Hiroaki Matsuzaki

Overexpression and secretion of cellulolytic enzymes by δ-sequence-mediated multicopy integration of heterologous DNA sequences into the chromosomes of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae transformants which secrete high levels of cellulolytic enzymes, with chromosome-integrated multicopies of heterologous DNA sequences encoding the cellulolytic enzymes were constructed. An expression construct of β-glucosidase and carboxymethyl cellulase directed by the GAP promoter was integrated into the chromosomes of the haploid S. cerevisiae using the δ sequence-mediated integration system. Southern blot analysis of the chromosomes prepared from various integrants and separated by pulse-field gel electrophoresis demonstrated that the integration occurred mainly in a particular chromosome and the copy number of the integration was variable. The amount of enzymes secreted by the transformants correlated with the copy number of integration. For each enzyme, the highest activity was about 1.4-fold that produced by the transformant harboring the same expression cassette on a YEp-type plasmid. The δ-integrated exogenous DNA was mitotically stable in rich medium. A haploid double transformant which coexpresses and secretes β-glucosidase and carboxymethyl cellulase was further constructed by genetic crossing of the haploid transformant that produces a high level of the enzyme, followed by meiotic segregation of the resulting diploid strain. The haploid double transformant, but neither of the single transformant, could grow on a plate containing carboxymethyl cellulose as a sole carbon source. It is suggested that the δ-sequence-mediated integration system is a very useful means for the genetic engineering of yeast, especially when overproduction and secretion of multiple heterologous enzymes are desired.

Extracellular formation of triglycerides from glucose by a mutant strain of Trichosporon

Abstract A strain, L-12, which secretes triglycerides (TG) was selected from low cell-density mutants of yeast Trichosporon sp. The strain was superior to the wild type strain in the extracellular production of TG from glucose; the production is over 75% of total TG accumulated.

Expression of the carboxymethylcellulase gene, CMC1, from Cryptococcus flavus in Saccharomyces cerevisiae

Abstract A cDNA copy of the carboxymethylcellulase (CMCase 1) gene, CMC1 , of the yeast Cryptococcus flavus was expressed in Saccharomyces cerevisiae under the control of the glyceraldehyde-3-phosphate dehydrogenase gene ( GAP ) promoter derived from S. cerevisiae . In glucose medium, extracellular production of a large amount of CMCase 1 was achieved by S. cerevisiae cells transformed with YEp-plasmid carrying CMC1 . Two major proteins (36 kDa and 34 kDa) secreted in the medium both had CMCase activity and immuno-responsibility to anti-CMCase 1-antibody. From an analysis of amino acid sequences, it was found that the 36 kDa protein is a pro-CMCase 1 having an N-terminal residue, Ala, the 19th amino acid of the peptide deduced from the nucleotide sequence of CMC1 on the cDNA, and that the 34 kDa protein is a mature CMCase 1. Alteration of the cell morphology of S. cerevisiae was induced by transformation with YEp-plasmid carrying CMC1 .

The Relationship between Chromosomal Positioning within the Nucleus and the SSD1 Gene in Saccharomyces cerevisiae

Eukaryotic cells are characterized by very large chromosomal DNAs efficiently packed within the nucleus. To identify the mechanism of chromosomal packaging based on the uniqueness of the centromere region in Saccharomyces cerevisiae, we isolated the HCH6 mutant, which shows 2.5-fold higher efficiency of site-specific recombination between the CEN5 and HIS3 loci than the wild-type CH53 strain. This mutant also displayed defects in cell integrity at high temperature. The SSD1 gene was perhaps responsible for this defect. The efficiency of site-specific recombination was decreased by the introduction of SSD1 in HCH6 cells and increased by disruption of SSD1 in the wild-type cells. Furthermore, the distances between the CEN5 and HIS3 loci and between the CEN5 locus and the spindle pole body (SPB) indicated that disrupting SSD1 caused a loss of the anchoring of the CEN5 locus near SPB. These results suggest Ssd1p-dependent cross-talk between chromosomal positioning within the nucleus and the positioning of cellular components within the cell.

Cell Death Caused by Excision of Centromeric DNA from a Chromosome in Saccharomyces cerevisiae

If genetically modified organisms (GMOs) are spread through the natural environment, it might affect the natural environment. To help prevent the spread of GMOs, we examined whether it is possible to introduce conditional lethality by excising centromeric DNA from a chromosome by site-specific recombination in Saccharomyces cerevisiae as model organism. First, we constructed haploid cells in which excision of the centromeric DNA from chromosome IV can occur due to recombinase induced by galactose. By this excision, cell death can occur. In diploid cells, cell death can also occur by excision from both homologous chromosomes IV. Furthermore, cell death can occur in the case of chromosome V. A small number of surviving cells appeared with excision of centromeric DNA, and the diploid showed greater viability than the haploid in both chromosomes IV and V. The surviving cells appeared mainly due to deletion of a recombination target site (RS) from the chromosome.