TAQing2.0 for genome reorganization of asexual industrial yeasts by direct protein transfection

Abstract

Genomic rearrangements often generate phenotypic diversification. We previously reported the TAQing system where genomic rearrangements are induced via conditional activation of a restriction endonuclease in yeast and plant cells to produce mutants with marked phenotypic changes. Here we developed the TAQing2.0 system based on the direct delivery of endonucleases into the cell nucleus by cell-penetrating peptides. Using the optimized procedure, we introduce a heat-reactivatable endonuclease TaqI into an asexual industrial yeast (torula yeast), followed by a transient heat activation of TaqI. TAQing2.0 leads to generation of mutants with altered flocculation and morphological phenotypes, which exhibit changes in chromosomal size. Genome resequencing suggested that torula yeast is triploid with six chromosomes and the mutants have multiple rearrangements including translocations having the TaqI recognition sequence at the break points. Thus, TAQing2.0 is expected as a useful method to obtain various mutants with altered phenotypes without introducing foreign DNA into asexual industrial microorganisms.

Gene mapping methodology powered by induced genome rearrangements

“…We further extended the TAQing system to many non-conventional industrial yeasts, including Candida utilis , an imperfect fungus that cannot undergo normal meiosis and cannot be improved by crossbreeding. For this, we developed a TAQing system of the protein transfection type (TAQing2.0) by directly delivering TaqI into fungal cells using the cell-penetrating peptide method 17 . Additionally, we employed restriction endonucleases, other than TaqI (e.g., MseI) in the TAQing system, to improve the frequency of genome rearrangement and phenotypic diversification in plants (extended-TAQing system, Ex-TAQing) 16 .…”

Gene mapping methodology powered by induced genome rearrangements

“…We further extended the TAQing system to many non-conventional industrial yeasts, including a strain of Cyberlindnera jadinii that cannot undergo normal meiosis and cannot be improved by crossbreeding. For this, we developed a TAQing system of the protein transfection type (TAQing2.0) by directly delivering TaqI into fungal cells using the cell-penetrating peptide method 17 . Additionally, we employed restriction endonucleases, other than TaqI (e.g., MseI) in the TAQing system, to improve the frequency of genome rearrangement and phenotypic diversification in plants (extended-TAQing system, Ex-TAQing) 16 .…”