Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.     

酿酒酵母Saccharomyces cerevisiae重组菌株木糖醇发酵的初步研究

木糖还原酶催化木糖为木糖醇的反应,是木糖代谢的第一步。将木糖还原酶的原因ＸＹＬ１引入酿酒酵母中,构建得到儿表达ＸＹＬ１基因的重组酿酒酵母菌株ＨＹＥＸ２,该重组菌株的木糖还原酶比活力为７．４７Ｕ／ｍｇ。研究表明,该菌株获得转化木糖产生木糖醇的能力,当辅助碳源葡萄糖的浓度为２％,并在发酵３０ｈ左右添加木糖,木糖醇的转化率可达到０．９７ｇ／ｇ。     

一种基因工程改造的NADH高亲和力木糖还原酶突变基因可促进酿酒酵母发酵木糖生成乙醇

在导入表达毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase,XR)和木糖醇脱氢酶(xylitol dehydrogenase,XDH)基因的重组酿酒酵母中,木糖还原酶活性主要依赖辅酶NADPH,木糖醇脱氢酶活性依赖辅酶 NAD+,两者的辅助因子不同导致细胞内电子氧化还原的不平衡,是造成木糖醇积累,影响木糖代谢和乙醇产量的主要原因之一.将经过基因工程改造获得的NADH高亲和力的木糖还原酶突变基因m1,与毕赤酵母木糖醇脱氢酶(PsXDH)基因xyl2共转染酿酒酵母AH109,以转染毕赤酵母木糖还原酶(PsXR)基因xyl1和xyl2重组质粒的酵母细胞为对照菌株,在SC/-Leu/-Trp营养缺陷型培养基中进行筛选,获得的阳性转化子分别命名为AH-M-XDH和AH-XR-XDH.重组酵母在限制氧通气条件下对木糖和葡萄糖进行共发酵摇瓶培养,HPLC检测发酵底物的消耗和代谢产物的产出情况.结果显示,与对照菌株AH-XR-XDH相比,AH-M-XDH的木糖利用率明显提高,乙醇得率增加了16%,木糖醇产生下降了41.4%.结果证实,通过基因工程改造的木糖代谢关键酶,可用于酿酒酵母发酵木糖生产乙醇,其能通过改善酿酒酵母细胞内氧化还原失衡的问题,提高木糖利用率和乙醇产率.     

A New Differentiation Inducer of Friend Leukemia Cells,Trichostatic Acid

FL-657B, which induced differentiation of Friend leukemia cells, was isolated from the culture fluid of Streptomyces sioyaensis and identified with trichostatic acid, a hydrolysis product of trichostatin A and C. FL-657B induced hemoglobin biosynthesis of both dimethyl sulfoxide-sensitive and -resistant Friend leukemia cells. FL-657B caused approximately 90% of the cells to be benzidine positive and reduced the growth to approximately 30~70% of the control at 2.42 μ/ml. Hemoglobin newly biosynthesized by the induction of FL-657B showed a UV absorption pattern similar to that from the normal mouse.     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

Induction of GLUT1 mRNA in response to azide and inhibition of protein synthesis

The biochemical behaviour of four commercial strains of Saccharomyces cerevisiae was studied in the presence of calcium ions, acrylamide and bisacrylamide. Calcium ions at a concentration of 300 µM induced an increase of NAD(P)⁺ reduction in commercial Turkish and American strains, while in Chilean and Brazilian commercial strains, it diminished NAD(P)⁺ reduction. On the other hand, polyacrylamide monomers (acrylamide and bisacrylamide) induced a decrease of NAD(P)⁺ reduction in all strains studied in this paper. When membrane potential () and oxygen consumption were measured in the presence of polyacrylamide monomers, a decrease of both was observed in all strains studied.     

4-Hydroxy-β-damascone and 4-Hydroxy-dihydro-β-damascone from Cigar Tobacco

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

酿酒酵母NAD(H)激酶Pos5p在细胞抵抗氧化胁迫中的作用

酿酒酵母线粒体NAD(H)激酶Pos5p显示出重要功能,其缺失将导致细胞抗氧化性能出现障碍。为了了解Pos5p的抗氧化作用机制及其与调节辅酶NAD(H)和NADP(H)之间的关系,比较了在不同类型的氧化胁迫试剂作用下,野生型BY4742、POS5基因缺失体pos5-及其回补体pos5-/POS5-YEp的生长表型,同时采用高效液相色谱测定细胞内辅酶含量。结果表明,在超氧生成试剂甲萘醌(VK3)、过氧化氢(H2O2)和GSH消耗试剂马来酸二乙酯(DEM)存在时,pos5-都表现出明显的生长缺陷,而各抗氧化基因缺失体只在其相应胁迫下表现出生长缺陷。在正常生长条件下,pos5-的NADPH含量降低,pos5-/POS5-YEp则提高,表明Pos5p对胞内NADPH的供应有重要作用。在VK3、H2O2和DEM胁迫下,BY4742、pos5-及pos5-/POS5-YEp的NADP(H)含量均有不同程度的下降,其中pos5-的NADP(H)/NAD(H)比率下降最为严重,而pos5-/POS5-YEp较pos5-有明显提高,这与其氧化胁迫表型相一致。因此,在细胞面临不同类型的氧化胁迫时,Pos5p都能有效行使其NAD(H)激酶活性,补充NADP(H)的损耗,从而对细胞起到抗氧化保护作用。     

木糖异构酶在酿酒酵母细胞表面的展示

将来源于嗜热细菌Thermus thermophilus的木糖异构酶基因xylA,与酿酒酵母(Sac-charomyces cerevisiae)a-凝集素表面展示载体pYD1的Aga2p亚基C端序列融合。编码融合蛋白的基因序列前接上半乳糖诱导型启动子。用LiAc完整细胞法转化酿酒酵母EBY100。含重组质粒的菌株EBY100/pYD-xylA经半乳糖诱导表达外源融合蛋白,免疫荧光显微镜结果显示外源蛋白被锚定在细胞壁上,木糖异构酶活性测定结果表明,细胞壁上酶活测定值为1.52U,木糖异构酶在酿酒酵母细胞壁上得到活性表达。     

Two-electron reduction of quinones by rat liver NAD(P)H:quinone oxidoreductase: quantitative structure-activity relationships

Mammalian NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) catalyzes the two-electron reduction of quinones and plays one of the main roles in the bioactivation of quinoidal drugs. In order to understand the enzyme substrate specificity, we have examined the reactions of rat NQO1 with a number of quinones with available potentials of single-electron (E(1)(7)) reduction and pK(a) of their semiquinones. The hydride transfer potentials (E(7)(H(-))) were calculated from the midpoint potentials of quinones and pK(a) of hydroquinones. Our findings imply that benzo- and naphthoquinones with a van der Waals volume (VdWvol) < or = 200 A(3) are much more reactive than glutathionyl-substituted naphthoquinones, polycyclic quinones, and FMN (VdWvol>200 A(3)) with the same reduction potentials. The entropies of activation (DeltaS(not equal)) in the reduction of "fast" oxidants are equal to -84 to -76 J mol(-1) K(-1), whereas in the reduction of "slow" oxidants Delta S(not equal)=-36 to -11 J mol(-1) K(-1). The large negative Delta S(not equal) in the reduction of fast oxidants may be explained by their better electronic coupling with reduced FAD or the formation of charge-transfer complexes, since fast oxidants bind at the dicumarol binding site, whereas the binding of some slow oxidants outside it has been demonstrated. The reactivity of quinones may be equally well described in terms of the three-step (e(-),H(+),e(-)) hydride transfer, using E(1)(7), pK(a)(QH*), and VdWvol as correlation parameters, or in terms of single-step (H(-)) hydride transfer, using E(7)(H(-)) and VdWvol in the correlation. The analysis of NQO1 reactions with single-electron acceptors and quinones using an "outer-sphere" electron transfer model points to the possibility of a three-step hydride transfer.     

氧化胁迫对酿酒酵母NAD(H)激酶突变体呼吸链活性的影响

【目的】了解酿酒酵母线粒体NAD(H)激酶Pos5p对呼吸链活性的维持是否与其抗氧化功能有关。【方法】比较在不同类型的氧化胁迫试剂作用下,野生菌BY4742、POS5基因缺失体pos5Δ及其回补体pos5Δ/POS5-YEp的呼吸链各个酶复合体的活性变化及细胞内活性氧水平变化。【结果】在非胁迫条件下,pos5Δ的各个复合体活性明显低于BY4742,而pos5Δ/POS5-YEp的活性有所恢复,这与它们的胞内活性氧水平相一致。在甲萘醌胁迫下,BY4742和pos5Δ的各个复合体活性都发生不同程度的下降,但pos5Δ/POS5-YEp的活性都升高。在H2O2、马来酸二乙酯胁迫下,除个别复合体外,BY4742、pos5Δ和pos5Δ/POS5-YEp的呼吸链复合体活性都降低,尤以pos5Δ的活性降低最为严重,BY4742的活性降低则较少,而pos5Δ/POS5-YEp在H2O2胁迫下的活性降低得到了缓解。说明甲萘醌、H2O2和马来酸二乙酯胁迫会造成酿酒酵母呼吸链各个复合体发生损伤,而过表达Pos5p则有助于缓解甲萘醌和H2O2引起的损伤。【结论】Pos5p对呼吸链的作用与其抗氧化功能有相关性。     

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.