Satoshi Harashima

Enhanced bio-ethanol production from cellulosic materials by semi-simultaneous saccharification and fermentation using high temperature resistant Saccharomyces cerevisiae TJ14

The capability of multi-stress-tolerant Saccharomyces cerevisiae diploid strain TJ14 for the production of cellulosic bio-ethanol by semi-simultaneous saccharification and fermentation (SSSF) technology was evaluated under high-temperature conditions. At 39°C, the TJ14 produced 45 g/l ethanol by SSSF of 100 g (w/v)/l cellulose - a significantly higher concentration than reported in prevailing literature.

Disruption of multiple genes whose deletion causes lactic-acid resistance improves lactic-acid resistance and productivity in Saccharomyces cerevisiae.

To create strains that have high productivity of lactic acid without neutralization, a genome-wide screening for strains showing hyper-resistance to 6% l-lactic acid (pH 2.6) was performed using the gene deletion collection of Saccharomyces cerevisiae. We identified 94 genes whose disruption led to resistance to 6% lactic acid in rich medium. We also found that multiple combinations of Δdse2, Δscw11, Δeaf3, and/or Δsed1 disruption led to enhanced resistance to lactic acid depending upon their combinations. In particular, the quadruple disruptant Δdse2Δscw11Δeaf3Δsed1 grew well in 6% lactic acid with the shortest lag phase. We then introduced an exogenous lactate dehydrogenase gene (LDH) into those single and multiple disruptants to evaluate their productivity of lactic acid. It was found that the quadruple disruptant displaying highest lactic-acid resistance showed a 27% increase of lactic-acid productivity as compared with the LDH-harboring wild-type strain. These observations suggest that disruption of multiple genes whose deletion leads to lactic-acid resistance is an effective way to enhance resistance to lactic acid, leading to high lactic-acid productivity without neutralization.

Cellular mechanisms contributing to multiple stress tolerance in Saccharomyces cerevisiae strains with potential use in high-temperature ethanol fermentation

High-temperature ethanol fermentation has several benefits including a reduction in cooling cost, minimizing risk of bacterial contamination, and enabling simultaneous saccharification and fermentation. To achieve the efficient ethanol fermentation at high temperature, yeast strain that tolerates to not only high temperature but also the other stresses present during fermentation, e.g., ethanol, osmotic, and oxidative stresses, is indispensable. The C3253, C3751, and C4377 Saccharomyces cerevisiae strains, which have been previously isolated as thermotolerant yeasts, were found to be multiple stress-tolerant. In these strains, continuous expression of heat shock protein genes and intracellular trehalose accumulation were induced in response to stresses causing protein denaturation. Compared to the control strains, these multiple stress-tolerant strains displayed low intracellular reactive oxygen species levels and effective cell wall remodeling upon exposures to almost all stresses tested. In response to simultaneous multi-stress mimicking fermentation stress, cell wall remodeling and redox homeostasis seem to be the primary mechanisms required for protection against cell damage. Moreover, these strains showed better performances of ethanol production than the control strains at both optimal and high temperatures, suggesting their potential use in high-temperature ethanol fermentation.

Nuclear Localization of Haa1, Which Is Linked to Its Phosphorylation Status, Mediates Lactic Acid Tolerance in Saccharomyces cerevisiae

ABSTRACT Improvement of the lactic acid resistance of the yeast Saccharomyces cerevisiae is important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened for S. cerevisiae genes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genes YGP1, GPG1, and SPI1, while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin gene MSN5 led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

Yeast protein phosphatases Ptp2p and Msg5p are involved in G1–S transition, CLN2 transcription, and vacuole morphogenesis

We previously reported that double disruption of protein phosphatase (PPase) genes PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes Ca2+ sensitive growth, whereas the single disruptions do not. This finding suggests that Ptp2p and Msg5p are involved in Ca2+-induced stress response in a redundant manner. To gain insight into the molecular mechanism causing calcium sensitivity of the ∆ptp2 ∆msg5 double disruptant, we performed fluorescence-activated cell sorting analysis and found a delayed G1 phase. This delayed G1 was consistent with the defect in bud emergence, and reduced CLN2 transcription upon addition of CaCl2. We also found that Slt2p is hyper-phosphorylated in the Δptp2 Δmsg5 double disruptant and that the vacuole of the Δptp2 Δmsg5 double disruptant is fragmented even in the absence of Ca2+. These findings suggest that both Ptp2p and Msg5p are involved in the G1 to S transition and vacuole morphogenesis possibly through their regulation of Slt2 pathway.

Strategy for preventing bacterial contamination by adding exogenous ethanol in solid-state semi-continuous bioethanol production.

A strategy for preventing reduction of ethanol yield in fermentation caused by bacterial contamination was developed. In solid-state ethanol fermentation in which saccharification, fermentation and ethanol recovery are performed simultaneously, the addition of exogenous ethanol to the fermentation mixture at the start of fermentation at 50 g kg(-1) prevented contamination, and the ethanol yield reached 0.50 g g(-1).

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.

Functionally redundant protein phosphatase genes PTP2 and MSG5 co-regulate the calcium signaling pathway in Saccharomyces cerevisiae upon exposure to high extracellular calcium concentration.

Reversible phosphorylation is one of the key post-translational modifications for the regulation of many essential cellular processes. We have previously reported that the disruption of two protein phosphatase (PPase) genes, PTP2 and MSG5, causes calcium sensitivity indicating that functional redundancy exists between the two PPases in response to high extracellular calcium. In this paper, we found that the inactivation of calcineurin by the disruption of the calcineurin regulatory subunit, CNB1 or treatment with a calcineurin inhibitor, FK506, can suppress the calcium-sensitive phenotype of the ptp2Δmsg5Δ double disruptant. In the wake of a calcium-induced, calcineurin-driven signaling pathway activation, the calcium sensitivity of the ptp2Δmsg5Δ double disruptant can be suppressed by regulating the SLT2 pathway through the disruption of the major kinases in the SLT2 signal cascade that include BCK1, MKK1 and SLT2. Also, we show that PTP2 and MSG5 are key regulatory PPases that prevent over-activation of the calcium-induced signaling cascade under the parallel control of the SLT2 and calcineurin pathways.

CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae.

PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function.

Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding δ12- and δ15-fatty acid desaturases in Hansenula polymorpha

Two fatty acid desaturase genes have been cloned: HpFAD2 and HpFAD3 encode Hansenula polymorpha Δ12-fatty acid desaturase (HpFad2) and Δ15-fatty acid desaturase (HpFad3), which are responsible for the production of linoleic acid (LA, C18:2, Δ9, Δ12) and α-linolenic acid (ALA, αC18:3, Δ9, Δ12, Δ15), respectively. The open reading frame of the HpFAD2 and HpFAD3 genes is 1215bp and 1239bp, encoding 405 and 413 amino acids, respectively. The putative amino acid sequences of HpFad2 and HpFad3 share more than 60% similarity and three conserved histidine-box motifs with other known yeast Fad homologs. Hpfad2Δ disruptant cannot produce C18:2 and αC18:3, while the deletion of HpFAD3 only causes the absence of αC18:3. Heterologous expression of either the HpFAD2 or the HpFAD3 gene in Saccharomyces cerevisiae resulted in the presence of C18:2 and αC18:3 when the C18:2 precursor was added. Taken together, these observations indicate that HpFAD2 and HpFAD3 indeed encode Δ12- and Δ15-fatty acid desaturases that function as the only ones responsible for desaturation of oleic acid (C18:1) and linoleic acid (C18:2), respectively, in H. polymorpha. Because a Fatty Acid Regulated (FAR) region and a Low Oxygen Response Element (LORE), which are responsible for regulation of a Δ9-fatty acid desaturase gene (ScOLE1) in S. cerevisiae, are present in the upstream regions of both genes, we investigated whether the transcriptional levels of HpFAD2 and HpFAD3 are affected by supplementation with nutrient unsaturated fatty acids or by low oxygen conditions. Whereas both genes were up-regulated under low oxygen conditions, only HpFAD3 transcription was repressed by an excess of C18:1, C18:2 and C18:3, while the HpFAD2 transcript level did not significantly change. These observations indicate that HpFAD2 expression is not controlled at the transcriptional level by fatty acids even though it contains a FAR-like region. This study indicates that HpFAD2 may be regulated by post-transcriptional mechanisms, whereas HpFAD3 may be mainly controlled at a transcriptional level.