Studies on the effect of an heterologous fatty acid-binding protein on acyl-CoA oxidase induction in Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Smaczynska I
PROVIDER: S-EPMC1137125 | biostudies-other | 1994 Jul
REPOSITORIES: biostudies-other
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Project description:Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae, we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA.
| S-EPMC7573125 | biostudies-literature
Project description:In the present study, we have used DNA microarray and quantitative real-time PCR analysis to examine the transcriptional changes that occur in response to cellular depletion of the yeast acyl-CoA-binding protein, Acb1p. Depletion of Acb1p resulted in the differential expression of genes encoding proteins involved in fatty acid and phospholipid synthesis (e.g. FAS1, FAS2, ACC1, OLE1, INO1 and OPI3), glycolysis and glycerol metabolism (e.g. GPD1 and TDH1), ion transport and uptake (e.g. ITR1 and HNM1) and stress response (e.g. HSP12, DDR2 and CTT1). In the present study, we show that transcription of the INO1 gene, which encodes inositol-3-phosphate synthase, cannot be fully repressed by inositol and choline, and UAS(INO1) (inositol-sensitive upstream activating sequence)-driven transcription is enhanced in Acb1p-depleted cells. In addition, the reduction in inositol-mediated repression of INO1 transcription observed after depletion of Acb1p appeared to be independent of the transcriptional repressor, Opi1p. We also demonstrated that INO1 and OPI3 expression can be normalized in Acb1p-depleted cells by the addition of high concentrations of exogenous fatty acids, or by the overexpression of FAS1 or ACC1. Together, these findings revealed an Acb1p-dependent connection between fatty acid metabolism and transcriptional regulation of phospholipid biosynthesis in yeast. Finally, expression of an Acb1p mutant which is unable to bind acyl-CoA esters could not normalize the transcriptional changes caused by Acb1p depletion. This strongly implied that gene expression is modulated either by the Acb1p-acyl-CoA ester complex directly or by its ability to donate acyl-CoA esters to utilizing systems.
| S-EPMC2049021 | biostudies-literature
Project description:Fatty acyl-coenzyme A oxidase 1 (ACOX1) knockout (ACOX1(-/-)) mice manifest hepatic metabolic derangements that lead to the development of steatohepatitis, hepatocellular regeneration, spontaneous peroxisome proliferation, and hepatocellular carcinomas. Deficiency of ACOX1 results in unmetabolized substrates of this enzyme that function as biological ligands for peroxisome proliferator-activated receptor-? (PPAR?) in liver. Here we demonstrate that sustained activation of PPAR? in ACOX1(-/-) mouse liver by these ACOX1 substrates results in endoplasmic reticulum (ER) stress. Overexpression of transcriptional regulator p8 and its ER stress-related effectors such as the pseudokinase tribbles homolog 3, activating transcription factor 4, and transcription factor CCAAT/-enhancer-binding protein homologous protein as well as phosphorylation of eukaryotic translation initiation factor 2?, indicate the induction of unfolded protein response signaling in the ACOX1(-/-) mouse liver. We also show here that, in the liver, p8 is a target for all three PPAR isoforms (-?, -?, and -?), which interact with peroxisome proliferator response elements in p8 promoter. Sustained activation of p8 and unfolded protein response-associated ER stress in ACOX1(-/-) mouse liver contributes to hepatocyte apoptosis and liver cell proliferation culminating in the development of hepatocarcinogenesis. We also demonstrate that human ACOX1 transgene is functional in ACOX1(-/-) mice and effectively prevents metabolic dysfunctions that lead to ER stress and carcinogenic effects. Taken together, our data indicate that progressive PPAR?- and p8-mediated ER stress contribute to the hepatocarcinogenesis in ACOX1(-/-) mice.
| S-EPMC3157234 | biostudies-literature
Project description:The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.
| S-EPMC2943281 | biostudies-literature
Project description:BackgroundBirds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters.ResultscDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland.ConclusionThe data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.
| S-EPMC3265415 | biostudies-literature
Project description:We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovine ACBP, i.e. the initiating methionine was removed and the following serine residue was N-acetylated. The expression of this protein did not affect the growth rate of the cells. Determination of the yeast acyl-CoA pool size showed a close positive correlation between the ACBP content of the cells and the size of the acyl-CoA pool. Thus ACBP can act as an intracellular acyl-CoA pool former. Possible physiological functions of ACBP in cells are discussed.
| S-EPMC1132282 | biostudies-other
Project description:Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.
| S-EPMC1148584 | biostudies-other
Project description:BackgroundLevopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction. This work aimed to apply synthetic biology to produce LA in yeast strains from a simple carbon source.ResultsLevopimaradiene (LP), the precursor of LA, was produced via LP synthase (LPS) expression in yeast. LPS was then modified by N-terminal truncating and site-directed mutagenesis. The strain containing t79LPSMM (79 N-terminal amino acid truncating and M593I/Y700F mutation) produced 6.92 mg/L of LP, which were 23-fold higher than the strain containing LPS. Next, t79LPSMM was expressed in a new metabolically engineered chassis, and the final LP production increased 164-folds to 49.21 mg/L. Three cytochrome P450 reductases (CPRs) were co-expressed with CYP720B1 (the enzyme responsible for LA production from LP) in yeast to evaluate their LA producing abilities, and the CPR from Taxus cuspidata (TcCPR) was found to be the best (achieved 23.13 mg/L of LA production). CYP720B1 and TcCPR genes overexpression in the multi-copy site of the S.cerevisiae genome led to a 1.9-fold increase in LA production to 45.24 mg/L in a shake-flask culture. Finally, LA production was improved to 400.31 mg/L via fed-batch fermentation in a 5-L bioreactor.ConclusionsThis is the first report to produce LA in a yeast cell factory and the highest titer of LA is achieved.
| S-EPMC6050663 | biostudies-literature
Project description:Background and aimsA 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-5β-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 resultsAmong 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.ConclusionsDysfunctional 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.
| S-EPMC9796151 | biostudies-literature
Project description:Malonyl-CoA is an energy-rich molecule formed by the ATP-dependent carboxylation of acetyl coenzyme A catalyzed by acetyl-CoA carboxylase. This molecule is an important precursor for many biotechnologically interesting compounds such as flavonoids, polyketides, and fatty acids. The yeast Saccharomyces cerevisiae remains one of the preferred cell factories, but has a limited capacity to produce malonyl-CoA compared to oleaginous organisms. We developed a new S. cerevisiae strain with a conditional allele of ACC1, the essential acetyl-CoA carboxylase (ACC) gene, as a tool to test heterologous genes for complementation. Yarrowia lipolytica is an oleaginous yeast with a higher capacity for lipid production than S. cerevisiae, possibly due to a higher capacity to produce malonyl-CoA. Measuring relative intracellular malonyl-CoA levels with an in-vivo biosensor confirmed that expression of Y. lipolytica ACC in S. cerevisiae leads to a higher accumulation of malonyl-CoA compared with overexpression of the native gene from an otherwise identical vector. The higher accumulation was generally accompanied by a decreased growth rate. Concomitant expression of both the homologous and heterologous ACC1 genes eliminated the growth defect, with a marginal reduction of malonyl-CoA accumulation.
| S-EPMC9582701 | biostudies-literature