Shigehito Ikushima

PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae

Chromosome engineering is playing an increasingly important role in the functional analysis of genomes. A simple and efficient technology for manipulating large chromosomal segments is key to advancing these analyses. Here we describe a simple but innovative method to split chromosomes in Saccharomyces cerevisiae, which we call PCR-mediated chromosome splitting (PCS). The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event) with the CreAoxP system for marker rescue. Using this novel method, chromosomes I (230 kb) and XV (1091 kb) of a haploid cell were split collectively into 10 minichromosomes ranging in size from 29-631 kb with high efficiency (routinely 80%) that were occasionally lost during mitotic growth in various combinations. These observations indicate that the PCS method provides an efficient tool to engineer the yeast genome and may offer a possible approach to identify minimal genome constitutions as a function of culture conditions through further splitting, followed by combinatorial loss of minichromosomes.

Identification and characterization of genes involved in glutathione production in yeast.

Glutathione is a major peptide protecting cells against oxidative stress. To study the cellular processes affecting intracellular glutathione production, we screened Saccharomyces cerevisiae mutant collections and identified new eight yeast deletion mutants that produced more than 1.2-fold higher levels of intracellular glutathione: chc1, cst6, ddc1, def1, pep12, rts1, ubp6, and yih1. Furthermore, overexpression of the DEF1 and CYS4 genes led to a higher production of glutathione, similar to overexpression of GSH1. A multiplier effect on activation of glutathione synthesis was observed by a combination of overexpression of GSH1 and deletion of one of the eight genes. Metabolome analysis of the def1, pep12, and ubp6 deletion mutant, and DEF1-overexpressing strains showed that levels of intracellular methionine and oxidized glutathione were higher than in the control strains, suggesting that methionine biosynthesis was activated and the oxidative stress response was increased in these glutathione-overproductive strains. Moreover, overexpression of GSH1, CYS4, and DEF1 also increased glutathione production in Candida utilis. Taken together, these results will significantly contribute to more effective industrial production of glutathione using yeasts.

Efficient production of L-lactic acid from xylose by a recombinant Candida utilis strain.

Efficient L-lactic acid production from xylose was achieved using a pyruvate decarboxylase-deficient Candida utilis strain expressing an L-lactate dehydrogenase, an NADH-preferring mutated xylose reductase (XR), a xylitol dehydrogenase and a xylulokinase. The recombinant strain showed 53% increased L-lactic acid production compared with the reference strain expressing native XR (NADPH-preferring).

Genome and Transcriptome Analysis of the Food-Yeast Candida utilis

The industrially important food-yeast Candida utilis is a Crabtree effect-negative yeast used to produce valuable chemicals and recombinant proteins. In the present study, we conducted whole genome sequencing and phylogenetic analysis of C. utilis, which showed that this yeast diverged long before the formation of the CUG and Saccharomyces/Kluyveromyces clades. In addition, we performed comparative genome and transcriptome analyses using next-generation sequencing, which resulted in the identification of genes important for characteristic phenotypes of C. utilis such as those involved in nitrate assimilation, in addition to the gene encoding the functional hexose transporter. We also found that an antisense transcript of the alcohol dehydrogenase gene, which in silico analysis did not predict to be a functional gene, was transcribed in the stationary-phase, suggesting a novel system of repression of ethanol production. These findings should facilitate the development of more sophisticated systems for the production of useful reagents using C. utilis.

Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes.

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.

Draft Genome Sequence of Lactococcus lactis subsp. lactis JCM 5805T, a Strain That Induces Plasmacytoid Dendritic Cell Activation

ABSTRACT Lactococcus lactis subsp. lactis JCM 5805T is a dairy lactic acid bacterium that induces plasmacytoid dendritic cell (pDC) activation. Here, we report the 2.55-Mb draft genome and annotation of Lactococcus lactis JCM 5805T. This genome information will provide further insights into the mechanisms underlying the immunomodulatory function of this strain.

Improvement of skin conditions by ingestion of Aspergillus kawachii (Koji) extract containing 14-dehydroergosterol in a randomized, double-blind, controlled trial

Purpose The present study examined the effect of ingestion of Koji extract containing 14-dehydroergosterol (14-DHE), prepared from Aspergillus kawachii NBRC4308, on improvement of skin conditions among healthy volunteers. Subjects and methods In a randomized, double-blind, placebo-controlled, parallel-group study, 70 healthy adult women who felt that their skin was dry ingested either a placebo dietary supplement or Koji extract (200 mg/day) supplement containing 0.1% 14-DHE for 12 weeks. Throughout the treatment period and for 4 weeks afterward, objective indicators – including moisture content of the stratum corneum, trans-epidermal water loss (TEWL), and skin wrinkles – were evaluated; in addition, the subjects answered a questionnaire on their skin conditions with ratings on a visual analog scale. Statistical analysis was conducted on the basis of differences from baseline scores. Results Compared with the placebo group, the Koji extract group showed significantly increased forearm moisture at 4, 8, and 16 weeks (p < 0.05 on unpaired t-test). The questionnaire survey showed a marked improvement in skin conditions, particularly crow’s feet, in the Koji extract group versus the placebo group at 8 weeks (p < 0.05 by unpaired t-test). Furthermore, the Koji extract group showed a trend (p < 0.10) toward improvement in skin moisture (at 4 weeks), dryness around the eyes/mouth (at 4 weeks), and overall skin condition (at 8 weeks) versus the placebo group. Conclusion Ingestion of Koji extract containing 14-DHE was demonstrated to have positive effects toward improving skin conditions – in particular, on increasing skin moisture in the stratum corneum.

Metabolomic and transcriptomic analysis for rate-limiting metabolic steps in xylose utilization by recombinant Candida utilis.

We have reported that a recombinant Candida utilis strain expressing a Candida shehatae xylose reductase K275R/N277D, a C. shehatae xylitol dehydrogenase, and xylulokinase from Pichia stipitis produced ethanol from xylose, but its productivity was low. In the present study, metabolomic (CE-TOF MS) and transcriptomic (microarray) analyses were performed to characterize xylose metabolism by engineered C. utilis and to identify key genetic changes contributing to efficient xylose utilization. The metabolomic analysis revealed that the xylose-fermenting strain accumulated more pentose phosphate pathway intermediates, more NADH, and more glycolytic intermediates upstream of glyceraldehyde 3-phosphate than the wild-type. Transcriptomic analysis of the strain grown on xylose indicated a significant increase in expression of the genes encoding tricarboxylic acid cycle enzymes, respiratory enzymes, and enzymes involved in ethanol oxidation. To decrease the NADH/NAD+ ratio and increase the ethanol yield of the fermentation of xylose, ADH1 encoding NADH-dependent alcohol dehydrogenase was overexpressed. The resulting strain exhibited a 17% increase in ethanol production and a 22% decrease in xylitol accumulation relative to control.

Multi-locus genotyping of bottom fermenting yeasts by single nucleotide polymorphisms indicative of brewing characteristics

Yeast plays a capital role in brewing fermentation and has a direct impact on flavor and aroma. For the evaluation of competent brewing strains during quality control or development of novel strains it is standard practice to perform fermentation tests, which are costly and time-consuming. Here, we have categorized DNA markers which enable to distinguish and to screen brewing strains more efficiently than ever before. Sequence analysis at 289 loci in the genomes of six bottom fermenting Saccharomyces pastorianus strains revealed that 30 loci contained single nucleotide polymorphisms (SNPs). By determining the nucleotide sequences at the SNP-loci in 26 other S. pastorianus strains and 20 strains of the top fermenting yeast Saccharomyces cerevisiae, almost all these strains could be discriminated solely on the basis of the SNPs. By comparing the fermentative phenotypes of these strains we found that some DNA markers showed a strong association with brewing characteristics, such as the production of ethyl acetate and hydrogen sulphide (H2S). Therefore, the DNA markers we identified will facilitate quality control and the efficient development of brewing yeast strains.