Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris)

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.     

Vacuolar (lysosomal) trehalase ofSaccharomyces cerevisiae

In the yeastSaccharomyces cerevisiae thePEP4 gene product, protease A, is responsible for activating all soluble vacuolar (lysosomal) enzymes. These vacuolar enzymes remain inactive inpep4 mutants. Vacuolar trehalase activity was diminished in such mutants as well. This suggests that the vacuolar (lysosomal) trehalase is processed in a manner similar to other vacuolar enzymes inS. cerevisiae.     

Comprehensive quantitative analysis of central carbon and amino‐acid metabolism in Saccharomyces cerevisiae under multiple conditions by targeted proteomics

Decades of biochemical research have identified most of the enzymes that catalyze metabolic reactions in the yeast Saccharomyces cerevisiae. The adaptation of metabolism to changing nutritional conditions, in contrast, is much less well understood. As an important stepping stone toward such understanding, we exploit the power of proteomics assays based on selected reaction monitoring (SRM) mass spectrometry to quantify abundance changes of the 228 proteins that constitute the central carbon and amino‐acid metabolic network in the yeast Saccharomyces cerevisiae, at five different metabolic steady states. Overall, 90% of the targeted proteins, including families of isoenzymes, were consistently detected and quantified in each sample, generating a proteomic data set that represents a nutritionally perturbed biological system at high reproducibility. The data set is near comprehensive because we detect 95–99% of all proteins that are required under a given condition. Interpreted through flux balance modeling, the data indicate that S. cerevisiae retains proteins not necessarily used in a particular environment. Further, the data suggest differential functionality for several metabolic isoenzymes.     

Biochemical characterization and evaluation of invertases produced from Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa for the production of fructooligosaccharides

Abstract

Invertases are used for several purposes; one among these is the production of fructooligosaccharides. The aim of this study was to biochemically characterize invertase from industrial Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa isolated from Cerrado soil. The optimum pH and temperature were 4.0 and 70?°C for Rhodotorula mucilaginosa invertase and 4.5 and 50?°C for Saccharomyces cerevisiae invertase. The pH and thermal stability from 3.0 to 10.5 and 75?°C for R. mucilaginosa invertase, respectively. The pH and thermal stability for S. cerevisiae CAT-1 invertase from 3.0 to 7.0, and 50?°C, respectively. Both enzymes showed good catalytic activity with 10% of ethanol in reaction mixture. The hydrolysis by invertases occurs predominantly when sucrose concentrations are ≤5%. On the other hand, the increase in the concentration of sucrose to levels above 10% results in the highest transferase activity, reaching about 13.3?g/L of nystose by S. cerevisiae invertase and 12.6?g/L by R. mucilaginosa invertase. The results demonstrate the high structural stability of the enzyme produced by R. mucilaginosa, which is an extremely interesting feature that would enable the application of this enzyme in industrial processes.     

A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.     

接种发酵和自然发酵中酿酒酵母菌株多样性比较

[目的]探究自然发酵和接种发酵两种发酵方式,对霞多丽葡萄发酵中酵母菌种多样性和酿酒酵母菌株遗传多样性的影响.[方法]以霞多丽葡萄为原料,分别进行自然发酵和接种不同酿酒酵母菌株(NXU 17-26、UCD522和UCD2610)的发酵,利用26S rDNA D1/D2区序列分析和Interdelta指纹图谱技术分别进行酵...     

Lack of carbon catabolite inactivation in a mutant of Saccharomyces cerevisiae with reduced hexokinase activity

Summary A mutant of Saccharomyces cerevisiae with reduced hexokinase activity and deficient in carbon catabolite inactivation is described. The reason for this lack of inactivation is not a lowered concentration of glycolysis metabolites or other low molecular effectors such as glucose, and ATP. The results point to the hexose phosphorylation step as initiator for carbon catabolite inactivation. It appears that one of the hexokinase isoenzymes, altered in the mutant, initiates the inactivation by conformational change. Repression of enzymes that are subject to carbon catabolite inactivation, is normal in the mutant. This indicates that inactivation and repression of those enzymes proceed in different ways, even though they may share common intermediate reactions.     

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

Resistance to 2-deoxyglucose in yeast: A direct selection of mutants lacking glucose-phosphorylating enzymes

Summary When strains of Saccharomyces cerevisiae carrying a single glucose-phosphorylating enzyme such as hexokinase P1 or hexokinase P2 or glucokinase, are subjected to the selection pressure against the toxic sugar 2-deoxyglucose, the majority of survivors are mutants lacking the respective enzymes. All the 2-deoxyglucose-resistant segregants recovered from backcrosses of these mutants to a wild type strain are glucose-negative and all the sensitive ones are glucose-positive. The hexokinase mutations are located in the same complementation groups as defined by the structural genes of hexokinase P1 and hexokinase P2. No interallelic complementation has been observed either in hexokinase P1 or in hexokinase P2 amongst a total of 4×64, and 5×60 different combinations of independent mutants at the hxk1 and hxk2 loci respectively. There appears to be neither a common genetic regulator controlling two or more of these glucose-phosphorylating enzymes nor a sugar carrier that can be dispensed with.     

Inhibition of glycolysis by furfural in Saccharomyces cerevisiae

Summary Furfural, a Maillard reaction product, was found to inhibit growth and alcohol production by Saccharomyces cerevisiae. Furfural concentrations above 1 mg ml^–1 significantly decreased CO₂ evolution by resuspended yeast cells. Important glycolytic enzymes such as hexokinase, phosphofructokinase, triosephosphate dehydrogenase, aldolase and alcohol dehydrogenase were assayed in presence of furfural. Dehydrogenases appeared to be the most sensitive enzymes and are probably responsible for the observed inhibition of alcohol production and growth.     

酿酒酵母芳樟醇耐受性的工程改造

【背景】芳樟醇具有特殊的香气和多种生物学活性,是食品、医药和化妆品行业的重要原料。随着合成生物学的高速发展,代谢改造微生物进行芳樟醇生物合成是当前研究的一大热点。然而在微生物的生物合成中,芳樟醇对底盘细胞的毒性是一大瓶颈问题,也是其他单萜物质生物合成的共性问题。【目的】建立合理的耐受性改造方法,以提高微生物宿主细胞对芳樟醇的耐受性。【方法】以酿酒酵母BY4741为研究对象,通过对ABC转运蛋白、活性氧调控相关酶及转录调控因子的过表达,考察它们对酿酒酵母芳樟醇耐受性的影响,并通过对酿酒酵母细胞进行定向驯化,筛选耐受性提高的酿酒酵母突变株。【结果】单独过表达ABC转运蛋白(Yor1、Snq2、Pdr5、Pdr15和Pdr18)、ROS调控相关酶(Gre2、Ctt1、Yhb1、Gpx2、Trr1、Trx2和Gsh2)及转录调控因子(Ino2、Yap1、Yap5和Stb5)并不能有效提高酿酒酵母的耐受性,但在传代适应性驯化过程中获得了两株耐受性提高的酿酒酵母突变株,将芳樟醇的致死浓度从430mg/L提高到了645mg/L以上。进一步通过基因组重测序分析揭示了驯化菌株突变位点。其中YBR074W...     

酿酒酵母表达和纯化功能性的铁载体合成蛋白PchE

【目的】利用酿酒酵母表达系统,通过乙醇脱氢酶启动子异源表达细菌源的铁载体合成蛋白PchE,并与来源于枯草芽孢杆菌的泛酰化酶Sfp同宿主共表达,探索真核表达体系表达具有生化活性的细菌源蛋白。【方法】从大肠杆菌BAP1染色体上扩增sfp基因,将pchE基因及串联的pchE与sfp基因分别构建到酵母-大肠杆菌穿梭质粒pXW55中,各自转化酿酒酵母BJ5464-npg A表达,经过亲和层析和离子交换层析纯化蛋白,利用HPLC检测细菌源与酵母源表达的PchE在体外重构生化反应中的催化活性。【结果】利用酿酒酵母表达系统可以获得高纯度的原核蛋白PchE。真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰PchE,合成中间产物HPT-Cys。【结论】在酿酒酵母Saccharomyces cerevisiae BJ5464-npgA表达系统中,首次证明真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰细菌源的非核糖体肽合酶。比较酵母和细菌宿主的目标蛋白表达,证明酵母表达的巨大蛋白PchE的纯度更高,非特异性条带减少,推测酵母宿主可能更适合表达纯化功能性的巨型蛋白质。     

The Positive Charge at Position 189 IS Essential for the Catalytic Activity of Iron-and Manganese-Containing Superoxide Dismutases

We have previously shown (C.L. Borders, Jr. el al., (1989) Archives of Biochemistry and Eiaphysics. 268, 74–80) that the iron-containing (FeSOD) and manganese-containing (MnSOD) superoxide dismutases from Eschericliia coli are extensively (≥98%) inactivated by treatment with phenylglyoxal. an arginine-specific reagent. Examination of the published primary sequences of these two enzymes shows that Arg-189 is the only conserved arginine. This arginine is also conserved in the three additional FeSODs and seven of the eight additional MnSODs sequenced to date, with the only exception king the MnSOD from Saccharomyces cerevisiae, in which it is conservatively replaced by lysine. Treatment of S. cerevisiae MnSOD with phenylglyoxal under the same conditions used for the E. coli enzymes gives very little inactivation. However, treatment with low levels of 2.4.6-trinitrobenzenesulfonate (TNBS) and acetic anhydride, two lysine-selective reagents that cause a maximum of 65–80% inactivation of the E. coli SODs, gives complete inactivation of the yeast enzyme. Total inactivation of yeast MnSOD with TNBS correlates with the modification of approximately 5 lysines per subunit, whereas 6–7 lysines per subunit are acylated with acetic anhydride on complete inactivation. It appears that the positive charge contributed by residue 189. lysine in yeast MnSOD and arginine in all other SODs. may be critical for the catalytic activity or MnSODs and FeSODs.     

Statins interfere with the attachment of S. cerevisiae mtDNA to the inner mitochondrial membrane

Abstract

The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used Saccharomyces cerevisiae as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in S.cerevisiae by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins.     

Debaryomyces hansenii as a new biocatalyst in the asymmetric reduction of substituted acetophenones

Chiral secondary alcohols are convenient mediator for the synthesis of biologically active compounds and natural products. In this study fifteen yeast strains belonging to three food originated yeast species Debaryomyces hansenii, Saccharomyces cerevisiae and Hanseniaspora guilliermondii were tested for their capability for the asymmetric reduction of acetophenone to 1-phenylethanol as biocatalyst microorganisms. Of these strains, Debaryomyces hansenii P1 strain showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to the corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high conversion rates. This is the first report on the enantioselective reduction of acetophenone by D. hansenii P1 from past?rma, a fermented Turkish meat product. The preparative scale asymmetric bio reduction of 3-methoxy acetophenone 1g by D. hansenii P1 gave (R)-1-(3-methoxyphenyl) ethanol 2g 82% yield, and >99% enantiomeric excess. Compound 2g can be used for the synthesis of (+)-NPS-R-568 [3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenly) ethyl] propan-1-amine] which have a great potential for the treatment of primary and secondary hyper-parathyroidism. In addition, D. hansenii P1 successfully reduced acetophenone derivatives. This study showed that this yeast can be used industrially to produce enantiomerically pure chiral secondary alcohols, which can be easily converted to different functional groups.     

Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products

A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from various fungi. These genes belong to different gene clusters and are involved in the biosynthesis of secondary metabolites. Ten of them were cloned and overexpressed in Escherichia coli and Saccharomyces cerevisiae and proven to be soluble proteins. They catalyse different prenyl transfer reactions onto indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. These enzymes showed broad substrate specificities towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by several prenyltransferases as substrates and converted to prenylated derivatives. This feature of substrate flexibility was successfully used for regiospecific and stereospecific synthesis of different indole derivatives.     

Qualitative yeast enzyme analysis by electrophoresis

Summary Baker's yeast (Saccharomyces cerevisiae) was cultivated under different intensities of aeration on glucose and on ethanol. Seventeen enzymes of the Embden-Meyerhof pathway and the TCA cycle or related reactions were then assayed by starch gel electrophoresis. There were both qualitative and quantitative differences in many enzymes, most notably in glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, and fumarase. Enzyme electrophoresis seems to offer a promising method for rapidly obtaining information about many yeast enzymes from a large number of samples.     

CRISPR-Cas9系统与mazF介导的大片段删减法在酿酒酵母染色体大片段删减中的比较

【目的】比较CRISPR-Cas9系统与maz F法这两种酿酒酵母染色体大片段删减方法。【方法】分别用上述两种方法删减了酿酒酵母长度为26.5 kb的染色体大片段YKL072W-YKL061W,并比较了两种方法的转化效率、敲除成功率。【结果】利用CRISPR-Cas9系统平均得到5个转化子,但正确率为100%;maz F法得到约100个转化子,正确率略低于前者,为93%。【结论】两种方法均能高效删减酿酒酵母染色体大片段,CRISPR-Cas9系统正确率较高,操作简便省时;maz F法相对稳定,对目的基因无PAM位点要求。     

Homology Modeling of Adenylosuccinate Synthetase from Saccharomyces Cerevisiae Reveals a Possible Binding Region for Single-Stranded ARS Sequences

Abstract

Adenylosuccinate synthetase from Saccharomyces cerevisiae was investigated in order to find a structural explanation for its ability to bind specifically to single-stranded ARS elements (autonomously replicating sequences). Using the E. coli enzyme as template, a model for the structure of adenylosuccinate synthetase from S. cerevisiae was generated and subsequently refined by molecular dynamics techniques.

The resulting three-dimensional structure offers an explanation for the DNA binding activity of the yeast enzyme by revealing a distinct basic region that is not present in the homologous enzymes from other organisms.

The model is also in good agreement with biochemical data available for a mutant protein in which Glycine 252 is replaced by Aspartate. On the basis of the model a significant structural distortion near the catalytic center was predicted for this mutant, corresponding well to the enzymatic inactivity observed. The mutant enzyme shows larger structural fluctuations than the wild-type protein according to the results of two independent molecular dynamics simulations.     

Leucine biosynthesis and its regulation in the basidiomycete Rhodosporidium toruloides

Complementation tests and enzyme analyses separated 29 leucine auxotrophs of the Basidiomycete Rhodosporidium toruloides into three groups, each deficient in one of the leucine biosynthetic enzymes. The following differences are suggested between the organization of the leucine pathway in R. toruloides and the Ascomycetes Saccharomyces cerevisiae and Neurospora crassa: (1) isopropylmalate, the product of the first enzymic reaction appears not to be an internal inducer of the later enzymes of the pathway; this is consistent with the apparent lack of mutants homologous to the leu3 class in N. crassa and S. cerevisiae; (2) as in S. cerevisiae, but unlike N. crassa, isopropylmalate synthase is under the control of a general cross pathway control system; (3) unlike S. cerevisiae, but like N. crassa, R. toruloides appears to possess only one gene encoding isopropylmalate synthase.Abbreviations IPM Isopropylmalate - EMS methanesulphonic acid, ethyl ester