Identification and molecular characterization of the calmodulin-binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae. An alpha-factor inducible gene.

A method has been developed for the rapid purification of yeast calmodulin in high yield. Using a 125I-labeled calmodulin SDS/PAGE gel overlay procedure with either yeast or bovine calmodulin, we show that the bovine and yeast proteins recognize the same proteins in total yeast extracts. However, yeast calmodulin does not bind to many of the proteins in vertebrate cells identified using bovine calmodulin. A lambda gt11 yeast genomic expression library was screened with yeast or bovine brain 125I-calmodulin to identify sequences derived from calmodulin binding proteins. Twelve clones were recovered, all containing a common DNA insert; all bound calmodulin in a Ca(2+)-dependent manner. The complete coding sequence was recovered and sequenced. The predicted protein sequence show greater than 50% identity to the A subunit of vertebrate protein phosphatase 2B. The gene was designated CMP1 and shown to reside on chromosome IV. Disruption or over-expression of CMP1 have no obvious phenotype; yeast appears to contain one or more CMP1-related genes. The protein product of the CMP1 gene is elevated by alpha-factor treatment, suggesting an involvement of protein phosphatase 2B in the mating response.     

CYP78A1 Preferentially Expressed in Developing Inflorescences of Zea mays Encoded a Cytochrome P450-Dependent Lauric Acid 12-Monooxygenase

Cytochrome P450 (P450 or CYP) monooxygenases play an important role in the oxidation of a number of lipophilic substrates including secondary metabolites in higher plants. Larkin reported that CYP78A1 was preferentially expressed in developing inflorescences of Zea mays (Larkin, Plant Mol. Biol. 25: 343-353, 1994). However, the enzymatic function of CYP78A1 hasn’t been clarified yet. To characterized the enzymatic activity of CYP78A1, in this study, CYP78A1 cDNA and tobacco or yeast NADPH-cytochrome P450 oxidoreductase (P450 reductase) was expressed in the yeast Saccharomyces cerevisiae AH22 cells under the control of alcohol dehydrogenase promoter I and terminator. The reduced CO-difference spectrum of a microsomal fraction prepared from the transformed yeast cells expressing CYP78A1 and yeast P450 reductase showed a peak at 449 nm. Based on the spectrum, the content of a P450 molecule was estimated to be 45 pmol P450 equivalent/mg of protein in the microsomal fraction. The recombinant yeast microsomes containing CYP78A1 and yeast P450 reductase were found to catalyze 12-monooxygenation of lauric acid. Based on these results, CYP78A1 preferentially expressed in developing inflorescences of Zea mays appeared to have participated in the monooxygenation of fatty acids.     

The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans

During ribosomal RNA (rRNA) maturation, cleavages at defined sites separate the mature rRNAs from spacer regions, but the identities of several enzymes required for 18S rRNA release remain unknown. PilT N-terminus (PIN) domain proteins are frequently endonucleases and the PIN domain protein Utp24 is essential for early cleavages at three pre-rRNA sites in yeast (A0, A1 and A2) and humans (A0, 1 and 2a). In yeast, A1 is cleaved prior to A2 and both cleavages require base-pairing by the U3 snoRNA to the central pseudoknot elements of the 18S rRNA. We found that yeast Utp24 UV-crosslinked in vivo to U3 and the pseudoknot, placing Utp24 close to cleavage at site A1. Yeast and human Utp24 proteins exhibited in vitro endonuclease activity on an RNA substrate containing yeast site A2. Moreover, an intact PIN domain in human UTP24 was required for accurate cleavages at sites 1 and 2a in vivo, whereas mutation of another potential site 2a endonuclease, RCL1, did not affect 18S production. We propose that Utp24 cleaves sites A1/1 and A2/2a in yeast and human cells.     

Rapid detection and quantification of yeast species during spontaneous wine fermentation by PCR-RFLP analysis of the rDNA ITS region

L. GRANCHI, M. BOSCO, A. MESSINI and M. VINCENZINI.1999.PCR–RFLP analysis of the rDNA–ITS (internal transcribed spacer) region was applied to 174 yeast strains belonging to 30 species of oenological significance and including 27 type strains in order to define a rapid identification protocol for yeast colonies. Dra I-or Hae III-PCR–RFLP patterns were species-specific with the exception of teleomorphic and anamorphic forms. An improved protocol taking about 30 h was used for the detection and quantification of yeast species occurring in the course of a spontaneous wine fermentation at industrial level. Wine samples were taken and plated daily on an agar medium and the developed colonies were analysed by PCR–RFLP after 24 h of incubation. A representative sample of these colonies was also identified by traditional methods. Both procedures gave identical results. However, PCR–RFLP analysis allowed a more precise enumeration of the yeast populations, proving to be a reliable and simple method for monitoring the development of the yeast community throughout wine fermentation.     

Changes in a hydropsychid guild downstream from a eutrophic impoundment

The production of unicellular algae is laborious and is a major constraint for the culturing of aquatic filter-feeders. Because of their small particle size and their high protein content yeasts are considered as a promising substitute for micro-algae. Furthermore, recent work has shown that baker's yeast can be converted into a digestible diet for Artemia by chemical treatment. The present study documents the use at laboratory scale of this manipulated yeast as an algal substitute for the culture of two anostracan species. The experiments were conducted with the brine shrimp artemia franciscana and the fairy shrimp Streptocephalus proboscideus. A similar experimental set-up was used for both species. The algal diet, consisting of Dunaliella tertiolecta for A. franciscana and Selenastrum capricornutum for S. proboscideus, was substituted at various levels by two types of treated baker's yeast: a fresh form and a dried product which was rich in highly unsaturated fatty acids (HUFA). The chemically-treated yeast offers promising possibilities as an algal substitute for Artemia; i.e. replacing 75% of the algae by the dried yeast resulted in similar survival and even higher growth rates in comparison with the reference algal diet; for the treated fresh yeast similar results could be achieved by up to 95% substitution. For S. proboscideus, a substitution of 75% by either of the yeast products resulted in good survival, though growth did not exceed 80% of the observed growth in the algal control. A diet consisting solely of yeast resulted in poor survival for larvae of both species. Experiments were run to investigate whether this was due to a sub-optimal feeding regime, nutritional deficiencies, or deterioration of the water quality.     

Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae

Aims: Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. Methods and Results: A dammarenediol‐producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT‐PCR. Fermentation was carried out in a 5‐L GRJY‐bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250 μg l^?1. However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Conclusions: Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. Significance and Impact of the Study: This study showed that the high‐value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene‐derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.     

Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation

A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase.     

Studies on the relationship between the estrous cycle of BALB/c mice and their resistance to Paracoccidioides brasiliensis infection

A relationship between the estrous cycle and non-specific host resistance to Paracoccidioides brasiliensis yeast cells was examined by using both sexes of adult BALB/c mice. They were divided into 6 groups, including a male group and females at proestrus, estrus, metestrus-I, metestrus-II and diestrus. The mice received yeast cells through three different inoculation routes; intravenous, intraperitoneal and intratracheal. In all of the inoculation routes, the clearance of the yeast cells was influenced by the estrous cycle. The female mice at estrus, which might have high blood estrogen levels, showed a marked clearance of the yeast cells from the blood, peritoneal cavity and lungs. These results suggested that non-specific host resistance to the yeast cells was enhanced by estrogen. All female groups inoculated by the three routes showed higher clearance of the yeast cells than the male group.     

Production of ethanol in repeated-batch fermentation with membrane-type bioreactor

The average ethanol content in sake is 14 wt%; continuous production of such a high ethanol content was found not to be stably maintained in a packed-bed bioreactor with immobilized yeast cells, used normally for production of an ethanol content of up to 10 wt%. However, use of repeated-batch ethanol fermentation incorporating a membrane filter for product separation enabled a high ethanol content and improved productivity to be achieved. In this bioreactor, the yeast cells were retained within the bioreactor and a high yeast concentration was possible. A filtrate containing 14 wt% ethanol was obtained steadily after each batchwise operation. At a yeast concentration of 110 g/l, an ethanol productivity of 3.5 g/l/h was attained, which is 9 times higher than that in conventional batch fermentation. A mathematical model is proposed for assessment of the repeated-batch fermentation process. The estimated results agreed well with the observed ones. With a view to the application of this system to sake production, the aroma components of the filtrate were assayed and compared with those of a commercial-grade sake.     

Activation of Intracellular Proteinases of Yeast

The existence of the inactive precursors of yeast proteinases B and C was confirmed in the autolysate of baker’s yeast and they were named as pro-proteinases B and C, respectively. The active and inactive forms of proteinase C were two distinct proteins, separable by chromatographical procedures. The two precursors were markedly activated by incubation at pH 5 or by treatment with denaturing agents, e.g. urea, dioxane, acetone and certain alcohols.

These activations were also observed with extracts from acetone-dried cells and from mechanically destructed cells, but the activation of proteinase A was not demonstrated under any conditions tested. Therefore, it was assumed that most of proteinases B and C exist in vivo as inactive precursors, whereas proteinase A originally exists in an active form.

Pro-proteinase C, the latent form of yeast proteinase C, was partially purified from the autolysate of baker’s yeast. It was strongly activated by incubation at pH 5 or by treatment with urea or dioxane. The former activation was prevented by treatment to inactivate yeast proteinase A, which co-existed with the pro-enzyme in the present preparation, but was promoted by addition of purified proteinase A. Thus, it was confirmed that A could activate pro-proteinase C. Furthermore, it was found that activation could be caused by extremes in pH or by heating to 55~60°C, accompanied by the simultaneous destruction of the enzyme produced. Pro-proteinase C was stable over a range of pH 5 to 8 after 60 min incubation at 50°C.     

On-line determination of flocculating Saccharomyces cerevisiae concentration and growth rate using a capacitance probe

During continuous alcoholic fermentation, Saccharomyces cerevisiae 38A floc concentration was monitored using an on-line impedance probe. Since the sensor response is linear and does not depend significantly on yeast particle size, an automatic technique of determining the yeast growth rate has been developed and validated against the conventional mass balance method.     

RAPYD--rapid annotation platform for yeast data

Lower eukaryotes of the kingdom Fungi include a variety of biotechnologically important yeast species that are in the focus of genome research for more than a decade. Due to the rapid progress in ultra-fast sequencing technologies, the amount of available yeast genome data increases steadily. Thus, an efficient bioinformatics platform is required that covers genome assembly, eukaryotic gene prediction, genome annotation, comparative yeast genomics, and metabolic pathway reconstruction. Here, we present a bioinformatics platform for yeast genomics named RAPYD addressing the key requirements of extensive yeast sequence data analysis. The first step is a comprehensive regional and functional annotation of a yeast genome. A region prediction pipeline was implemented to obtain reliable and high-quality predictions of coding sequences and further genome features. Functions of coding sequences are automatically determined using a configurable prediction pipeline. Based on the resulting functional annotations, a metabolic pathway reconstruction module can be utilized to rapidly generate an overview of organism-specific features and metabolic blueprints. In a final analysis step shared and divergent features of closely related yeast strains can be explored using the comparative genomics module. An in-depth application example of the yeast Meyerozyma guilliermondii illustrates the functionality of RAPYD. A user-friendly web interface is available at https://rapyd.cebitec.uni-bielefeld.de.     

Isolation of a psychrotolerant Debaryomyces hansenii strain from fermented tea plant (Camellia sinensis) leaves

Abstract

A psychrotolerant, halotolerant and alkalophilic yeast was isolated from fermented leaves of Camellia sinensis Kuntze, the tea plant. The yeast strain, named Tea-Y1, was both phenotypically and genotypically identified as belonging to the species Debaryomyces hansenii. This assignment was confirmed by scanning and transmission electron microscopy. The analysis of growth curves demonstrated the ability this yeast strain to grow in a temperature range between 4°C and 28°C, with an optimum of 23°C. The ecology of this yeast in the C. sinensis phyllosphere, as well as its possible role in tea fermentation and storage, with particular reference to iced tea, are discussed.     

Functional expression of amine oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> (AO-I) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.     

Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

The human RAD18 gene product interacts with HHR6A and HHR6B

During DNA replication, lesion bypass is an important cellular response to unrepaired damage in the genome. In the yeast Saccharomyces cerevisiae, Rad6 and Rad18 are required for both the error-free and error-prone lesion bypass mechanisms. Furthermore, Rad6–Rad18 interaction is thought to be critical at an early step during lesion bypass in yeast. Two closely related human homologs of yeast Rad6 have been identified as HHR6A and HHR6B. Here, we report a full-length cDNA coding for the human homolog of yeast Rad18. The human RAD18 gene codes for a protein of 484 amino acid residues with a calculated molecular weight of 54 804 Da, and the gene is localized to chromosome 3 between reference intervals D3S3591 and D3S1283. Human RAD18 protein (hRAD18) was found to interact with HHR6A and HHR6B. When co-expressed in yeast cells, stable hRAD18–HHR6A and hRAD18–HHR6B protein complexes were identified and purified to near homogeneity. Thus, through interaction and complex formation with HHR6A and HHR6B, RAD18 protein may play an important role in lesion bypass mech­anisms in humans. Consistent with its role as a fundamental lesion bypass protein, the RAD18 gene is ubiquitously expressed in various human tissues.     

A new method for yeast recovery in batch ethanol fermentations: Filter aid filtration followed by separation of yeast from filter aid using hydrocyclones

In the Melle-Boinot process for alcohol production, centrifuges are normally used for yeast recovery at the end of a batch fermentation. Centrifuges are expensive equipment and represent an impressive part of the equipment costs in alcohol industries. In the present work, an alternative method for yeast recovery using less expensive equipment was studied. Instead of using centrifuges, yeast was separated from the fermented broth by filter aid filtration, followed by separation of yeast from the filter aid using hydrocyclones. A stainless steel plate-and-frame filter of filtration area 1.14 m² and two 30 mm hydrocyclones, which followed the Bradley and Rietema recommended proportions, were used in this work. The filter aid was perlite. Tests of direct separation of yeast from the fermented broth using the Bradley hydrocyclone proved to be completely unfeasible, since the maximal reduced total efficiency obtained was only 1%. When the hydrocyclones were used to separate perlite from the resuspended filtration cake, the perlite total separation efficiency obtained in the underflow was as high as 95% when using the Bradley hydrocyclone with an underflow diameter of 3 mm. To show the feasibility of the proposed new method of yeast recovery, a complete cycle of experiments, which included fermentation, yeast separation, and new fermentation using the recycled cells, was performed with good results.     

Alternate Procedure for the Preparation of the Coenzyme A-Synthesizing Protein Complex of Bakers' Yeast

Abstract

The coenzyme A-synthesizing protein complex (CoA-SPC) of Bakers' yeast synthesizes coenzyme A in an in vitro system from the precursors ATP, D-pantothenic acid and L-cysteine. CoA-SPC has been produced on a small scale by freezing Bakers' yeast cells in a mixture of diethyl ether and solid CO₂. followed by a thawing period, and subsequent removal of the diethyl ether by vacuum. The resulting yeast lysate was then stirred for 18 h in the presence of t-Factor to solubilize CoA-SPC. When a greater quantity of CoA-SPC was needed, the danger associated with the use of a large volume of diethyl ether was apparent. Therefore, the freezing step involving diethyl ether and solid CO₂ has been replaced by a process of slowly drying fresh Bakers' yeast until approximately 34% of the initial weight of the yeast remained as dry solids. The yeast solids were ground to further disrupt the cell wall and membrane structure. The grinding step was followed by rehydration of the dry yeast solids with deionized H₂O and stirring the rehydrated yeast for 18 h to solubilize CoA-SPC.     

RNA Recognition Motif 2 of Yeast Pab1p Is Required for Its Functional Interaction with Eukaryotic Translation Initiation Factor 4G

The eukaryotic mRNA 3′ poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.