scholarly journals Saccharomyces cerevisiae Δ9‐desaturase Ole1 Interacts with Lipid Biosynthetic Enzymes that Produce Storage Lipid, Phospholipid, and Sterol‐esters

2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Brianna Greenwood ◽  
David Stuart
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biosynthetic Enzymes ◽  
Sterol Esters ◽  
Storage Lipid ◽  
Δ9 Desaturase

scholarly journals Combined Biosynthetic Pathway Engineering and Storage Pool Expansion for High-Level Production of Ergosterol in Industrial Saccharomyces cerevisiae

2021 ◽  
Vol 9 ◽  
Author(s):  
Zhi-Jiao Sun ◽  
Jia-Zhang Lian ◽  
Li Zhu ◽  
Yi-Qi Jiang ◽  
Guo-Si Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biosynthetic Pathway ◽  
Metabolic Flux ◽  
Feeding Strategy ◽  
Pathway Engineering ◽  
Ergosterol Biosynthesis ◽  
Sterol Esters ◽  
Storage Pool ◽  
Ergosterol Content ◽  
Dry Cell Weight

Ergosterol, a terpenoid compound produced by fungi, is an economically important metabolite serving as the direct precursor of steroid drugs. Herein, ergsosterol biosynthetic pathway modification combined with storage capacity enhancement was proposed to synergistically improve the production of ergosterol in Saccharomyces cerevisiae. S. cerevisiae strain S1 accumulated the highest amount of ergosterol [7.8 mg/g dry cell weight (DCW)] among the wild-type yeast strains tested and was first selected as the host for subsequent metabolic engineering studies. Then, the push and pull of ergosterol biosynthesis were engineered to increase the metabolic flux, overexpression of the sterol acyltransferase gene ARE2 increased ergosterol content to 10 mg/g DCW and additional overexpression of a global regulatory factor allele (UPC2-1) increased the ergosterol content to 16.7 mg/g DCW. Furthermore, considering the hydrophobicity sterol esters and accumulation in lipid droplets, the fatty acid biosynthetic pathway was enhanced to expand the storage pool for ergosterol. Overexpression of ACC1 coding for the acetyl-CoA carboxylase increased ergosterol content from 16.7 to 20.7 mg/g DCW. To address growth inhibition resulted from premature accumulation of ergosterol, auto-inducible promoters were employed to dynamically control the expression of ARE2, UPC2-1, and ACC1. Consequently, better cell growth led to an increase of ergosterol content to 40.6 mg/g DCW, which is 4.2-fold higher than that of the starting strain. Finally, a two-stage feeding strategy was employed for high-density cell fermentation, with an ergosterol yield of 2986.7 mg/L and content of 29.5 mg/g DCW. This study provided an effective approach for the production of ergosterol and other related terpenoid molecules.

scholarly journals Establishment of Strigolactone-Producing Bacterium-Yeast Consortium

2021 ◽  
Author(s):  
Sheng Wu ◽  
Xiaoqiang Ma ◽  
Anqi Zhou ◽  
Alex Valenzuela ◽  
Yanran Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Plant Growth ◽  
Production Process ◽  
Growth And Development ◽  
Pathway Engineering ◽  
Biosynthetic Enzymes ◽  
Functional Identification ◽  
Plant Growth And Development ◽  
Microbial System

Strigolactones (SLs) are a class of phytohormones playing diverse roles in plant growth and development, yet the limited access to SLs is largely impeding SL-based foundational investigations and applications. Here, we developed Escherichia coli-Saccharomyces cerevisiae consortia to establish a microbial biosynthetic platform for the synthesis of various SLs, including carlactone, carlactonic acid, 5-deoxystrigol (5DS), 4-deoxyorobanchol (4DO), and orobanchol (OB). The SL-producing platform enabled us to conduct functional identification of CYP722Cs from various plants as either OB or 5DS synthase. It also allowed us to quantitatively compare known variants of plant SL biosynthetic enzymes in the microbial system. The titer of 5DS was further enhanced through pathway engineering to 0.0473 mg/L. This work provides a unique platform for investigating SL biosynthesis and evolution and lays the foundation for developing SL microbial production process.

scholarly journals Composition of the protoplast membrane from Saccharomyces cerevisiae

1968 ◽  
Vol 108 (3) ◽  
pp. 401-412 ◽  
Author(s):  
R. P. Longley ◽  
A. H. Rose ◽  
B. A. Knights
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Magnesium Chloride ◽  
Membrane Lipids ◽  
Dry Weight ◽  
Exponential Phase ◽  
Gas Liquid Chromatography ◽  
Liquid Chromatography Mass Spectrometry ◽  
Sterol Esters ◽  
Whole Cells ◽  
Fatty Acid Compositions

1. Protoplasts of Saccharomyces cerevisiae N.C.Y.C. 366 were prepared by incubating washed exponential-phase cells in buffered mannitol (0·8m) containing 10mm-magnesium chloride and snail gut juice (about 8mg. of protein/ml. of reaction mixture). Protoplast membranes were obtained by bursting protoplasts in ice-cold phosphate buffer (pH7·0) containing 10mm-magnesium chloride. 2. Protoplast membranes accounted for 13–20% of the dry weight of the yeast cell. They contained on a weight basis about 39% of lipid, 49% of protein, 6% of sterol (assayed spectrophotometrically) and traces of RNA and carbohydrate (glucan+mannan). 3. The principal fatty acids in membrane lipids were C16:0, C16:1 and C18:1 acids. Whole cells contained a slightly greater proportion of C16:0 and a somewhat smaller proportion of C18:1 acids. Membrane and whole-cell lipids included monoglycerides, diglycerides, triglycerides, sterols, sterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine. Phosphorus analyses on phospholipid fractions from membranes and whole cells showed that membranes contained proportionately more phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine than whole cells, which in turn were richer in phosphatidylcholine. Phospholipid fractions from membranes and whole cells had similar fatty acid compositions. 4. Membranes and whole cells contained two major and three minor sterol components. Gas–liquid chromatography, mass spectrometry and u.v. and i.r. spectra indicated that the major components were probably Δ5,7,22,24(28)-ergostatetraen-3β-ol and zymosterol. The minor sterol components in whole cells were probably episterol (or fecosterol), ergosterol and a C29 di-unsaturated sterol. 5. Defatted whole cells contained slightly more glutamate and ornithine and slightly less leucine and isoleucine than membranes. Otherwise, no major differences were detected in the amino acid compositions of defatted whole cells and membranes.

Inositol regulates phosphatidylglycerolphosphate synthase expression in Saccharomyces cerevisiae

1988 ◽  
Vol 8 (11) ◽  
pp. 4773-4779
Author(s):  
M L Greenberg ◽  
S Hubbell ◽  
C Lam
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biosynthetic Enzymes ◽  
Phospholipid Synthesis ◽  
Wild Type ◽  
Mitochondrial Inner Membrane ◽  
Regulatory Circuit ◽  
The Media ◽  
Constitutive Synthesis ◽  
First Time ◽  
Phosphatidylcholine Synthesis

The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the committed step in the synthesis of cardiolipin, a phospholipid found predominantly in the mitochondrial inner membrane. To determine whether PGPS is regulated by cross-pathway control, we analyzed PGPS expression under conditions in which the regulation of general phospholipid synthesis could be examined. The addition of inositol resulted in a three- to fivefold reduction in PGPS expression in wild-type cells in the presence or absence of exogenous choline. The reduction in enzyme activity in response to inositol was seen in minutes, suggesting that inactivation or degradation of the enzyme plays an important role in inositol-mediated repression of PGPS. In cho2 and opi3 mutants, which are blocked in phosphatidylcholine synthesis, inositol-mediated repression of PGPS did not occur unless choline was added to the media. Three previously identified genes that regulate general phospholipid synthesis, INO2, INO4, and OP11, did not affect PGPS expression. Thus, ino2 and ino4 mutants, which are unable to derepress biosynthetic enzymes involved in general phospholipid synthesis, expressed wild-type levels of PGPS activity under derepressing conditions. PGPS expression in the opi1 mutant, which exhibits constitutive synthesis of general phospholipid biosynthetic enzymes, was fully repressed in the presence of inositol and partially repressed even in the absence of inositol. These results demonstrate for the first time that an enzymatic step in cardiolipin synthesis is coordinately controlled with general phospholipid synthesis but that this control is not mediated by the same genetic regulatory circuit.

scholarly journals Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae

2015 ◽  
Vol 26 (9) ◽  
pp. 1601-1615 ◽  
Author(s):  
Harsha Garadi Suresh ◽  
Aline Xavier da Silveira dos Santos ◽  
Wanda Kukulski ◽  
Jens Tyedmers ◽  
Howard Riezman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Metabolic Flux ◽  
Fatty Acid Synthetase ◽  
Growth Conditions ◽  
Lipid Homeostasis ◽  
Biosynthetic Enzymes ◽  
Glucose Depletion ◽  
Key Enzyme ◽  
Quantitative Changes

Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes.

Use of aromatic thia fatty acids as active site mapping agents for a yeast Δ9 desaturase

1994 ◽  
Vol 72 (1) ◽  
pp. 176-181 ◽  
Author(s):  
Peter H. Buist ◽  
Dale M. Marecak
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Active Site ◽  
Optical Purity ◽  
Shift Reagent ◽  
Chiral Shift Reagent ◽  
Site Mapping ◽  
Δ9 Desaturase ◽  
Conformational Model ◽  
Thia Fatty Acids

The Δ9 desaturating system of Saccharomyces cerevisiae can sulfoxidize methyl S-benzyl-8-mercaptooctanoate as well as its naphthyl analogue but the corresponding isomers where sulfur is bonded to the aromatic ring do not function as substrates. We have accounted for these results in terms of a conformational model. We have also accumulated some evidence that suggests that dealkylation of these thia analogues does not compete with sulfoxidation. Our recently discovered chiral shift reagent (S)-(+)-α-methoxyphenylacetic acid (MPAA) can be used to determine the optical purity and absolute configuration of all sulfoxides prepared in this work.

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