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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.
Highlights
IntroductionWe 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
AG9 had aneuploidy, i.e., ChrIII was tetrasomy, and the right arm of ChrI had an additional copy. These results showed that the TAQing2.0 system, which is based on direct protein delivery into the cell nucleus, induced TaqI-mediated large-scale genomic rearrangements in C. utilis39 (Cu) and enabled the generation of mutants with an altered flocculation phenotype
We upgraded the TAQing system to the TAQing2.0 system, which can induce genomic rearrangements even in non-sporulating industrial yeasts without introducing any foreign DNA (Fig. 3a). This technology is characterized by delivering restriction endonuclease proteins directly into the cell nucleus by the cell-penetrating peptide (CPP)-mediated protein transfection method (Fig. 3b, c)
Summary
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. One cutting-edge example[4–6] is the synthetic chromosome rearrangements and modifications by loxP-mediated evolution (SCRaMbLE) system, a core technology of the synthetic yeast genome project, Sc2.0 (Sc is an abbreviation for the budding yeast Saccharomyces cerevisiae)[7]. A loxPsym site, which is the target sequence of the site-specific recombinant enzyme Cre recombinase, is inserted into the 3′ untranslated region of each of ~5000 nonessential genes Chromosomal rearrangements, such as deletions, duplications, and translocations of genes between loxP sequences, are caused by induction of the Cre enzyme introduced by plasmid, thereby enabling the creation of a mutant pool with a diverse genetic constitution and characteristics. The SCRaMbLE system has fundamental challenges, such as the rearrangement site being limited to the 3′ region of nonessential genes, and a more general-purpose technology has been awaited
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