Development and Application of Novel Genome Engineering Technologies in Saccharomyces cerevisiae

Abstract

Various genome engineering technologies have been developed in the yeast Saccharomyces cerevisiae. One such key technology is PCR-mediated chromosome-splitting technology, designated PCS. The aim of PCS is to cut a chromosome at any chosen site into two smaller pieces and to make these “newly generated chromosomes” behave as functional chromosomes. PCS splits a chromosome very efficiently (more than 70 %) and allows repeated splitting because the built-in Cre/loxP site-specific recombination system facilitates the use of marker recycling. Subsequently, PCR-mediated chromosome deletion (PCD) technology was developed as a derivative technology of PCS. PCD facilitates the deletion of any chromosomal region, irrespective of an internal or terminal location. Genome reorganization (GReO) technology was also developed on the basis of PCS. In GReO, a huge variety of genomic constitutions are generated from a master strain harboring a few dozen mini-chromosomes constructed by PCS when it undergoes combinatorial mini-chromosome loss during mitosis. PCD and GReO technology have been exploited in the breeding of yeast. Several strains with a large-scale deletion ranging from 400 to 500 kb were constructed by PCD. Some of these strains produced twofold more ethanol and glycerol than the parental strain. The gene expression profiles revealed that the physiological adjustment from fermentative to oxidative metabolism, including stimulation of mitochondrial function, does not occur in these strains. GReO technology has been successfully used to create ethanol-tolerant strains showing a more than tenfold-higher specific growth rate in the presence of 11 % ethanol as compared with the parental strain. Thus, these genome engineering technologies provide a new tool for breeding novel yeasts useful for industrial applications.