Production of Agarase Using Yeast Artificial Chromosome and Cellular Physiological Analysis

Yeast artificial chromosomes (YACs) with expression clusters of three genes (AGAH71, AGAG1, and NABH558) responsible for agar degradation were constructed using chromosome manipulation technique.The three agar-degrading genes were stably introduced into chromosome II of the Saccharomyces cerevisiae strain, confirming efficient production and secretion of various agarases.For efficient chromosome manipulation, each splitting fragment (DNA module) was transformed, and yeast chromosome II (825 kb) was successfully split 270 kb-YAC, 38 kb-mini YAC and 524 kb-YAC through two-rounds chromosome splitting, resulting in the construction of the YKY180 strain, which has a total of 18 chromosomes.Splitting efficiency for chromosome manipulation was 27~100% and the expression level of foreign genes on 38 kb-mini YAC and enzyme activity were indistinguishable from those of the host strain.This means that the structural alteration of the chromosome did not affect gene expression level.The 38 kb-mini YAC demonstrated stable mitotic stability for up to 200 generations, and it was capable of producing various metabolites from agar degradation.This suggests that the artificially manipulated mini-YAC could have diverse industrial applications.