l-Tryptophan-starved cultivation enhances S-allyl-l-cysteine synthesis in various food-related microorganisms.

S-Allyl-l-cysteine (SAC) has received much interest due to its beneficial effects on human health. To satisfy the increasing demand for SAC, this study aims to develop a valuable culturing method for microbial screening synthesizing SAC from readily available materials. Although tryptophan synthase is a promising enzyme for SAC synthesis, its expression in microorganisms is strictly regulated by environmental l-tryptophan. Thus, we constructed a semisynthetic medium lacking l-tryptophan using casamino acids. This medium successfully enhanced the SAC-synthesizing activity of Lactococcus lactis ssp. cremoris NBRC 100676. In addition, microorganisms with high SAC-synthesizing activity were screened by the same medium. Food-related Klebsiella pneumoniae K-15 and Pantoea agglomerans P-3 were found to have a significantly increased SAC-synthesizing activity. The SAC-producing process established in this study is shorter in duration than the conventional garlic aging method. Furthermore, this study proposes a promising alternative strategy for producing food-grade SAC by microorganisms.

Multiple pathways for the formation of the γ-glutamyl peptides γ-glutamyl-valine and γ- glutamyl-valyl-glycine in Saccharomyces cerevisiae.

The role of glutathione (GSH) in eukaryotic cells is well known. The biosynthesis of this γ-glutamine tripeptide is well studied. However, other γ-glutamyl peptides were found in various sources, and the pathways of their formation were not always clear. The aim of the present study was to determine whether Saccharomyces cerevisiae can produce γ-glutamyl tripeptides other than GSH and to identify the pathways associated with the formation of these peptides. The tripeptide γ-Glu-Val-Gly (γ-EVG) was used as a model. Wild-type yeast cells were shown to produce this peptide during cultivation in minimal synthetic medium. Two different biosynthetic pathways for this peptide were identified. The first pathway consisted of two steps. In the first step, γ-Glu-Val (γ-EV) was produced from glutamate and valine by the glutamate-cysteine ligase (GCL) Gsh1p or by the transfer of the γ-glutamyl group from GSH to valine by the γ-glutamyltransferase (GGT) Ecm38p or by the (Dug2p-Dug3p)2 complex. In the next step, γ-EV was combined with glycine by the glutathione synthetase (GS) Gsh2p. The second pathway consisted of transfer of the γ-glutamyl residue from GSH to the dipeptide Val-Gly (VG). This reaction was carried out mainly by the (Dug2p-Dug3p)2 complex, whereas the GGT Ecm38p did not participate in this reaction. The contribution of each of these two pathways to the intracellular pool of γ-EVG was dependent on cultivation conditions. In this work, we also found that Dug1p, previously identified as a Cys-Gly dipeptidase, played an essential role in the hydrolysis of the dipeptide VG in yeast cells. It was also demonstrated that γ-EV and γ-EVG could be effectively imported from the medium and that γ-EVG was imported by Opt1p, known to be a GSH importer. Our results demonstrated that γ-glutamyl peptides, particularly γ-EVG, are produced in yeast as products of several physiologically important reactions and are therefore natural components of yeast cells.

Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering in Saccharomyces cerevisiae.

Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected-a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.

Preparation of a γ-glutamylcysteine-enriched yeast extract from a newly developed GSH2-deficient strain.

Gamma-glutamylcysteine (γ-GC), the precursor of glutathione (GSH), may have significant health benefits as a dietary supplement, but there are few cost-effective methods available for its large-scale production. We developed an efficient method for producing γ-GC in a mutant yeast strain using a three-step breeding procedure and a unique cultivation process. In the first breeding step, we prepared a glutathione synthetase (GSH2)-deficient yeast mutant. In the second step, selenate (SeO(4)(2-)) sensitivity was introduced by crossing the GSH2-deficient mutant with a strain harboring the met30 mutation. In the final step, pantothenic acid auxotrophy was introduced by ethyl methanesulfonate mutagenesis. The isolated strain displayed significantly enhanced cellular γ-GC when cultivated in synthetic medium without pantothenic acid, reaching a maximum level of 4.39% of dry cell weight. Using this strain, we were able to prepare a yeast extract containing approximately 13% γ-GC (w/w), which is markedly higher than the reported value (0.3%) of commercially available yeast extracts. The present method may facilitate large-scale γ-GC production for investigating the nutritive value and other benefits of dietary γ-GC.

A combination of flow cytometry and traditional screening using chemicals to isolate high glutathione-producing yeast mutants.

Traditional screening using chemicals or flow cytometry (FCM) alone is not sufficient to isolate the high glutathione (GSH)-producing yeast strains used in food production. Therefore, to improve screening efficiency, we investigated a combination of both methods. A mutated Saccharomyces cerevisiae strain was labeled with 5-chloromethylfluorescein diacetate and sorted by FCM according to emitted fluorescence intensity. Moderate GSH (1%-2%)-producing mutants were isolated, whereas high GSH (>2%)-producing mutants were not. Traditional screening using cerulenin resulted in similar findings, but a combination of both methods resulted in a 40% increase in the screening yield of high GSH-producing mutants. An analysis of model strains indicated that the ratio of high GSH-producing cells in a sample affected the FCM results. By combining FCM with traditional screening using chemicals, we succeeded in isolating high GSH-producing mutants from several parental strains.

A new method for repeated "self-cloning" promoter replacement in Saccharomyces cerevisiae.

A method for repeated PCR-mediated promoter replacement in the yeast Saccharomyces cerevisiae is described. It was proposed to use the DNA fragment comprising the marker gene that enables both positive and negative selection (a selectable/counter-selectable marker) surrounded by direct repeats of the desired promoter as a promoter replacement cassette. This fragment is integrated upstream of the target gene because of PCR-added terminal sequences for homologous recombination with the target locus. Subsequent marker excision via homologous recombination between the copies of the two promoters leaves one copy of the desired promoter upstream of the target genes, without any heterologous scar sequence. To test this method, a set of plasmids bearing the S. cerevisiae URA3 gene surrounded by two copies of the ADH1 or PGK1 promoter was constructed. Using these cassettes, the native promoters of the GSH1 and GSH2 genes were replaced in the ura3Δ0 recipient strains. The proposed method is useful for research applications due to simple marker excision, and for construction of "self-cloning" industrial strains, because no heterologous DNA is retained in the genome of the resulting strain after marker excision.

Deciphering cellular functions of protein phosphatases by comparison of gene expression profiles in Saccharomyces cerevisiae.

Expression profiles of protein phosphatase (PPase) disruptants were analyzed by use of Pearson's correlation coefficient to find profiles that correlated with those of 316 Reference Gene (RG) disruptants harboring deletions in genes with known functions. Twenty-six Deltappase disruptants exhibited either a positive or negative correlation with 94 RG disruptants when the p value for Pearson's correlation coefficient was >0.2. Some of the predictions that arose from this analysis were tested experimentally and several new Delta ppase phenotypes were found. Notably, Delta sit4 and Delta siw14 disruptants exhibited hygromycin B sensitivity, Delta sit4 and Delta ptc1 disruptants grew slowly on glycerol medium, the Delta ptc1 disruptant was found to be sensitive to calcofluor white and congo red, while the Delta ppg1 disruptant was found to be sensitive to congo red. Because on-going analysis of expression profiles of Saccharomyces cerevisiae disruptants is rapidly generating new data, we suggest that the approach used in the present study to explore PPase function is also applicable to other genes.

Advances in molecular methods to alter chromosomes and genome in the yeast Saccharomyces cerevisiae.

A fundamental issue in biotechnology is how to breed useful strains of microorganisms for efficient production of valuable biomaterials. On-going and more recent developments in gene manipulation technologies and chromosomal and genomic modifications in particular have facilitated important contributions in this area. "Chromosome manipulation technology" as an outgrowth of "gene manipulation technology" may provide opportunities for creating novel strains of organisms with a variety of genomic constitutions. A simple and rapid chromosome splitting technology called "PCR-mediated chromosome splitting" (PCS) that we recently developed has made it possible to manipulate chromosomes and genomes on a large scale in an industrially important microorganism, Saccharomyces cerevisiae. This paper focuses on recent advances in molecular methods for altering chromosomes and genome in S. cerevisiae featuring chromosome splitting technology. These advances in introducing large-scale genomic modifications are expected to accelerate the breeding of novel strains for biotechnological purposes, and to reveal functions of presently uncharacterized chromosomal regions in S. cerevisiae and other organisms.

Conditional chromosome splitting in Saccharomyces cerevisiae using the homing endonuclease PI-SceI.

A novel chromosome engineering technology is described which enables conditional splitting of natural chromosomes in haploid cells of the yeast Saccharomyces cerevisiae. The technology consists of introduction of a recognition sequence for the homing endonuclease PI-SceI into the S. cerevisiae genome and conditional expression of the gene encoding the PI-SceI enzyme under the control of the MET3 promoter. To test the technology, we split chromosome V upstream of GLC7 by use of the autonomously replicating sequence (ARS)-added polymerase-chain-reaction-mediated chromosome-splitting (ARS-PCS) method that we recently developed. A recognition sequence for PI-SceI was subsequently introduced downstream of the GLC7 locus. Splitting was analyzed following induction of the PI-SceI-encoding gene. Approximately 50% of the clones tested had the expected minichromosome harboring only the GLC7 gene, suggesting that any desired chromosomal region may be converted into a new chromosome by use of this method. Because this technology allows initial construction of a strain harboring multiple constructs prior to subsequent induction of random chromosome loss events under specific selective conditions, we propose that this technology may be applicable to reconstructing the S. cerevisiae genome by means of combinatorial loss of minichromosomes.

Construction and characterization of single-gene chromosomes in Saccharomyces cerevisiae.

Based on a previously developed PCR-mediated chromosome splitting method, a genome engineering technique was developed in haploid Saccharomyces cerevisiae for incorporating any desired chromosomal region into a chromosome that carries a single gene. Based on the viability of cells carrying an essential gene in such a construct, close physical proximity of two telomeres and a centromere does not appear to compromise gene function. Spontaneous loss of constructed single-gene chromosomes during vegetative growth was high (0.2-0.4 per cell division), suggesting the possibility of creating novel cells carrying single-gene chromosomes derived from various chromosomal regions in a variety of combinations by exploiting combinatorial loss.

A versatile and general splitting technology for generating targeted YAC subclones.

Yeast artificial chromosomes (YAC) splitting technology was developed as a means to subclone any desired region of eukaryotic chromosomes from one YAC into new YACs. In the present study, the conventional YAC splitting technology was improved by incorporating PCR-mediated chromosome splitting technique and by adding autonomously replicating sequence (ARS) to the system. To demonstrate the performance of the improved method, a 60-kb region from within a 590-kb YAC (clone CIC9e2 from Arabidopsis thaliana chromosome 5) that could not be subcloned using the original method was split to convert into a replicating YAC. Two template plasmids, pSK-KCA and pSKCLY, were used to generate two splitting fragments by PCR. Two splitting fragments consisted of telomeric (C(4)A(2))(6) repeats, 400-bp target region, CEN4, H4ARS and Km(r) (selective marker for plant transformants), or CgLEU2. These splitting fragments were introduced into Saccharomyces cerevisiae harboring the 100-kb split YAC generated by splitting of the 590-kb YAC and containing the 60-kb region. Among 12 Leu(+) transformants, four exhibited the expected karyotype in which two newly split 40- and 60-kb chromosomes were generated. These results demonstrate that the improved method can convert a targeted region of a eukaryotic chromosome within a YAC into a replicating YAC.

Conservation of translation initiation sites based on dinucleotide frequency and codon usage in Escherichia coli K-12 (W3110): non-random distribution of A/T-rich sequences immediately upstream of the translation initiation codon.

Dinucleotide frequencies are useful for characterizing consensus elements as a minimum unit of nucleotide sequence because the neighborhood relations of nucleotide sequences are reflected in dinucleotides. Using a consensus score based on dinucleotide frequencies and intra-species codon usage heterogeneity, denoted by the Z1 parameter, we report the relationship between nucleotide conservation at the translation initiation sites of genes in the Escherichia coli K-12 genome (W3110) and codon usage in its downstream genes. Significant positive correlations were obtained in three regions centered at -13, -4, and +7, which correspond to the Shine-Dalgarno element, the A + T element immediately upstream of the translation initiation site, and the downstream box, respectively.