Saccharomyces cerevisiae fatty acid synthase complex with bound gamma subunit
Ontology highlight
ABSTRACT:
SUBMITTER: Holger Stark
PROVIDER: EMPIAR-10454 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Discovery of a Regulatory Subunit of the Yeast Fatty Acid Synthase.
Singh Kashish K Graf Benjamin B Linden Andreas A Sautner Viktor V Urlaub Henning H Tittmann Kai K Stark Holger H Chari Ashwin A
Cell 20200310 6
Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α<sub>6</sub>-β<sub>6</sub>S. cerevisiae FAS, which is shown by high-resolution structures to stabiliz ...[more]
Similar Datasets
Project description: Not available
| EMPIAR-10470 | biostudies-other
Project description:BackgroundSaccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate.ResultsOverexpression of the PDH complex significantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD+-dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The best fatty acid producing strain PDH7 with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acids (FFAs) in shake flask, which was 83.2% higher than the control strain YJZ08. Profile analysis of free fatty acid suggested the cytosolic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1).ConclusionsWe demonstrated that cytosolic PDH pathway enabled more efficient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cytosolic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways.
| S-EPMC7730738 | biostudies-literature
Project description:Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr approximately 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the beta-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 A of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH2-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1-1,297; Mr approximately 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296-2,504; Mr approximately 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure-function relationship of FAS in animal tissues.
| S-EPMC24928 | biostudies-literature
Project description:Methylation of histone H3 lysine 4 (H3K4) is catalyzed by the multi-component COMPASS or COMPASS-like complex, which is highly conserved from yeast to human, and plays essential roles in gene expression and transcription, cell cycle progression, and DNA repair. Here we present a cryo-EM map of the complete S. cerevisiae COMPASS complex. Through tag or Fab labeling strategy combined with cryo-EM 3D reconstruction and cross-linking and mass spectrometry (XL-MS) analysis, we uncovered new information on the subunit arrangement: Cps50, Cps35, and Cps30 were determined to group together to form the face region in the head of the complex, and Cps40 and the N-terminal portion of Set1 reside on the top of the head. Our map reveals the location of the active center and a canyon in the back of the head. Together, our study provides the first snapshot of the complete architecture of yeast COMPASS and a picture of its subunit interaction network, which could facilitate our understanding of the COMPASS machinery and its functionality.
| S-EPMC6258710 | biostudies-literature
Project description:Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h-1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.
| S-EPMC10320613 | biostudies-literature
Project description:Acyl carrier protein (ACP) is the work horse of polyketide (PKS) and fatty acid synthases (FAS) and acts as a substrate shuttling domain in these mega enzymes. In fungi, FAS forms a 2.6 MDa symmetric assembly with six identical copies of FAS1 and FAS2 polypeptides. However, ACP spatial distribution is not restricted by symmetry owing to the long and flexible loops that tether the shuttling domain to its corresponding FAS2 polypeptide. This symmetry breaking has hampered experimental investigation of substrate shuttling route in fungal FAS. Here, we develop a protein engineering and expression method to isolate asymmetric fungal FAS proteins containing odd numbers of ACP domains. Electron cryomicroscopy (cryoEM) observation of the engineered complex reveals a non-uniform distribution of the substrate shuttling domain relative to its corresponding FAS2 polypeptide at 2.9 Å resolution. This work lays the methodological foundation for experimental study of ACP shuttling route in fungi.
| S-EPMC10786820 | biostudies-literature
Project description:Terminally hydroxylated fatty acids or dicarboxylic acids are industrially relevant compounds with broad applications. Here, we present the proof of principle for the de novo biosynthesis of 8-hydroxyoctanoic acid from glucose and ethanol in the yeast Saccharomyces cerevisiae. Toxicity tests with medium-chain length ?-hydroxy fatty acids and dicarboxylic acids revealed little or no growth impairments on yeast cultures even at higher concentrations. The ability of various heterologous cytochrome P450 enzymes in combination with their cognate reductases for ?-hydroxylation of externally fed octanoic acid were compared. Finally, the most efficient P450 enzyme system was expressed in a yeast strain, whose fatty acid synthase was engineered for octanoic acid production, resulting in de novo biosynthesis of 8-hydroxyoctanoic acid up to 3 ?mg/l. Accumulation of octanoic acid revealed that cytochromes P450 activities were limiting 8-hydroxyoctanoic acid synthesis. The hydroxylation of both externally added and intracellularly produced octanoic acid was strongly dependent on the carbon source used, with ethanol being preferred. We further identified the availability of heme, a cofactor needed for P450 activity, as a limiting factor of 8-hydroxyoctanoic acid biosynthesis.
| S-EPMC6906673 | biostudies-literature
Project description:BackgroundThe ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.ResultsThe previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L-1 in a 72-h fermentation. The additional accumulation of 90 mg L-1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L-1. However, in growth tests concentrations even lower than 50.0 mg L-1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.ConclusionsBy providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.
| S-EPMC5984327 | biostudies-literature
Project description:De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the Type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT-malonyl-/acetyltransferase) and the condensation (KS-β-ketoacyl synthase) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate-binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.
| S-EPMC6954729 | biostudies-literature
Project description:The Gpg1 protein is a Ggamma subunit mimic implicated in the G-protein glucose-signaling pathway in Saccharomyces cerevisiae, and its function is largely unknown. Here we report that Gpg1 blocks the maintenance of [PSI(+)], an aggregated prion form of the translation termination factor Sup35. Although the GPG1 gene is normally not expressed, over-expression of GPG1 inhibits propagation of not only [PSI(+)] but also [PIN(+)], [URE3] prions, and the toxic polyglutamine aggregate in S. cerevisiae. Over-expression of Gpg1 does not affect expression and activity of Hsp104, a protein-remodeling factor required for prion propagation, showing that Gpg1 does not target Hsp104 directly. Nevertheless, prion elimination by Gpg1 is weakened by over-expression of Hsp104. Importantly, Gpg1 protein is prone to self-aggregate and transiently colocalized with Sup35NM-prion aggregates when expressed in [PSI(+)] cells. Genetic selection and characterization of loss-of-activity gpg1 mutations revealed that multiple mutations on the hydrophobic one-side surface of predicted alpha-helices of the Gpg1 protein hampered the activity. Prion elimination by Gpg1 is unaffected in the gpa2Delta and gpb1Delta strains lacking the supposed physiological G-protein partners of Gpg1. These findings suggest a general inhibitory interaction of the Gpg1 protein with other transmissible and nontransmissible amyloids, resulting in prion elimination. Assuming the ability of Gpg1 to form G-protein heterotrimeric complexes, Gpg1 is likely to play a versatile function of reversing the prion state and modulating the G-protein signaling pathway.
| S-EPMC2630093 | biostudies-literature