High expression of unsaturated fatty acid synthesis gene OLE 1 in sake yeasts

Gene Filters and Northern blot analysis revealed that the sake yeast strain Kyokai no. 7 (K 7) showed a higher expression level of OLE 1, which encodes a Delta-9 fatty acid desaturase gene, compared with the laboratory yeast strain X 2180-1A. Other sake yeasts also showed a high expression level of OLE 1. Unsaturated fatty acid concentrations in strain K 7 are higher than that in strain X 2180-1A, suggesting that the higher expression level of OLE 1 in sake yeasts increases the unsaturated fatty acid content in the cell membrane. Experiments using OLE 1 promoter:lacZ fusion reporter genes revealed that both the cis element of the OLE 1 promoter and trans factors are involved in the increased expression of OLE 1 in sake yeasts.     

Construction of recombinant sake yeast containing a dominant FAS2 mutation without extraneous sequences by a two-step gene replacement protocol

A novel two-step gene replacement protocol was developed to construct a recombinant industrial yeast free of bacterial and drug-resistant marker sequences. A yeast strain exhibiting cerulenin resistance conferred by a dominant mutation of FAS2 was previously shown to produce high levels of a flavor component of Japanese sake. A N- and C-terminally truncated portion of the mutant FAS2 gene was subcloned to an integrating plasmid containing an aureobasidin A-resistant transformation marker and a galactose-inducible growth inhibitory sequence (GAL10p::GIN11). The plasmid was targeted into the chromosomal FAS2 locus of sake yeast Kyokai no. 7, resulting in a tandem repeat of inactive FAS2 sequences surrounding the integrated plasmid sequences. Cells containing the integrated plasmid were unable to grow on galactose medium due to the inhibitory effect of GAL10p::GIN11. This growth inhibition allowed efficient counter-selection for cells that had undergone homologous recombination between the FAS2 repeats by their growth on galactose medium. This recombination event resulted in loss of the integrated plasmid sequences and the resulting strains should contain a single copy of either wild-type or cerulenin-resistant FAS2. The selected cerulenin-resistant strains produced approximately 3.7-fold more ethyl caproate, a flavor component, than the Kyokai no. 7 strain. Southern blot and sequence analyses confirmed the presence of the FAS2 mutation and the absence of integrated plasmid sequences in the genome of the selected strain. This gene replacement method provides a straightforward approach for the construction of recombinant industrial yeasts free of undesirable DNA sequences.     

Tolerance mechanism of the ethanol-tolerant mutant of sake yeast

Several ethanol-tolerant mutants have been bred from industrial sake yeasts, but the mechanism of ethanol tolerance in these mutants has not been elucidated. After the determination of the entire genome sequence of Saccharomyces cerevisiae, various methods to monitor the whole-gene expression of the yeast have been developed. In this study, we used a commercially available nylon membrane on which virtually every gene of S. cerevisiae was spotted to compare expression profiles between the ethanol-tolerant mutant and its parent sake yeast to investigate the mechanism of ethanol tolerance in this mutant. As a result, we found that several genes were highly expressed only in the ethanol-tolerant mutant but not in the parent strain. These genes were known to be induced in cells that were exposed to various stresses, such as ethanol, heat, and high osmolarity, or at the stationary-phase but not at the log-phase. In the ethanol-tolerant mutant, the expression level of these stress-responsive genes was further increased after exposure to ethanol. We also found that substances such as catalase, glycerol and trehalose that may have protective roles under stressful conditions were accumulated in high amounts in the ethanol-tolerant mutant. The ethanol-tolerant mutant also exhibited resistance to other stresses including heat, high osmolarity and oxidative stress in addition to ethanol tolerance. These results indicate that the mutant exhibits multiple stress tolerance because of elevated expression of stress-responsive genes, resulting in accumulation of stress protective substances.     

Visual expression analysis of the responses of the alternative oxidase gene (aox1) to heat shock, oxidative, and osmotic stresses in conidia of citric acid-producing Aspergillus niger

The citric acid-producing filamentous fungus Aspergillus niger WU-2223L shows cyanide-insensitive respiration catalyzed by alternative oxidase in addition to the cytochrome pathway. Sequence analysis of the 5' flanking region of the alternative oxidase gene (aox1) revealed a potential heat shock element (HSE) and a stress response element (STRE). We have previously confirmed aox1 expression in conidia. In this study, to confirm whether the upstream region of aox1 responds to various stresses, we used a visual expression analysis system for single-cell conidia of the A. niger strain AOXEGFP-1. This strain harbored a fusion gene comprising aox1 and egfp, which encodes the enhanced green fluorescent protein (EGFP). The fluorescence intensity of EGFP increased in conidia of A. niger AOXEGFP-1 that were subjected to heat shock at 35-45 °C, oxidative stress by exposure to 5mM paraquat or 1 mM t-butylhydroperoxide, or osmotic stresses by exposure to 0.5 M KCl or 1.0 M mannitol. These results indicate that the putative HSE and STRE in the upstream region of aox1 directly or indirectly respond to heat shock, oxidative, and osmotic stresses.     

The level of cAMP-dependent protein kinase A activity strongly affects osmotolerance and osmo-instigated gene expression changes in Saccharomyces cerevisiae

The influence of cAMP-dependent protein kinase (PKA) on protein expression during exponential growth under osmotic stress was studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The responses of isogenic strains (tpk2Deltatpk3Delta) with either constitutively low (tpk1(w1)), regulated (TPK1) or constitutively high (TPK1bcy1Delta) PKA activity were compared. The activity of cAMP-dependent protein kinase (PKA) was shown to be a major determinant of osmotic shock tolerance. Proteins with increased expression during growth under sodium chloride stress could be grouped into three classes with respect to PKA activity, with the glycerol metabolic proteins GPD1, GPP2 and DAK1 standing out as independent of PKA. The other osmotically induced proteins displayed a variable dependence on PKA activity; fully PKA-dependent genes were TPS1 and GCY1, partly PKA-dependent genes were ENO1, TDH1, ALD3 and CTT1. The proteins repressed by osmotic stress also fell into distinct classes of PKA-dependency. Ymr116c was PKA-independent, while Pgi1p, Sam1p, Gdh1p and Vma1p were fully PKA-dependent. Hxk2p, Pdc1p, Ssb1p, Met6p, Atp2p and Hsp60p displayed a partially PKA-dependent repression. The promotors of all induced PKA-dependent genes have STRE sites in their promotors suggestive of a mechanism acting via Msn2/4p. The mechanisms governing the expression of the other classes are unknown. From the protein expression data we conclude that a low PKA activity causes a protein expression resembling that of osmotically stressed cells, and furthermore makes cells tolerant to this type of stress.     

Molecular cloning and application of a gene complementing pantothenic acid auxotrophy of sake yeast Kyokai no. 7

Kyokai no. 7 is the most widely used yeast in sake brewing. This yeast is a pantothenic acid auxotroph at 35 degrees C, and this phenotype has been used to distinguish Kyokai no. 7 from other sake yeasts. We cloned a DNA fragment complementing the pantothenic acid auxotrophy from a genomic library of a Saccharomyces cerevisiae laboratory strain. DNA sequence analysis revealed that the DNA fragment encodes ECM31, the deletion of which had previously been identified as a calcofluor white-sensitive mutation. The ECM31 product is similar to the Escherichia coli ketopantoate hydroxymethyltransferase. Disruption of ECM31 in a laboratory S. cerevisiae strain resulted in pantothenic acid auxotrophy, indicating that ECM31 is also involved in pantothenic acid synthesis in yeast. A hybrid of a Kyokai no. 7 haploid and the ecm31 disruptant required pantothenic acid at 35 degrees C for its growth, suggesting that Kyokai no. 7 possesses a temperature-sensitive allele of ECM31. Thus, the ECM31 gene can be used as a selective marker in the transformation of Kyokai no. 7.     

MSN4基因的过表达对酿酒酵母耐受性的影响研究

以实验室现有菌种AY12a为出发菌株,URA3基因作为筛选标记,利用胞内重组,在MSN4基因的N端加上强启动子PGK1p以实现基因的过表达,最终通过多聚酶链式反应（PCR）验证,成功构建突变株AY12a-msn4。结果表明,该突变株具有一定的耐高温性能,在55 ℃条件下热击后仍能正常生长。同时将突变株AY12a-msn4与出发菌株AY12a进行玉米高温浓醪发酵,并测定发酵完成后的酒精度、残糖、48 h细胞存活率、CO2失重及发酵时间。结果表明,突变株AY12a-msn4发酵液酒精度提高3.85%,48 h细胞存活率上升,残糖含量下降14.5%。