Accumulation of a recombinant Aspergillus oryzae lipase artificially localized on the Bacillus subtilis cell surface

cutL cDNA encoding an extracellular lipase, L1, from Aspergillus oryzae was fused to the cell wall-binding domain (CWB) region of a plasmid, pHCB3R. SDS-polyacrylamide gel electrophoresis (PAGE) and zymography of proteins extracted from the cell surface of Bacillus subtilis 168 harboring a fused lipase plasmid (pHCB3RCL) revealed that the fused gene product, CWB-CutL, was localized in the B. subtilis cell wall and retained lipase activity. B. subtilis WASD (wprA sigD), recently used for the accumulation of CWB-LipB (the CWB protein fused with B. subtilis lipase B), was also a suitable host for the accumulation of CWB-CutL, the amount being 10% of the total proteins extracted from the cell surface.     

Effect of gene disruption of succinate dehydrogenase on succinate production in a sake yeast strain

Succinate dehydrogenase (SDH) of Saccharomyces cerevisiae consists of four subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. We determined the effect of SDH deficiency on the productivity of organic acids in a sake yeast strain Kyokai no. 9. The SDH activity of single disruptants was retained at 30-90% of that of the wild-type strain, but the activity disappeared in double disruptants of the SDH1 and SDH2 or SDH1b (the SDH1 homologue) genes. Two double disruptants showed no growth on a medium containing glycerol as the sole carbon source, while the single disruptants could utilize glycerol. These results indicate that double disruption of the SDH1 and SDH2 or SDH1b genes is required for complete loss of SDH activity and that the SDH1b gene compensates for the function of the SDH1 gene. The sdh1 sdh1b disruptant showed a marked increase in succinate productivity of up to 1.9-fold along with a decrease in malate productivity relative to the wild-type strains under shaking conditions. Under both static and sake brewing conditions, the productivity of these organic acids in the disruptants was virtually unchanged from that in the wild-type strain. Furthermore, SDH activity was undetectable in the wild-type and the disrupted strains under static conditions. These results suggest that SDH activity contributes to succinate production under shaking conditions, but not under static and sake brewing conditions.     

Cloning of Aspergillus oryzae Aovps5 gene,homologous to vacuolar protein sorting associated gene VPS5 and construction of the disruptant

Aovps5 gene was isolated from Aspergillus oryzae as a homologue to S. cerevisiae VPS5 gene which encodes a polypeptide consisting of 451 amino acids that is nearly 32% homologous to Vps5p. Three Aovps5 gene disruptants were generated and they showed higher activity of tripeptidyl peptidase, which is mainly detected in vacuoles, in their culture medium. Higher amount of nitrogenous constituent was found in the filtrate of wheat gluten degraded by addition of culture medium of these disruptants than that of wild type strain. These results suggest that disruption of Aovps5 may contribute to production of fermented foods.     

Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins

One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host-specific proteases. As an initial solution to this problem, we constructed a protease-deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine- and/or cysteine-type proteases in the culture medium. These findings also demonstrated that construction of a protease-deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required.     

A series of double disruptants for protein phosphatase genes in Saccharomyces cerevisiae and their phenotypic analysis

Thirty-two protein phosphatase (PPase) genes were identified in Saccharomyces cerevisiae based on the nucleotide sequences of the entire genome. In an effort to understand the role of PPases and their functional redundancy in the cellular physiology of one of the reference eukaryotic organisms, a series of single and double PPase gene disruptants were constructed in the W303 strain background. Two single disruptants for the CDC14 and GLC7 genes were lethal. Double disruptants for 30 non-essential PPase genes were constructed in all possible 435 combinations. No double disruptant showed synthetic lethality. Several phenotypes of the viable 30 single and 435 double disruptants were examined; temperature-sensitive growth, utilization of carbon sources and sensitivity to cations and drugs. Four double disruptants exhibited synthetic phenotypes in addition to eight single ones: the pph21 pph22 double disruptant showed slow growth on complete medium, as did the sit4 and yvh1 single ones. In addition to the ptc1, ynr022c and ycr079w single disruptants, the ppz1 ppz2 double disruptant showed temperature-sensitive slow growth. The msg5 ptp2 double disruptant, like the ynr022c single one, did not grow on complete medium containing 0.3 M CaCl(2). The double msg5 ptc2 disruptant failed to grow on medium containing 1.0 M NaCl and, like the ynr022c single deletion, also could not grow on medium containing 0.3 M CaCl(2). The synthetic phenotypes in the two latter cases where each of the PPases is categorized in a different phosphatase family led us to discuss the novel mechanism involved in the functional redundancy of the PPases.     

Molecular breeding of the Mureka-non-forming sake koji mold from Aspergillus oryzae by the disruption of the mreA gene

Mureka-non-forming sake koji molds were constructed from an Aspergillus oryzae industrial strain by the disruption of the mreA gene using a host-vector system with the ptrA gene as a dominant selectable marker. All of the mreA gene disruptants obtained retained the advantages of the host strain in terms of the brewing characteristics, while their isoamyl alcohol oxidase (IAAOD) activities were significantly lower than that of the host strain. Sake brewing was successfully carried out using the koji prepared with the disruptants, followed by storage of the resultant non-pasteurized sake (nama-shu). The isovaleraldehyde (i-Val) concentration in the sake brewed the host strain increased rapidly and reached the threshold values for mureka, 1.8 ppm and 2.6 ppm after storage at 20 degrees C for 42 d and 63 d, respectively, while those of the disruptants were less than 0.5 ppm even after storage at 20 degrees C or 30 degrees C for 63 d. In the sensory evaluation of the sake stored at 20 degrees C or 30 degrees C for 63 d, all members of the panel recognized the strong mureka flavor of the sake brewed with the host strain, while they did not detect this flavor in the sake brewed with the disruptants. Thus, we concluded that the mreA gene disruptants can be used for the production of sake in which mureka is not formed.     

Differential expression of genes associated with lipid metabolism in longissimus dorsi of Korean bulls and steers

The objective of this study was to compare expression of genes associated with lipid deposition and removal between bulls and steers in the longissimus dorsi muscle (LM) tissue of Korean cattle. Castration increased the expression of lipid uptake lipoprotein lipase, fatty acid translocase, and fatty acid transport protein 1 in LM. Castration increased lipogenic gene expression of both acetyl-CoA carboxylase and fatty acid synthase. In contrast, castration downregulated lipolytic gene expression of both adipose triglyceride lipase (ATGL) and monoglyceride lipase. Steers showed higher expression levels of insulin signaling phospho-v-akt murine thymoma viral oncogene homolog 1 than bulls but lower protein levels of nuclear Forkhead box O 1 (FoxO1) than bulls, suggesting that increased insulin signaling following castration decreases nuclear FoxO1 levels, leading to downregulation of ATGL gene expression. These findings suggest that castration contributes to increases in lipid uptake and lipogenesis and a decrease in lipolysis, resulting in improved marbling.     

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 Δppase 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 Δppase phenotypes were found. Notably, Δsit4 and Δsiw14 disruptants exhibited hygromycin B sensitivity, Δsit4 and Δptc1 disruptants grew slowly on glycerol medium, the Δptc1 disruptant was found to be sensitive to calcofluor white and congo red, while the Δppg1 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.     

Chr4, a Schizosaccharomyces pombe homologue of the Saccharomyces cerevisiae Chs4p/Skt5p protein, is related to septum formation and is required for the proper localization of Chs2

In Saccharomyces cerevisiae, Chs4p directly interacts with chitin synthase III (Chs3p) to act as a post-translational regulator of the Chs3p complex. We identified four Chs4p homologous proteins in Schizosaccharomyces pombe which we named Chr1, Chr2, Chr3 and Chr4 (putative chitin synthase regulatory factor). We assessed the functions of these proteins and found that while overproduction of Chr1, Chr2 or Chr3 did not affect the cellular morphology of wild-type Sz. pombe cells, overproduction of Chr4 caused the cells to form multi-septa and delayed their growth. All multiple disruptants of chr1, chr2, chr3 and chr4 grew normally under a variety of growth conditions. However, while chitin synthase II (Chs2) normally localizes exclusively at the septum, in many chr4-disrupted cells it was found in the cytoplasm and the septa. Chs2 did localize at the abnormal septa caused by the overproduction of chr4+. Chr4-13Myc expression was unaffected by the different media or growth conditions in both wild-type and the chs2 disruptant. Chs2 expression was also unaltered by the absence of Chr4. Moreover, Chr4-13Myc localized mostly at the tips and the septum during vegetative growth in chs2, chr1, chr2 and chr3 disruptants as well as in wild-type. Thus, chr4+ is involved in septum formation and is required for the proper localization of Chs2 at the septum in Sz. pombe.     

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.     

Disruption of six ORFs on Saccharomyces cerevisiae chromosome X: the YJL069c gene of unknown function is essential to cell viability.

The short flanking homology PCR strategy (Wach et al., 1994) was used to disrupt six open reading frames (ORFs) on chromosome X of diploid strains (FY1679 and W303) of the yeast Saccharomyces cerevisiae. Two of the six ORFs analysed (YJL069c and YJL066c) display no similarity to known sequences. Three others (YJL065c, YJL068c, and YJL070c) are similar to those respectively encoding the DNA polymerase epsilon subunit c, human esterase D and rat AMP deaminase 1. YJL071w has recently been identified as the ARG2 gene coding for acetylglutamate synthase. Inactivation of the YJL069c gene proved lethal and the yjl071w haploid disruptants were auxotrophic for arginine. For the four other gene inactivations, neither the heterozygous deletion diploids nor the corresponding haploid deletion mutants displayed any special phenotype when grown on rich glycerol or glucose medium or on synthetic minimal medium at three different temperatures, or on media containing compounds interfering with nucleic acid or protein synthesis. Mating and sporulation efficiencies were the same for the viable disruptants as for wild-type cells. The six kanMX4 disruption cassettes were cloned into the pUG7 vector and each of the cognate wild-type genes was inserted into the pRS416 centromeric plasmid. All strains and plasmids have been deposited in the EUROFAN collection (EUROSCARF, K. -D. Entian, Frankfurt, Germany).