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.
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Dillon PJ, Rosen CA (1990) A rapid method for the construction of synthetic genes using the polymerase chain reaction. Biotechniques 9:298–299
Güldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524
Kim YH, Sugiyama M, Kaneko Y, Fukui K, Kobayashi A, Harashima S (2005) A versatile and general splitting technology for generating targeted YAC subclones. Appl Microbiol Biotechnol 69:65–70
Kim YH, Sugiyama M, Kaneko Y, Fukui K, Kobayashi A, Harashima S (2006) A polymerase chain reaction-mediated yeast artificial chromosome-splitting technology for generating targeted yeast artificial chromosomes subclones. Methods Mol Biol 349:103–115
Murakami K, Tao E, Ito Y, Sugiyama M, Kaneko Y, Harashima S, Sumiya T, Nakamura A, Nishizawa M (2007) Large scale deletions in the Saccharomyces cerevisiae genome create strains with altered regulation of carbon metabolism. Appl Microbiol Biotechnol 75:589–597
Murray AW, Claus TB, Szostak JW (1988) Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae. Mol Cell Biol 8:4642–4650
Park AH, Sugiyama M, Harashima S, Kim YH (2012) Creation of an ethanol-tolerant yeast strain by genome reconstruction based on chromosome splitting technology. J Microbiol Biotechnol 22:184–189
Sugiyama M, Ikushima S, Nakazawa T, Kaneko Y, Harashima S (2005) PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae. Biotechniques 38:909–914
Sugiyama M, Yamamoto E, Mukai Y, Kaneko Y, Nishizawa Y, Harashima S (2006) Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 72:947–952
Sugiyama M, Nakazawa T, Murakami K, Sumiya T, Nakamura A, Kaneko Y, Nishizawa M, Harashima S (2008) PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae. Appl Microbiol Biotechnol 80:545–553
Sugiyama M, Yamagishi K, Kim YH, Kaneko Y, Nishizawa M, Harashima S (2009) Advances in molecular methods to alter chromosomes and genome in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 84:1045–1052
Surosky RT, Newlon CS, Tye BK (1986) The mitotic stability of deletion of chromosome III in yeast. Proc Natl Acad Sci USA 83:414–418
Ueda Y, Ikushima S, Sugiyama M, Matoba R, Kaneko Y, Matsubara K, Harashima S (2012) Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes. J Biosci Bioeng 113:675–682
Yamagishi K, Sugiyama M, Kaneko Y, Nishizawa M, Harashima S (2008) Construction and characterization of single-gene chromosomes in Saccharomyces cerevisiae. J Biosci Bioeng 106:563–567
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© 2014 Springer Japan
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Sasano, Y., Sugiyama, M., Harashima, S. (2014). Development and Application of Novel Genome Engineering Technologies in Saccharomyces cerevisiae . In: Anazawa, H., Shimizu, S. (eds) Microbial Production. Springer, Tokyo. https://doi.org/10.1007/978-4-431-54607-8_5
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DOI: https://doi.org/10.1007/978-4-431-54607-8_5
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