ACO) to promote lipolysis and ß-oxidation. Moreover, polyphenols greatly enhanced mitochondrial biogenesis in adipocytes, as demonstrated by the expression of Nrf1 and Tfam were up-regulated. Furthermore, polyphenols treatment greatly up-regulated the browning program in adipocytes by increased brown-specific genes and proteins uncoupling protein 1 (UCP-1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and PR domain containing 16 (PRDM16), as well as beige-specific genes (Tmem26, Tbx1, CD137, Cited1), especially engeletin. Further research found that the brown-specific markers were decreased by antagonist treatment of AMPK or ß3-AR, but polyphenols treatment reversed the effect of antagonists and improved the expression of UCP-1, PRDM16 and PGC-1α. In conclusion, these results indicated that polyphenols stimulate browning in adipocytes via activation of the ß3-AR/AMPK signaling pathway, and SCLP and its monomer may be worth investigating to prevent obesity.

Pex20p functions as the receptor for non-PTS1/non-PTS2 acyl-CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica.

Most soluble proteins targeted to the peroxisomal matrix contain a C-terminal peroxisome targeting signal type 1 (PTS1) or an N-terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl-CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co-receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2-containing protein 3-ketoacyl-CoA thiolase (Pot1p) but also the non-PTS1/non-PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C-terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C-terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non-PTS1/non-PTS2 Aox isozymes into peroxisomes.

Carbohydrate response element-binding protein regulates lipid metabolism via mTOR complex1 in diabetic nephropathy.

Lipid deposition caused by the disorder of renal lipid metabolism is involved in diabetic nephropathy (DN). Carbohydrate response element-binding protein (ChREBP) is a key transcription factor in high glucose-induced cellular fat synthesis. At present, the regulation and mechanism of ChREBP on fat metabolism in diabetic kidneys are still unclear. In this study, we showed that lack of ChREBP significantly improved renal injury, inhibited oxidative stress, lipid deposition, fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC) and thioredoxin-interacting protein (TXNIP) expression, as well as the activity of mammalian target of rapamycin complex 1 (mTORC1) in diabetic kidneys. Meanwhile, ChREBP deficiency upregulated the expression of peroxisome proliferator-activated receptor-α (PPARα), carnitine palmitoyltransferaser 1A (CPT1A) and acyl-coenzyme A oxidase 1 (ACOX1) in diabetic kidneys. In vitro, knockdown of ChREBP attenuated lipid deposition, mTORC1 activation, and expression of FASN and ACC, increased PPARα, CPT1A, and ACOX1 expression in HK-2 cells and podocytes under high glucose (HG) conditions. Moreover, HG-induced lipid deposition, increased expression of FASN and ACC and decreased expression of PPARα, CPT1A, and ACOX1 were reversed by rapamycin, a specific inhibitor of mTORC1, in HK-2 cells. These results indicate that ChREBP deficiency alleviates diabetes-associated renal lipid accumulation by inhibiting mTORC1 activity and suggest that reduction of ChREBP is a potential therapeutic strategy to treat DN.

Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies.

Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+ Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5'-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family.

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion.

Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in ß-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8-fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories.

Effect of Continuous Feeding of Ayu-Narezushi on Lipid Metabolism in a Mouse Model of Metabolic Syndrome.

Ayu-narezushi, a traditional Japanese fermented food, comprises abundant levels of lactic acid bacteria (LAB) and free amino acids. This study aimed to examine the potential beneficial effects of ayu-narezushi and investigated whether ayu-narezushi led to improvements in the Tsumura Suzuki obese diabetes (TSOD) mice model of spontaneous metabolic syndrome because useful LAB are known as probiotics that regulate intestinal function. In the present study, the increased body weight of the TSOD mice was attenuated in those fed the ayu-narezushi-comprised chow (ayu-narezushi group) compared with those fed the normal rodent chow (control group). Serum triglyceride and cholesterol levels were significantly lower in the Ayu-narezushi group than in the control group at 24 weeks of age. Furthermore, hepatic mRNA levels of carnitine-palmitoyl transferase 1 and acyl-CoA oxidase, which related to fatty acid oxidation, were significantly increased in the ayu-narezushi group than in the control group at 24 weeks of age. In conclusion, these results suggested that continuous feeding with ayu-narezushi improved obesity and dyslipidemia in the TSOD mice and that the activation of fatty acid oxidation in the liver might contribute to these improvements.

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα.

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid ß-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal ß-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid ß-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid ß-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.