Application of Genome Editing Method on Kluyveromyces marxianus 17694-DH2 using CRISPR-Cas9 System for Enhanced Xylose Utilization

The third-generation gene editing technology, CRISPR-Cas9 system, is derived from the bacterial immune system. These CRISPR-Cas9 systems have recently been used to mutate yeast gene or replace cleavage sites with other DNA. In this study, CRISPR-Cas9 system was applied to delete PHO13 gene of Kluyveromyces marxianus. As a result, only one strain of three transformants, that the CRISPR-Cas9 system was applied, was confirmed partial deletion of PHO13 gene. Sequencing of the PHO13 gene revealed the deletion of cytosine at position 457, which resulted in premature termination of translation as compared to that from the parental strain. The ΔPHO13 strain, K. marxianus 17694-DH2, showed 18.29% and 21.28% improvement in xylose consumption and ethanol production from xylose, respectively, as compared to those form the parental strain.