acetyl-CoA Synthetase Gene Research Articles

Saccharomyces cerevisiae cellular engineering for the production of FAME biodiesel

The unsustainable and widespread utilization of fossil fuels continues to drive the rapid depletion of global supplies. Biodiesel has emerged as one of the most promising alternatives to conventional diesel, leading to growing research interest in its production. Microbes can facilitate the de novo synthesis of a type of biodiesel in the form of fatty acid methyl esters (FAMEs). In this study, Saccharomyces cerevisiae metabolic activity was engineered to facilitate enhanced FAME production. Initially, free fatty acid concentrations were increased by deleting two acetyl-CoA synthetase genes (FAA1, FAA4) and an acyl-CoA oxidase gene (POX1). Intracellular S-adenosylmethionine (SAM) levels were then enhanced via the deletion of an adenosine kinase gene (ADO1) and the overexpression of a SAM synthetase gene (SAM2). Lastly, the S. cerevisiae strain overproducing free fatty acids and SAM were manipulated to express a plasmid encoding the Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). Using this combination of engineering approaches, a FAME concentration of 5.79 ± 0.56 mg/L was achieved using these cells in the context of shaking flask fermentation. To the best of our knowledge, this is the first detailed study of FAME production in S. cerevisiae. These results will provide a valuable basis for future efforts to engineer S. cerevisiae strains for highly efficient production of biodiesel.

Transcriptomic analysis of the metabolic regulatory mechanism of Schizochytrium limacinum B4D1 using sodium acetate to produce DHA

The aim of the study was to evaluate the potential of acetate as a carbon source by examining the fermentation performance of Schizochytrium limacinum B4D1 and its effects on metabolism using physiology and transcriptomics studies. The results demonstrated that, while the metabolic rate of acetate in S. limacinum B4D1 was lower than glucose, it increased the content of docosahexaenoic acid (DHA) in the total fatty acids. Transcriptomic analysis indicated that the use of acetate as a carbon source significantly upregulated the acetyl-CoA synthetase gene, which increased the source of acetyl-CoA for fatty acid biosynthesis. Moreover, the upregulation of polyunsaturated fatty acid enzymes promoted the polyketide synthase (PKS) pathway of fatty acid synthesis, resulting in an increase in DHA content. Meanwhile, the upregulation of genes such as long-chain acyl-CoA synthetase and 3-hydroxyacyl-CoA dehydrogenase enhanced the β-oxidation process of saturated fatty acids in mitochondria, while the downregulation of acyl-CoA oxidase and catalase genes resulted the β-oxidation process of polyunsaturated fatty acids in peroxisomes was inhibited, leading to an increased proportion of DHA in the total fatty acids. Thus, these changes in gene expression contributed to the increased proportion of DHA in the total fatty acids.

Exploring a High-Efficiency Genetic Transformation System for Engineering Saccharopolyspora pogona ASAGF58 To Improve Butenyl-Spinosyn Production

Butenyl-spinosyn is a potent insecticide potentially useful as a broad-spectrum pesticide. Because it is relatively nontoxic to mammals and does not damage the environment, there has been considerable interest in the use of butenyl-spinosyn for increasing agricultural production. However, genetically engineering Saccharopolyspora pogona ASAGF58 to increase its relatively low butenyl-spinosyn content remains challenging because it cannot be transformed efficiently. In this study, genes encoding novel methyltransferases (04455 and 28970) were identified in the Sa. pogona ASAGF58 genome through a bioinformatic-based analysis. Additionally, the transformation efficiency increased by 5.8- and 16.4-fold when foreign DNA was pre-methylated in ET-28970 and ET-04455, respectively, through bypassing of the restriction–modification system. A comparative proteomic analysis of Sa. pogona and Saccharopolyspora spinosa revealed that acetyl-CoA synthetase may be useful for improving butenyl-spinosyn production. The fermentation results indicated that compared with the wild-type butenyl-spinosyn content, overexpressing the acetyl-CoA synthetase gene increased butenyl-spinosyn production by 2-fold. The findings presented herein suggest that the strategy employed in this study may be applicable for the genetic engineering of other nonmodel Saccharopolyspora strains and increase target product yields.

Development of a new quantification method of Sarcocystis cruzi through detection of the acetyl-CoA synthetase gene.

Sarcocystis cruzi is a member of the genus Sarcocystis, infecting bovine animals such as cattle and bison as intermediate hosts, and canids such as dogs and raccoon dogs as definitive hosts. Acute sarcocystosis of S. cruzi causes occasional symptoms in cattle, including weight loss, reduced milk production, abortions, and death, and similar to other Sarcocystis species can potentially cause food poisoning in humans when raw or undercooked infected cattle meat is consumed. Despite these issues, genetic information on S. cruzi is scarce, and there is no specific quantitative method for the detection and quantification of the parasite in infected cattle. In this study, we aimed to develop a method based on high-throughput sequencing of S. cruzi genome and transcriptome that specifically and quantitatively detects the S. cruzi acetyl-CoA synthetase gene (ScACS). Cardiac muscles were collected from slaughterhouses in Saitama Prefecture to obtain sarcocysts from which DNA and RNA were extracted for the high-throughput sequencing. Using the sequences, we developed a specific quantitative PCR assay which could distinguish S. cruzi ACS from that of Toxoplasma gondii by taking advantage of the differences in their exon/intron organizations and validated the assay with the microscopic counting of the S. cruzi bradyzoites. Thus, this assay will be useful for future studies of S. cruzi pathogenesis in cattle and for the surveillance of infected animals, thereby easing public health concerns.

Effect of low expression level of acetyl coenzyme A synthetase gene on secondary metabolite in Monascus

Acetyl-coenzyme A (CoA) is a key metabolite produced by the acetyl-CoA synthetase (ACS) gene in energy metabolism and biosynthetic pathways. ACS is speculated to be the branching site of monacolin K (MK) and citrinin production and related to the metabolite production of Monascus. In this study, the ACS expression was inhibited by ribonucleic acid interference (RNAi). T7 was selected for a follow-up analysis of the lowest ACS expression, which was 0.401 times higher than that of the wild-type strain. The effects on the colony morphology of Monascus were determined. The morphological characteristics of mycelia and spores were observed under a scanning electron microscope. The contents of secondary metabolites, namely, MK and citrinin, were determined through high performance liquid chromatography (HPLC). Colour values were measured with a spectrophotometer. Results showed that the low ACS expression could inhibit the growth of Monascus colonies and the hypha and affect the formation and morphology of Monascus M1 spores. It could also inhibit the production of the main secondary metabolites, namely, MK, citrinin, and pigment.

Development of a 2-pyrrolidone biosynthetic pathway in Corynebacterium glutamicum by engineering an acetyl-CoA balance route.

2-Pyrrolidone is widely used in the textile and pharmaceutical industries. Here, we established a 2-pyrrolidone biosynthesis pathway in Corynebacterium glutamicum, by expressing glutamate decarboxylase (Gad) mutant and β-alanine CoA transferase (Act) which activates spontaneous dehydration cyclization of GABA to form 2-pyrrolidone. Also, the 5' untranslated regions (UTR) strategy was used to increase the expression of protein. Furthermore, considering the importance of acetyl-CoA in the 2-pyrrolidone synthesis pathway, the acetyl-CoA synthetase (acsA) gene was introduced to convert acetate into acetyl-CoA thus achieving the recyclability of the economy. Finally, the fed-batch fermentation of the final strain in a 5L bioreactor produced 10.5g/L 2-pyrrolidone within 78h, which increased by 42.5% by altering the level of gene expression. This is the first time to build the basic chemical 2-pyrrolidone from glucose in one step in C. glutamicum.

Characterization of Microalgal Acetyl-CoA Synthetases with High Catalytic Efficiency Reveals Their Regulatory Mechanism and Lipid Engineering Potential.

Acetyl-CoA synthetase (ACS) plays a key role in microalgal lipid biosynthesis and acetyl-CoA industrial production. In the present study, two ACSs were cloned and characterized from the oleaginous microalga Chromochloris zofingiensis. In vitro kinetic analysis showed that the Km values of CzACS1 and CzACS2 for potassium acetate were 0.99 and 0.81 mM, respectively. Moreover, CzACS1 and CzACS2 had outstanding catalytic efficiencies (kcat/Km), which were 70.67 and 79.98 s-1 mM-1, respectively, and these values were higher than that of other reported ACSs. CzACS1 and CzACS2 exhibited differential expression patterns at the transcriptional level under various conditions. Screening a recombinant library of 52 transcription factors (TFs) constructed in the present study via yeast one-hybrid assay pointed to seven TFs with potential involvement in the regulation of the two ACS genes. Expression correlation analysis implied that GATA20 was likely an important regulator of CzACS2 and that ERF9 could regulate two CzACSs simultaneously.

Acetyl-coenzyme A synthetase gene ChAcs1 is essential for lipid metabolism, carbon utilization and virulence of the hemibiotrophic fungus Colletotrichum higginsianum.

Acetyl-coenzyme A (acetyl-CoA) is a key molecule that participates in many biochemical reactions in amino acid, protein, carbohydrate and lipid metabolism. Here, we genetically dissected the distinct roles of two acetyl-CoA synthetase genes, ChAcs1 and ChAcs2, in the regulation of fermentation, lipid metabolism and virulence of the hemibiotrophic fungus Colletotrichum higginsianum. ChAcs1 and ChAcs2 are both highly expressed during appressorial development and the formation of primary hyphae, and are constitutively expressed in the cytoplasm throughout development. We found that C.higginsianum strains without ChAcs1 were non-viable in the presence of most non-fermentable carbon sources, including acetate, ethanol and acetaldehyde. Deletion of ChAcs1 also led to a decrease in lipid content of mycelia and delayed lipid mobilization in conidia to developing appressoria, which suggested that ChAcs1 contributes to lipid metabolism in C.higginsianum. Furthermore, a ChAcs1 deletion mutant was defective in the switch to invasive growth, which may have been directly responsible for its reduced virulence. Transcriptomic analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that ChAcs1 can affect the expression of genes involved in virulence and carbon metabolism, and that plant defence genes are up-regulated, all demonstrated during infection by a ChAcs1 deletion mutant. In contrast, deletion of ChAcs2 only conferred a slight delay in lipid mobilization, although it was highly expressed in infection stages. Our studies provide evidence for ChAcs1 as a key regulator governing lipid metabolism, carbon source utilization and virulence of this hemibiotrophic fungus.

Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered acetyl-CoA metabolism

As a bioactive triterpenoid, squalene is widely used in the food industry, cosmetics, and pharmacology. Squalene’s major commercial sources are the liver oil of deep-sea sharks and plant oils. In this study, we focused on the enhancement of squalene biosynthesis in Yarrowia lipolytica, with particular attention to the engineering of acetyl-CoA metabolism based on genome-scale metabolic reaction network analysis. Although the overexpression of the rate-limiting endogenous ylHMG1 (3-hydroxy-3-methylglutaryl-CoA reductase gene) could improve squalene synthesis by 3.2-fold over that by the control strain, the availability of the key intracellular precursor, acetyl-CoA, was found to play a more significant role in elevating squalene production. Analysis of metabolic networks with the newly constructed genome-scale metabolic model of Y. lipolytica iYL_2.0 showed that the acetyl-CoA pool size could be increased by redirecting carbon flux of pyruvate dehydrogenation towards the ligation of acetate and CoA or the cleavage of citrate to form oxaloacetate and acetyl-CoA. The overexpression of either acetyl-CoA synthetase gene from Salmonella enterica (acs*) or the endogenous ATP citrate lyase gene (ylACL1) resulted in a more than 50% increase in the cytosolic acetyl-CoA level. Moreover, iterative chromosomal integration of the ylHMG1, asc*, and ylACL1 genes resulted in a significant improvement in squalene production (16.4-fold increase in squalene content over that in the control strain). We also found that supplementation with 10 mM citrate in a flask culture further enhanced squalene production to 10 mg/g DCW. The information obtained in this study demonstrates that rationally engineering acetyl-CoA metabolism to ensure the supply of this key metabolic precursor is an efficient strategy for the enhancement of squalene biosynthesis.

High Expression Level of mok E Enhances the Production of Monacolin K in Monascus

ABSTRACTMonacolin K (MK) is a type of cholesterol-lowering agent produced by Monascus spp. The expression level of the mok E gene, a gene of the MK biosynthesis gene cluster, was previously speculated to be positively correlated with the production of MK. In this study, mok E was overexpressed in Monascus fuliginosusis CG-6. The expression levels were analyzed by RT-qPCR. Genome walking was used to analyze the insertion site of the exogenous gene. Results showed that MK production in transformants T-mok E1 (4083.2 μg/g), T-mok E2 (6186.3 μg/g), and T-mok E3 (4527.6 μg/g) was 1.7-fold, 2.5-fold, and 1.8-fold higher than that of the wild strain CG-6 (2469.8 μg/g), respectively. The expression level of mok E in T-mok E1, T-mok E2, and T-mok E3 was 1.83-fold, 4.82-fold, and 2.22-fold higher than that in the wild-type strain (WS CG-6), respectively. The AAA ATPase gene located upstream and acetyl-CoA synthetase gene located downstream of the insertion were obtained by genome walking. These results indicate that the expression of the mok E gene was positively correlated with the production of MK. The expression levels of the AAA ATPase gene and acetyl-CoA synthetase gene were enhanced by the insertion of an exogenous gene.

Cardiolipin deficiency causes triacylglycerol accumulation in Saccharomyces cerevisiae.

In yeast, the synthesis of cardiolipin (CL) and phosphatidylethanolamine (PE) occurs mainly in mitochondria. CL and PE have overlapping functions, and they are required for mitochondrial function. PE is physiologically linked with triacylglycerol (TAG) metabolism in Saccharomyces cerevisiae, involving an acyl-CoA-independent pathway through the phospholipid:diacylglycerol acyltransferase activity of the Lro1 protein. There is no report on the physiological link between CL and TAG metabolism. Here we report a metabolic link between CL and TAG accumulation in the S. cerevisiae. Our data indicated that CL deficiency causes TAG accumulation, involving an acyl-CoA-dependent pathway through the diacylglycerol acyltransferase activity of the Dga1 protein with no changes in the TAG molecular species. The DGA1 gene deletion from the CL-deficient strains reduced the TAG levels. Data from in vitro and in vivo analyses showed that CL did not affect the enzymatic activity of Dga1. Our data also showed that CL deficiency leads to the up-regulation of acetyl-CoA synthetase genes (ACS1 and ACS2) of the cytosolic pyruvate dehydrogenase bypass pathway. This study establishes a physiological link between CL and TAG metabolism in S. cerevisiae.

Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell.In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization.As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions.

Enzymatic characterization of two acetyl-CoA synthetase genes from Populus trichocarpa.

The acetyl-CoA synthetase (ACS) family is a subfamily of adenylate-forming enzymes, which has a close evolutionary relationship with the 4-coumarate:CoA ligase (4CL) family. In this study, two ACS genes were cloned from Populus trichocarpa and were named PtrACS1 and PtrACS2. Bioinformatics characterization of PtrACS1 and PtrACS2 showed that they contained the key ACS residues and a putative peroxisome targeting sequence 1 (PTS1) at the end of the C-terminal sequence. Real-time PCR results showed that PtrACS1 and PtrACS2 were expressed in the phloem, xylem, leaves, and roots of one-year-old P. trichocarpa, but were expressed primarily in the leaves. The ACS enzyme activity was higher in leaves than other tissues in P. trichocarpa. Two overexpressed recombinant proteins showed no catalytic activity toward the substrates of 4CL, but did have notable catalytic activity toward sodium acetate and substrates of ACS. The relative activities of PtrACS1 and PtrACS2 were 194.16 ± 11.23 and 422.25 ± 21.69 μM min−1 mg−1, respectively. The Km and Vmax of PtrACS1 were 0.25 mM and 698.85 μM min−1 mg−1, while those for PtrACS2 were 0.72 mM and 245.96 μM min−1 mg−1, respectively. Our results revealed that both proteins belong to the ACS family, and provide a theoretical foundation for the identification and functional analysis of members of the adenylate-forming enzyme superfamily.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2532-7) contains supplementary material, which is available to authorized users.

Role of AcsR in expression of the acetyl-CoA synthetase gene in Vibrio vulnificus.

BackgroundVarS/VarA is one of the global factors regulating diverse aspects of the metabolism and virulence of bacteria including pathogenic Vibrio spp. An experiment to identify the VarS/VarA-regulon in V. vulnificus revealed that a putative LuxR-type transcriptional regulator was down-regulated in ΔvarA mutant. To investigate the roles of this regulatory cascade, the target gene regulated by a LuxR-regulator was identified and its expression was characterized.ResultsTranscriptomic analysis of the mutant deficient in this LuxR-type regulator showed that the acsA gene encoding acetyl-CoA synthetase was down-regulated. Thus, this regulator was named AcsR for “regulator of acetyl-CoA synthetase”. A putative histidine kinase gene, acsS, was located five ORFs downstream of the acsR gene. Expression of an acsA::luxAB transcriptional fusion was decreased in both ΔacsR and ΔacsS mutants. Similar to a ΔacsA mutant, strains carrying deletions either in acsR or acsS grew slowly than wild type in a minimal medium with acetate as a sole carbon source. Growth defect of the ΔacsR strain in acetate-minimal medium was restored by complementation. To investigate if AcsR directly regulates acsA expression, in vitro-gel shift assays were performed using the recombinant AcsR and the regulatory region of the acsA gene, showing that AcsR specifically bound the upstream region of the acsA ORF.ConclusionThis study indicates that the VarS/VarA system plays a role in V. vulnificus metabolism via regulating AcsR, which in turn controls acetate metabolism by activating the transcription of the acetyl-CoA synthetase gene.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0418-4) contains supplementary material, which is available to authorized users.

Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium

High acetate accumulation was produced during glucose fermentation in high cell density cultures, which is harmful to cell growth. In order to reduce the negative impact of acetate accumulation on the fermentation products, we introduced the Escherichia coli acetyl-CoA synthetase (ACS) gene into the marine microalga Schizochytrium sp. TIO1101, generating genetically modified ACS transformants. The results of PCR and blotting analyses showed that the exogenous ACS gene was incorporated into the genome and successfully expressed. The engineered Schizochytrium increased the pH value and reduced the acetate concentration in the final fermentation medium significantly. Furthermore, the ACS transformants exhibited faster growth and glucose consumption rates than the wild-type strain. The biomass and fatty acid proportion of ACS transformants increased by 29.9 and 11.3 %, respectively. Taken together, the data suggest that ACS overexpression in Schizochytrium might improve the utilization of carbon resource and decrease the production of acetate byproduct. These results demonstrate that application of ACS in metabolic genetic engineering could improve the properties of Schizochytrium significantly.

Enhanced α-ketoglutarate production in Yarrowia lipolytica WSH-Z06 by alteration of the acetyl-CoA metabolism

α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid (TCA) cycle and has an important role in the regulation of the balance between carbon and nitrogen metabolism in most microorganisms. In previous research, a thiamine-auxotrophic yeast for α-KG overproduction was screened and named as Yarrowia lipolytica WSH-Z06. To enhance α-KG production and reduce by-product (mainly pyruvate) accumulation, the cofactor metabolism was regulated to redistribute the carbon flux from pyruvate to α-KG. The acetyl-CoA synthetase gene, ACS1, from Saccharomyces cerevisiae and the ATP-citrate lyase gene, ACL, from Mus musculus were expressed to regulate the acetyl-CoA metabolism in Y. lipolytica WSH-Z06. The resultant strains were designated as Y. lipolytica-ACS1 and Y. lipolytica-ACL, respectively. Both of the ACS1 and ACL genes could increase the level of acetyl-CoA and enhance the α-KG production. In a 3-L jar fermenter, the highest yield of α-KG in Y. lipolytica-ACL reached up to 56.5gL−1 with an obvious decrease of pyruvate accumulation from 35.1gL−1 to 20.2gL−1. This study demonstrated that enhancing the acetyl-CoA availability could effectively increase the α-KG production in Y. lipolytica.

Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.Electronic supplementary materialThe online version of this article (doi:10.1007/s00203-012-0829-7) contains supplementary material, which is available to authorized users.

E. coli hypoxia-inducible factor ArcA mediates lifespan extension in a lipoic acid synthase mutant by suppressing acetyl-CoA synthetase

We have previously shown that both the hypoxia-inducible transcription factor ArcA and the PoxB/Acs bypass of the pyruvate dehydrogenase complex contribute to extended lifespan in Escherichia coli. In agreement with studies in higher eukaryotes, we also demonstrated that long-lived E. coli mutants, including LipA-deficient cells, are stress resistant. Here, we show that ArcA contributes to the enhanced lifespan and heat shock resistance of the lipA mutant by suppressing expression of the acetyl-CoA synthetase (acs) gene. The deletion of acs reversed the reduced lifespan of the lipA arcA mutant and promoted the accumulation of extracellular acetate, indicating that inhibition of carbon source uptake contributes to survival extension. However, Acs also sensitized cells lacking ArcA to heat shock, in the absence of extracellular acetate. These results provide evidence for the role of Acs in regulating lifespan and/or stress resistance by both carbon source uptake-dependent and -independent mechanisms.

Transcriptional regulation of the acetyl-CoA synthetase gene acsA in Pseudomonas aeruginosa

Pseudomonas aeruginosa ATCC 17933 is able to oxidize ethanol to acetate under aerobic conditions. The P. aeruginosa acetyl-CoA synthetase (ACS) gene acsA was previously identified, and the ACS enzyme described to be required for growth on ethanol as the sole source of carbon and energy. Here, we investigated the transcriptional regulation of the acsA gene using an acsA::lacZ fusion. Transcription of acsA was regulated by the carbon source, and expression was maximal on ethanol, acetate and propionate. In addition, the induction depended on the response regulator ErdR, which also regulates hierarchically arranged genes for ethanol oxidation. Transcription of the acsA gene was repressed by addition of succinate to an ethanol-containing medium. This repression required Crc, the product of the catabolite repression control gene crc.

The Role of Nuclear Receptor NHR-64 in Fat Storage Regulation in Caenorhabditis elegans

Nuclear hormone receptors (NHRs) play vital roles in the regulation of metabolism, reproduction, and development. We found that inactivation of a C. elegans HNF4 homologue nhr-64 by RNA interference (RNAi) suppresses low fat stores in stearoyl-CoA desaturase-deficient fat-6;fat-7 double mutants and sterol regulatory element binding protein (SREBP) sbp-1 mutants. Furthermore, inactivation of nhr-64 improves the growth rate of the fat-6;fat-7and sbp-1 strains. While nhr-64RNAi subtly affects fatty acid composition and fat storage in wild-type C. elegans, its effects on lipid metabolism are most apparent in the background of stearoyl-CoA desaturase or SREBP deficiency. NHR-64 displays transcriptional activating activity when expressed in yeast, and inactivation of nhr-64 affects the expression of at least 14 metabolic genes. Wild-type worms treated with nhr-64 RNAi display increased expression of acetyl-CoA carboxylase as well as increased abundance of de novo synthesized monomethyl branched chain fatty acids, suggesting an increase in fat synthesis. However, reduced expression of the acetyl-CoA synthetase gene acs-2 and an acyl-CoA oxidase gene indicates that a key role of NHR-64 may be to promote fatty acid oxidation in mitochondria and peroxisomes. These studies reveal that NHR-64 is an important regulator of fat storage in C. elegans.