“Malonate Uptake and Metabolism in Saccharomyces cerevisiae”

Malonyl-CoA plays an important role in the synthesis and elongation of fatty acids in yeast Saccharomyces cerevisiae. Malonyl-CoA is at a low concentration inside the cell and is produced mainly from acetyl-CoA through the enzyme acetyl-CoA carboxylase. It would be beneficial to find an alternative source of malonyl-CoA to increase its intracellular concentration and overall synthesis of the fatty acids. MatB gene from the bacteria Rhizobium leguminosarium bv. trifolii encodes for a malonyl-CoA synthetase which catalyzes the formation of the malonyl-CoA directly from malonate and CoA. However, results from high-performance liquid chromatography (HPLC) proved that Saccharomyces cerevisiae itself does not contain enough cytoplasmic malonate within them and is unable to uptake exogenously supplied malonate in the form of malonic acid. A dicarboxylic acid plasma membrane transporter with the ability to uptake exogenous malonic acid was identified from another species of yeast known as Schizosaccharomyces pombe and the gene encoding this transporter is identified as the mae1 gene. From the experiments thus far, the mae1 gene had been successfully cloned and transformed into Saccharomyces cerevisiae. The expression and functional ability of the encoded plasma membrane dicarboxylic acid transporter were also demonstrated and verified using specialized technologies such as RT-PCR, yeast immunofluorescence, HPLC, and LC-MS.     

Comparative Performance of Decoupled Input–Output Linearizing Controller and Linear Interpolation PID Controller: Enhancing Biomass and Ethanol Production in Saccharomyces cerevisiae

A decoupled input–output linearizing controller (DIOLC) was designed as an alternative advanced control strategy for controlling bioprocesses. Simulation studies of its implementation were carried out to control ethanol and biomass production in Saccharomyces cerevisiae and its performance was compared to that of a proportional–integral–derivative (PID) controller with parameters tuned according to a linear schedule. The overall performance of the DIOLC was better in the test experiments requiring the controllers to respond accurately to simultaneous changes in the trajectories of the substrate and dissolved oxygen concentration. It also exhibited better performance in perturbation experiments of the most significant parameters q _S,max, q _O2,max, and k _s , determined through a statistical design of experiments involving 730 simulations. DIOLC exhibited a superior ability of constraining the process when implemented in extreme metabolic regimes of high oxygen demand for maximizing biomass concentration and low oxygen demand for maximizing ethanol concentration.     

Zinc Oxide Nanoparticles Modulates the Production of β-Glucosidase and Protects its Functional State Under Alcoholic Condition in Saccharomyces cerevisiae

In the present investigation, we have investigated the effect of zinc oxide nanoparticles (ZnONP) on the production of β-glucosidase (BGL) in Saccharomyces cerevisiae under various conditions. ZnONP was synthesized chemically and characterized using various standard techniques. The results revealed that yeast culture administered with 5 mM ZnONP enhanced the intracellular BGL activity up to 28 % compared to control with simultaneous growth of cells. However, at a higher dose of ZnONP (10 and 15 mM), both the activity of the enzyme and yeast growth was dropped. When yeast cells were grown in alcoholic medium (2, 5, and 10 % ethanol), the growth was found inhibited with substantial reduction of intracellular BGL activity. Interestingly, the administration of ZnONP further inhibited the cell growth, however, suppressed the alcoholic effect on enzyme activity. Moreover, under the same condition, ZnONP enhanced the biological activity of the enzyme in cells, indicated a higher yield of BGL production. When the mechanism of ZnONP-mediated cell growth inhibition was investigated, N-acetylcysteine (NAC)-based cell growth study proved that reactive oxygen species (ROS) was not the sole cell death mechanism induced by ZnONP, indicating a second mechanism of cell death. Our findings provide a new insight on the potential application of ZnONP as an external supplement to enhance the active production of BGL like important industrial enzyme in S. cerevisiae in both normal and alcohol stressed condition as well as to produce baker’s yeast in higher amount.     

SYNTHESIS AND SECRETION OF HUMAN ATRIAL NATRIURETIC PEPTIDE IN Saccharomyces cerevisiae

A chemically synthesized α-hANP gene was inserted into plasmid YFD18, which was an expression-secretion vector of yeast. The recombinant then transformed in the yeast Y33. The expression level of yeast transformants was about 700 μg ANP/L detected by RIA. More than 99% of expression products were secreted in the culture medium. N-terminal analysis of purified product showed that the first 4 amino acid residues of α-hANP were deleted.     

Intrazelluläre Lokalisierung und Funktion von NADPH-Cytochromc-Reduktase in Saccharomyces cerevisiae

Zusammenfassung Das Flavoprotein NADPH-Cytochromc-Reduktase aus Hefe ist im Gegensatz zu bisherigen Anschauungen kein Enzym der mitochondralen Atmungskette, sondern an bisher unbekannte subzelluläre Partikel gebunden, die nach Homogenisieren der Hefezellen von den Mitochondrien abgetrennt werden können. Die Beteiligung von Cytochromb ₂ oder einer Transhydrogenase-reaktion an der Elektronenübertragung von NADPH auf Cytochromc in einem Hefehomogenat konnte ausgeschlossen werden. Die Natur der neuen Hefepartikel sowie die physiologische Funktion des Enzyms NADPH-Cytochromc-Reduktase werden diskutiert.Abkürzungen NADPH reduziertes Nikotinamid-adenin-dinukleotidphosphat - NADH reduziertes Nikotinamid-adenin-dinukleotid - Tris Tris-(hydroxymethyl)aminomethan - BAL 1,2-Dimercapto-3-hydroxypropan - RNase Ribonuklease - ATPase Adenosintriphosphatase - EDTA Äthylendiamintetraessigsäure Mit 2 Abbildungen     

Enzymatic Hydrolysis of Sodium Dodecyl Sulphate (SDS)—Pretreated Newspaper for Cellulosic Ethanol Production by Saccharomyces cerevisiae and Pichia stipitis

Fermentation of enzymatic hydrolysate of waste newspaper was investigated for cellulosic ethanol production in this study. Various nonionic and ionic surfactants were applied for waste newspaper pretreatment to increase the enzymatic digestibility. The surfactant-pretreated newspaper was enzymatically digested in 0.05 M sodium citrate buffer (pH 4.8) with varying solid content, filter paper unit loading (FPU/g newspaper), and ratio of filter paper unit/β-glucosidase unit (FPU/CBU). Newspaper pretreated with the anionic surfactant sodium dodecyl sulphate (SDS) demonstrated the highest sugar yield. The addition of Tween-80 in the enzymatic hydrolysis process enhanced the enzymatic digestibility of newspaper pretreated with all of the surfactants. Enzymatic hydrolysis of SDS-pretreated newspaper with 15% solid content, 15 FPU/g newspaper, and FPU/CBU of 1:4 resulted in a newspaper hydrolysate conditioning 29.07 g/L glucose and 4.08 g/L xylose after 72 h of incubation at 50 °C. The fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae, Pichia stipitis, and their co-culture produced 14.29, 13.45, and 14.03 g/L of ethanol, respectively. Their corresponding ethanol yields were 0.43, 0.41, and 0.42 g/g.     

Biotransformation of 3-azidomethyl-4-phenyl-3-buten-2-one and analogs by Saccharomyces cerevisiae: New evidence for an SN2′ mechanism

(Z)-3-XCH₂-4-(C₆H₅)-3-buten-2-one enones (X = SCN, N₃, SO₂Me, OC₆H₅) were synthesized and submitted to biotransformations using whole Saccharomyces cerevisiae cells. The enone (X = SCN) produced (R)-4-(phenyl)-3-methylbutan-2-one (R)-6 with 93% ee and enones (X = N₃, SO₂Me, OC₆H₅) yielded a mixture of (R)-6 and the corresponding CC bond reduction products. Biotransformation with enone (X = N₃) mediated by Saccharomyces cerevisiae resulted in two products via two different routes: (i) the ketone (R)-4-azido-3-benzylbutan-2-one in 28% yield and with >99% ee by CC bond reduction; (ii) ketone (R)-6 in 51% yield and with 95% ee via cascade reactions beginning with azido group displacement by the formal hydride from flavin mononucleotide in an S_N2′ type reaction followed by reduction of the newly formed CC bond.     

人表皮生长因子受体cDNA在酵母(Saccharomyces cerevisiae)中表达

人表皮生长因子受体(human epidermal growth factor receptor,简称hEGFR)是一个170kD的穿膜糖蛋白。利用酵母诱导型Gall启动子,hEGFR cDNA被成功地在酵母细胞中表达,其转录产物(mRNA)大小约为3.5kb。免疫荧光显微实验显示该mRNA的翻译产物(hEGFRy)是位于酵母细胞质膜上,并且hEGFRy具有与EGF结合的功能。本文还进一步证明了酵母2μm质粒的2kb的片段的确具有转录终止的功能。     

CLONING, EXPRESSION AND CHARACTERIZATION OF Pro3 GENE OF YEAST Saccharomyces cerevisiae

The proline biosynthetic pathway and Pro genes in Saccharomyces cerevisiae have just begun to be studied recently. In our laboratory, Pro2 gene of S. cerevisiae had been cloned in yeast. As described in this paper, yeast Pro3 gene was also cloned, which can complement yeast Pro3 mutants, and be expressed efficiently in E. coli. The high activities of this gene product, L-pyrroline-5-carboxylate (P5C) reductase, can be detected in both organisms. The activity of the Pro3 gene product in multiple copy plasmids is not higher than that of single copy genes in chromosomes in both yeast and E. coll. The preliminary characterization of the gene is also reported.     

Determination of Cu, Zn and Cd in water by FAAS after preconcentration by baker’s yeast (Saccharomyces cerevisiae) immobilized on sepiolite

By using the adsorbent Saccharomyces cerevisiae immobilized on sepiolite an adsorption-elution method was developed for the preconcentration of Cu, Zn, and Cd followed by flame atomic absorption spectrometry (FAAS). Recoveries were 98.3 ± 0.4% for Cu, 94.2 ± 0.3% for Zn, and 99.04 ± 0.04% for Cd at 95% confidence level obtained by the column method. The influence of sea water matrix elements on the separation of the trace elements was also assessed by using the column procedure. The breakthrough capacities were found to be 74 μmol/g for copper, 128 μmol/g for zinc and 97 μmol/g for cadmium. After optimization the proposed method was applied to the trace metal determination in sea and river water. Received: 8 June 1998 / Revised: 8 September 1998 / Accepted: 16 September 1998     

Determination of Cu, Zn and Cd in water by FAAS after preconcentration by baker’s yeast (Saccharomyces cerevisiae) immobilized on sepiolite

By using the adsorbent Saccharomyces cerevisiae immobilized on sepiolite an adsorption-elution method was developed for the preconcentration of Cu, Zn, and Cd followed by flame atomic absorption spectrometry (FAAS). Recoveries were 98.3 ± 0.4% for Cu, 94.2 ± 0.3% for Zn, and 99.04 ± 0.04% for Cd at 95% confidence level obtained by the column method. The influence of sea water matrix elements on the separation of the trace elements was also assessed by using the column procedure. The breakthrough capacities were found to be 74 μmol/g for copper, 128 μmol/g for zinc and 97 μmol/g for cadmium. After optimization the proposed method was applied to the trace metal determination in sea and river water.     

Metabolic engineering of Saccharomyces cerevisiae for efficient production of pure l−(+)−lactic acid

We developed a metabolically engineered Saccharomyces cerevisiae, which produces optically pure L-lactic acid efficiently using cane juice-based medium. In this recombinant, the coding region of pyruvate decarboxylase (PDC)1 was completely deleted, and six copies of the bovine L-lactate dehydrogenase (L-LDH) genes were introduced on the genome under the control of the PDC1 promoter. To confirm optically pure lactate production in low cost medium, cane juice-based medium was used in fermentation with neutralizing conditions. L-lactate production reached 122 g/L, with 61% of sugar being transformed into L-lactate finally. The optical purity of this L-lactate, that affects the physical characteristics of poly-L-lactic acid, was extremely high, 99.9% or over.     

Advances and Developments in Strategies to Improve Strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and Processes to Obtain the Lignocellulosic Ethanol−A Review

The conversion of biomass into ethanol using fast, cheap, and efficient methodologies to disintegrate and hydrolyse the lignocellulosic biomass is the major challenge of the production of the second-generation ethanol. This revision describes the most relevant advances on the conversion process of lignocellulose materials into ethanol, development of new xylose-fermenting strains of Saccharomyces cerevisiae using classical and modern genetic tools and strategies, elucidation of the expression of some complex industrial phenotypes, tolerance mechanisms of S. cerevisiae to lignocellulosic inhibitors, monitoring and strategies to improve fermentation processes. In the last decade, numerous engineered pentose-fermenting yeasts have been developed using molecular biology tools. The increase in the tolerance of S. cerevisiae to inhibitors is still an important issue to be exploited. As the industrial systems of ethanol production operate under non-sterile conditions, microbial subpopulations are generated, depending on the operational conditions and the levels of contaminants. Among the most critical requirements for production of the second-generation ethanol is the reduction in the levels of toxic by-products of the lignocellulosic hydrolysates and the production of low-cost and efficient cellulosic enzymes. A number of procedures have been established for the conversion of lignocellulosic materials into ethanol, but none of them are completely satisfactory when process time, costs, and efficiency are considered.     

Mutation of isoleucine 705 of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae affects lanosterol's C/D-ring cyclization and 17α/β-exocyclic side chain stereochemistry

Site-saturated substitution in Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase at Ile705 position produced three chair-boat-chair (C-B-C) truncated tricyclic compounds, two 17α-exocyclic protosteryl intermediates, two protosteryl C-17 truncated rearranged intermediates and the normal biosynthetic product, lanosterol. These results indicated the importance of the Ile705 residue in affecting lanosterol's C/D ring stabilization including 6-6-5 tricyclic and protosteryl C-17 cations and 17α/β-exocyclic side chain stereochemistry.     

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cells in Dual Matrices

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.     

IMMOBILIZATION OF Saccharomyces Cerevisiae USING POLY（ACRYLAMIDE） GEL FOR ASYMMETRIC SYNTHESIS OF R（-）-MANDELIC ACID

In this paper, the poly(acrylamide) hydrogel used to immobilize saccharomyces cerevisiae for asymmetric synthesis of R(-)-mandelic acid was prepared with free radical ploymerization in deionized water at room temperature under nitrogen atmosphere. The influence of the composition of hydrogel, loading amount of cells and culture conditions on the asymmetric synthesis was investigated. Results show that PAAm hydrogel is a feasible carrier for immobilization of cells which is a potential alternative method to prepare enantiomerically pure R(-)-mandelic acid.     

IMMOBILIZATION OF Saccharomyces Cerevisiae USING POLY(ACRYLAMIDE)GEL FOR ASYMMETRIC SYNTHESIS OF R(-)-MANDELIC ACID

1. INTRODUCTIONPoly(acrylamide) hydrogel (PAAm gel), which is able to swell, but cannot be dissolved in the aqueous environment, is a three-dimension network with repeating hydrophilic units -CONH2. Since PAAm gel has a good biocompatibility as well as a high mechanical strength, and can be easily separated from reaction medium, PAAm gel has often been employed to immobilize enzymes and cells so as to catalyze various of chemical and biochemical reactions [1,2]. So far, biosynthesis …     

Secretion of Geobacillus Thermoleovorans IT-08 α-L-Arabinofuranosidase (AbfA) in Saccharomyces Cerevisiae by Fusion with HM-1 Signal Peptide

Two different expression vectors were constructed to investigate two signal peptides on secretion of Geobacillus thermoleovorans IT-08 α-L-arabinofuranosidase (AbfA) in Saccharomyces cerevisiae. They were designed to direct the secretion of AbfA by the aid of one of the following signal peptides, the αF signal peptide in the plasmid YEpFLAG1-Af and HM-1 signal peptide in the plasmid pYHM1-Af. Although some successful results have been reported in proteins secretion with αF leader sequence, in this research no α-L-arabinofuranosidase activity could be observed in recombinants S. cerevisiae YEpFLAG1-Af. The HM-1 leader sequence, originated from Hansenula mrakii IFO 0895 killer toxin, showed the capability to AbfA secretion.     

Synthesis and characterization of di- and trivalent pyrazolylborate β-diketonates and cyanometalates

The syntheses, structures, and magnetic properties of a series of di- and trivalent hydridotris(3,5-dimethylpyrazol-1-yl)borate (Tp*) cyanomanganates are described. Treatment of tris(acetylacetonate)manganese(III) [Mn(acac)(3)] with KTp* and tetra(ethyl)ammonium cyanide affords [NEt(4)][(Tp*)Mn(II)(κ(2)-acac)(CN)] (1), as the first monocyanomanganate(II) complex; attempted oxidation of 1 with iodine affords {(Tp*)Mn(II)(κ(2)-acac(3-CN))}(n) (2) as a one-dimensional chain and bimetallic {[NEt(4)][(Tp*)Mn(II)(κ(2)-acac(3-CN))](2)(μ-CN) (3) as the major and minor products, respectively. A fourth complex, [NEt(4)][(Tp*)Mn(II)(η(2)-acac(3-CN))(η(1)-NC-acac)] (4), is obtained via treatment of Mn(acac(3-CN))(3) with KTp* and [NEt(4)]CN, while [NEt(4)](2)[Mn(II)(CN)(4)] (5) was prepared from manganese(II) trifluoromethanesulfonate and excess [NEt(4)]CN. Tricyanomanganate(III) complexes, [cat][(Tp*)Mn(III)(CN)(3)] [cat = NEt(4)(+), 7; PPN(+), 8], are prepared via sequential treatment of Mn(acac(3-CN))(3) with KTp*, followed by [NEt(4)]CN, or [cat](3)[Mn(III)(CN)(6)] with (Tp*)SnBu(2)Cl. Magnetic measurements indicate that 1, 2, and 4 contain isotropic Mn(II) (S = (5)/(2); g = 2.00) centers, and no long-range magnetic ordering is found above 1.8 K. Compounds 7 and 8 contain S = 1 Mn(III) centers that adopt singly degenerate spin ground states without orbital contributions to their magnetic moments.