A Short Region from the LEU2 Gene of Saccharomyces cerevisiae Functions as an ARS in the Yeast Saccharomyces exiguus Yp74L-3

We examined the autonomously replicating sequence (ARS) activity of some fragments derived from the LEU2 region of Saccharomyces cerevisiae onto Saccharomyces exiguus Yp74L-3. A DNA fragment functioning as an ARS in S. exiguus, but not in S. cerevisiae, was shown to exist. The ARS activity for S. exiguus was reduced by the 2-μm plasmid origin of S. cerevisiae when both elements coexisted on a single circular plasmid. Analysis of ARS activity with the PCR products from the fragment revealed that the ARS-acting sequence was located in the 3′-terminal area of the transcribed region of the LEU2 gene of S. cerevisiae. It is suggested that the ARS recognition system in S. exiguus is significantly different from that of S. cerevisiae. Received: 16 June 1998 / Accepted: 3 August 1998     

Construction of an effective host-vector system for the yeast Saccharomyces exiguus Yp74L-3.

An effective host-vector system specific to the yeast Saccharomyces exiguus Yp74L-3 was constructed to promote the molecular genetic analyses for the yeast. To obtain a stable reversionless host strain, we constructed an S. exiguus strain carrying leu2::ScURA3 by disrupting the S. exiguus LEU2 gene with the S. cerevisiae URA3 gene. A vector plasmid unique to S. exiguus was subsequently developed by inserting both the LEU2 gene and an ARS cloned from S. exiguus into an Escherichia coli phagemid, pUC119. The vector constructed, pTH119 was able to transform the S. exiguus leu2::ScURA3 strain to Leu+ efficiently. The stability of the vector in the S. exiguus host cells resembled that of a YRp-type vector in S. cerevisiae.     

Physical Characterization of the Chromosomal DNA of the Yeast Saccharomyces exiguus

The number of chromosomes in the yeast Saccharomyces exiguuswas determined to be thirteen by two-dimensional pulsed-fieldgel electrophoresis. The thirteen chromosomes ranged in DNAsize from 520 to 2,600 kbp, with a total length of approximately14 Mbp. Numbers I to XIII were assigned to the chromosomes indecreasing order of DNA length. Southern hybridization analysisusing total DNAs from S. exiguus and S. cerevisiae as probesshowed that there was no significant homology between the chromosomalDNAs of the two species, except in the case of the chromosomalDNA that included rDNA. When rDNA and genes LEU2, TRP1, URA3and HO of S. cerevisiae were used as hybridization probes, itwas apparent that S. exiguus had DNA sequences homologous tothe rDNA and to the LEU2 and HO genes. In S. exiguus, rDNA-likeand LEU2-like DNAs were located on chromosomes I and IX, respectively,and HO-like DNA was located on chromosome VI or VII. (Received May 17, 1993; Accepted July 15, 1993)     

Control of ATP-Dependent Binding of Saccharomyces cerevisiae Origin Recognition Complex to Autonomously Replicating DNA Sequences

Eukaryotic origin recognition complexes (ORCs) play pivotal roles in the initiation of chromosomal DNA replication. ORC from the yeast, Saccharomyces cerevisiae, recognizes and binds replication origins in the late G1 phase and the binding has profound implications in the progression of the cell cycle to the S-phase. Therefore, we have quantitatively analyzed the mechanism of recognition and interaction of the yeast ORC with various elements of a yeast origin of DNA replication, the autonomously replicating sequence 1 (ARS1). ORC bound all four individual A and B elements of ARS1 with reasonably high affinities. However, the highest affinity binding was observed with a DNA sequence containing both the A and B1 elements. In addition, ATP and ADP significantly modulated the binding of ORC to the combined elements as well as modulating the binding of ORC to the element A alone or in combination with the B1 element. However, binding of ORC to individual B1, B2, and B3 elements was not responsive to nucleotides. Thus, the consensus ARS sequence in element A appeared to play a pivotal role in the ATP-dependent binding of ORC to ARS1 and likely in other ARSs or origins of DNA replication.     

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.     

Single-strand-binding factor(s) which interact with ARS1 of Saccharomyces cerevisiae

Since plasmids containing autonomously replicating sequence(s) (ARS) can transform Saccharomyces cerevisiae cells at high frequency, ARS are considered to be the replication origins of chromosomes. To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors which interact with the ARS1 region located near the centromere of chromosome IV in S. cerevisiae. Using the gel-shift assay, we found protein factors which bound to a single-stranded, 97-bp fragment of the ARS1 region containing the core consensus. Competition experiments with various oligodeoxyribonucleotides (oligos) suggest that a site recognized by the factor(s) was within the element containing the core consensus and adjacent close matches to the core consensus of the minus strand. Indeed, when the oligo containing the minus strand of this element was used as a probe, two oligo-protein complexes were detected. Mutations in the core consensus reduced these binding activities. When the plus-strand oligo of the same region was used as a probe, a retarded band was also detected, but with less specific binding. Considering the fact that the core consensus and close matches to the core consensus are important for ARS function, these results imply that the protein factors detected in this experiment may participate in DNA replication.     

Mechanisms Controlling the Integrity of Replicating Chromosomes in Budding Yeast

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.     

De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae)

Vanillin is one of the world''s most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world''s vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker''s yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin β-d-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.In 2007, the global market for flavor and fragrance compounds was an impressive $20 billion, with an annual growth of 11 to 12%. The isolation and naming of vanillin (3-methoxy-4-hydroxybenzaldehyde) as the main component of vanilla flavor in 1859 (8), and the ensuing chemical synthesis in 1874 (41), in many ways marked the true birth of this industry, and this compound remains the global leader in aroma compounds. The original source of vanillin is the seed pod of the vanilla orchid (Vanilla planifolia), which was grown by the Aztecs in Mexico and brought to Europe by the Spaniards in 1520. Production of natural vanillin from the vanilla pod is a laborious and slow process, which requires hand pollination of the flowers and a 1- to 6-month curing process of the harvested green vanilla pods (37). Production of 1 kg of vanillin requires approximately 500 kg of vanilla pods, corresponding to the pollination of approximately 40,000 flowers. Today, only about 0.25% (40 tons out of 16,000) of vanillin sold annually originates from vanilla pods, while most of the remainder is synthesized chemically from lignin or fossil hydrocarbons, in particular guaiacol. Synthetically produced vanillin is sold for approximately $15 per kg, compared to prices of $1,200 to $4,000 per kg for natural vanillin (46).An attractive alternative is bioconversion or de novo biosynthesis of vanillin; for example, vanillin produced by microbial conversion of the plant constituent ferulic acid is marketed at $700 per kilogram under the trade name Rhovanil Natural (produced by Rhodia Organics). Ferulic acid and eugenol are the most attractive plant secondary metabolites amenable for bioconversion into vanillin, since they can be produced at relatively low costs: around $5 per kilogram (37). For the bioconversion of eugenol or ferulic acid into vanillin, several microbial species have been tested, including gram-negative bacteria of the Pseudomonas genus, actinomycetes of the genera Amycolatopsis and Streptomyces, and the basidiomycete fungus Pycnoporus cinnabarinus (19, 23, 25, 27, 31, 34, 35, 36, 45, 48). In experiments where the vanillin produced was absorbed on resins, Streptomyces cultures afforded very high vanillin yields (up to 19.2 g/liter) and conversion rates as high as 55% were obtained (15). Genes for the responsible enzymes from some of these organisms were isolated and expressed in Escherichia coli, and up to 2.9 g/liter of vanillin were obtained by conversion of eugenol or ferulic acid (1, 3, 32, 49).Compared to bioconversion, de novo biosynthesis of vanillin from a primary metabolite like glucose is much more attractive, since glucose costs less than $0.30/kilogram (42). One route for microbial production of vanillin from glucose was devised by Frost and coworker Li (6, 20), combining de novo biosynthesis of vanillic acid in E. coli with enzymatic in vitro conversion of vanillic acid to vanillin. 3-Dehydroshikimic acid is an intermediate in the shikimate pathway for biosynthesis of aromatic amino acids, and the recombinant E. coli was engineered to dehydrate this compound to form protocatechuic acid (3,4-dihydroxybenzoic acid) and methylate this to form vanillic acid. The vanillic acid was subsequently converted into vanillin in vitro using carboxylic acid reductase isolated from Neurospora crassa. The main products of the in vivo step were protocatechuic acid, vanillic acid, and isovanillic acid in an approximate ratio of 9:4:1, indicating a bottleneck at the methylation reaction and nonspecificity of the OMT (O-methyltransferase) enzyme for the meta-hydroxyl group of protocatechuic acid. Serious drawbacks of this scheme are the lack of an in vivo step for the enzymatic reduction of vanillic acid, demanding the addition of isolated carboxylic acid reductase and costly cofactors such as ATP, NADPH, and Mg²⁺, and the generation of isovanillin as a contaminating side product.In this study, we have genetically engineered single-recombination microorganisms to synthesize vanillin from glucose, according to the metabolic route depicted in Fig. ​Fig.1.1. To avoid the synthesis of isovanillin as an undesired side product, a large array of OMTs was screened for the desired high substrate specificity, and an appropriate enzyme was identified. A synthetic version of an aromatic carboxylic acid reductase (ACAR) gene, optimized for yeast codon usage, was introduced to achieve the reduction step. The vanillin pathway was introduced into both Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast, and significant levels of vanillin production were obtained in both organisms. Vanillin β-d-glucoside is the form in which vanillin accumulates and is stored in the fresh pod of the vanilla orchid (Vanilla planifolia). During the “curing” process of the pod, β-glucosidases are liberated and facilitate a partial conversion of the vanillin β-d-glucoside into vanillin. Upon consumption or application, the conversion of vanillin β-d-glucoside into free vanillin by enzymes in the saliva or in the skin microflora can provide for a slow-release effect that prolongs and augments the sensory event, as is the case for other flavor glycosides investigated, such as menthol glucoside (14, 16). In addition to the increased value of vanillin β-d-glucoside as an aroma or flavor compound, production of the glucoside in yeast may offer several advantages. Vanillin β-d-glucoside is more water soluble than vanillin, but most importantly, compounds such as vanillin in high concentrations are toxic to many living cells (4). It has been shown that glucosides of toxic compounds are less toxic to yeasts (24). We found this to be the case with vanillin and S. cerevisiae yeast as well. Thus, to facilitate storage and accumulation of higher vanillin yields, we introduced a step for vanillin glucosylation in S. pombe.Open in a separate windowFIG. 1.Biosynthetic scheme for de novo biosynthesis of vanillin in Schizosaccharomyces pombe and outline of the different vanillin catabolites and metabolic side products observed in different yeast strains and constructs. Gray arrows, primary metabolic reactions in yeast; black arrows, enzyme reactions introduced by metabolic engineering; diagonally striped arrows, undesired inherent yeast metabolic reactions.     

Recombination Among Three Mitochondrial Genes in Yeast (Saccharomyces cerevisiae)

Eight crosses involving three different mitochondrial genes were made in Saccharomyces cerevisiae. All three genes were linked, but an unambiguous linkage map could not be constructed.     

A Photometric Method for Determining the Growth Rate of Yeast (Saccharomyces cereviciae)

There have been several investigations into students' conceptions of animal classification. Previous research has generally failed to study the criteria of classification used by the students. This study shows that students prefer to classify creatures along the criteria of habitat and locomotion (method of movement). They continue using these criteria even after learning the categories of biological taxonomy. The results lead to the assumption of an implicit theory of natural kinship of animals. The educational consequences for biology instruction, especially with regard to biological taxonomy, biodiversity, and evolution, are discussed.     

The uptake of L-(methyl- 3 H) carnitine by the rat epididymis

The uptake of radioactivity by the epididymis and other tissues was measured following administration of L-[methyl-³H]carnitine to male rats. Rapid uptake occurred in both the caput and cauda epididymides. This radioactivity was shown to be present in carnitine and was located almost exclusively within the epididymal lumen.     

A 140-Bp-Long Palindromic Sequence Induces Double-Strand Breaks during Meiosis in the Yeast Saccharomyces Cerevisiae

Palindromic sequences have the potential to form hairpin or cruciform structures, which are putative substrates for several nucleases and mismatch repair enzymes. A genetic method was developed to detect such structures in vivo in the yeast Saccharomyces cerevisiae. Using this method we previously showed that short hairpin structures are poorly repaired by the mismatch repair system in S. cerevisiae. We show here that mismatches, when present in the stem of the hairpin structure, are not processed by the repair machinery, suggesting that they are treated differently than those in the interstrand base-paired duplex DNA. A 140-bp-long palindromic sequence, on the contrary, acts as a meiotic recombination hotspot by generating a site for a double-strand break, an initiator of meiotic recombination. We suggest that long palindromic sequences undergo cruciform extrusion more readily than short ones. This cruciform structure then acts as a substrate for structure-specific nucleases resulting in the formation of a double-strand break during meiosis in yeast. In addition, we show that residual repair of the short hairpin structure occurs in an MSH2-independent pathway.     

Detection of Active Yeast Cells (Saccharomyces cerevisiae) in Frozen Dough Sections

A new method based on fluorescence microscopy was developed to detect active yeast cells in cryosections of wheat dough. The sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) and counterstained with Evans blue. The active yeast cells in the sections appeared brilliant yellow and were readily distinguished from the red dough matrix. The dead cells allowed penetration of the Evans blue through the cell membrane, which interfered with the DAPI staining and caused the dead cells to blend into the red environment. The number of active yeast cells in fermenting dough sections containing different proportions of living and dead yeast cells correlated well with the gas-forming capability of the yeast in the dough but not with the results of the conventional plate count method. The new method allows the study of yeast activity not only during the different stages of frozen dough processing but also during the fermentation of doughs.     

用丰富培基作为选择压力在酿酒酵母PRO+转化子中稳定遗传的分泌型表达载体

构建了一个含酿酒酵母ＰＲＯ３基因的分泌型表达载体ｐＣＢｙ３１０,在ｐＣＢｙ３１０上插入 βＨＣＧ（人绒毛膜促性腺激素β亚基）－ｃＤＮＡ以形成一个重组质粒ｐＣＢｙ３１４。利用酿酒酵母脯 氨酸合成酶基因缺陷株的独特属性,即它们不能在丰富培基中生长,ｐＣＢｙ３１４在酿酒酵母 Ｐｒｏ３~－缺陷株（ＭＢ２９９－Ａ）的转化子可以在丰富培基中生长,而且保持有丝分裂稳定性。在优 化条件（但尚非最佳条件）下,βＨＣＧ在培液中的产量是６５０μｇ／Ｌ。说明ＹＰＤ可以用为选择培 基,而且酿酒酵母在ＹＰＤ中比在选择培基中生长更旺盛,可以提高表达产物的产量,加之 ＹＰＤ的成分简单又便宜。因此得出结论,ＰＲＯ基因可以广泛地用于构建在酿酒酵母中克隆和 表达外源基因的载体,以便用丰富培基作为选择压力直接筛选。     

The Yeast Protein Database (YPD): a curated proteome database for Saccharomyces cerevisiae.

The Yeast Protein Database (YPD) is a curated database for the proteome of Saccharomyces cerevisiae . It consists of approximately 6000 Yeast Protein Reports, one for each of the known or predicted yeast proteins. Each Yeast Protein Report is a one-page presentation of protein properties, annotation lines that summarize findings from the literature, and references. In the past year, the number of annotation lines has grown from 25 000 to approximately 35 000, and the number of articles curated has grown from approximately 3500 to >5000. Recently, new data types have been included in YPD: protein-protein interactions, genetic interactions, and regulators of gene expression. Finally, a new layer of information, the YPD Protein Minireviews, has recently been introduced. The Yeast Protein Database can be found on the Web at http://www.proteome.com/YPDhome. html