Press release
March 30, 2022
Mitsubishi Corporation Life Sciences Limited
Developing the technology TAQing2.0 for reorganizing the genome structure
without introducing foreign DNA and RNA

Research on “TAQing2.0 for genome reorganization of asexual industrial yeasts by direct protein transfection”
was published in Communications Biology. This study has been performed by a collaborative research group ledby Mitsubishi Corporation Life Sciences Limited (head office: Chiyoda-ku, Tokyo; representative director &president: Hiroshi Fujiki) and Professor Kunihiro Ohta and coworkers at the University of Tokyo (address:Bunkyo-ku, Tokyo; president: Teruo Fujii).

[Highlights]
We developed the breeding method TAQing2.0 without introducing DNA and RNA by updating the existing
technology of the TAQing system, which induces large-scale genome rearrangement and thereby improves
rapidly quantitative traits controlled by a complex gene network.

This technology is characterized by delivering endonuclease TaqI directly into the cell nucleus by using acommercially available cell-penetrating peptide. We can markedly enhance the frequency of genome
restructuring events, which naturally occur in the process of crossbreeding, in a simple manner where thecells are gently warmed at a specific temperature to regulate the activity of TaqI.

By using TAQing2.0 for torula yeast, one of the non-sporulating industrial yeasts, we confirmed theoccurrence of large-scale chromosomal rearrangements at multiple sites using computational genomeanalyses. Moreover, we succeeded in generating a more accurate reference genome sequence of the wild
type torula yeast.

We demonstrated the potential capability of TAQing2.0, which renders increased genetic diversity within aspecies, contributing to the efficient acquisition of microorganisms of high industrial utility. Strains, obtainedby TAQing2.0, can be handled in the same manner as naturally occurring mutants because any incoming
DNA and RNA have not been contained in the strains, as well as in this technical process. Therefore, we arespeculating that this may relieve the difficulties of public acceptance toward genetically modified organisms.

[Announcement document]
Since ancient times, people have adopted a crossbreeding technique, the representative utility for sexual
reproduction, for domestic animals or plants, and then improved their traits so as to obtain more useful ones forthemselves. In this method, genomic DNA(1) happened to mutate through generational change not throughintentionally introducing an exogenous gene. For improvement of species, therefore, fundamental issues must be
overcome, such as the need for a very long period. Although problems have arisen in applying crossbreeding
methods to asexual organisms, we induced mutagenesis by using radiation or drugs. In these approaches,nevertheless, the same problems mentioned above, in other words, the need for time-consuming processes,
remained to be addressed.
Professor Kunihiro Ohta and coworkers recently developed a large-scale genome restructuring technology called
the TAQing system(2) (Muramoto et al., Nat. Commun. 2018) and applied the technology to cells of budding yeast
and the plant Arabidopsis thaliana. In this technology, gene coding for the restriction enzyme(3), TaqI or MboI, isgenetically transfected into cells using vector-mediated tools, and then up-regulated transiently under the control
of an inducible promoter. Subsequently, the cells are gently warmed to partially and temporarily activate TaqI,
which simultaneously induces multiple random DNA double-strand breaks (DSBs) at TCGA sites throughout thegenome and rejoins them via DNA repair. As a result, large-scale genomic rearrangements are rapidly induced,
and variants with altered and improved phenotypes can be obtained in a very short period, such as the yeast
mutant with higher bioethanol fermentative ability.
In this original TAQing system, however, the genetic engineering approach is necessary to deliver exogenous
plasmid harboring TaqI gene into cells. Therefore, in the practical fields where we seek to apply the TAQing
system, there remain several issues to resolve; the TAQing system cannot be applied to species for which a generecombination assay has not been established or where a newly generated variant could be subject torecombinant DNA regulation if it contains incoming DNA.
Now researchers in this study have succeeded in resolving these challenges explained above by submitting theestablished technology of TAQing2.0.
In this experiments, we used torula yeast(4) (Cu), which is one of the non-sporulating industrial yeasts. Acell-penetrating peptide (CPP, commercially available) and the TaqI complex were introduced into live Cu cells ina buffer, and then the cells were warmed at mild temperatures for a constant time to temporarily and partially
activate TaqI. According to these simple procedures, we confirmed that the intracellular events, random DNADSBs and DNA repairs throughout the genome, efficiently took place, contributing to large-scale chromosomal
restructurings (Figure 1). Here, we also established an easy and versatile method for transfecting various foreign
active proteins in a minimally invasive and highly efficient manner by using CPP for budding yeasts. As the resultof the various trials, we optimized the transfection condition in which two factors turned out to be important: theuse of cells in the early exponential growth phase and the employment of a buffer condition.
By using the TAQing2.0 method one time, Cu mutants with altered phenotypes, for example, aggregability orstress resistance (unpublished data), were generated, and genome resequencing demonstrated that thehyper-flocculated mutants have multiple rearrangements with the TaqI recognition sequence (TCGA) at breakpoints (Figure 2). In this study, moreover, we generated a more accurate reference genome sequence of wild typeCu that has been roughly unveiled and rather controversial.
Co-researchers in this group named this updated method TAQing2.0 and are now attempting to evaluate thepracticality of this technology in practical and industrial fields. At our company, TAQing2.0 would be an additional
and an effective breeding approach to obtaining naturally occurring strains, and we aim to submit a high-ordermicrobial platform to flexibly meet customer and market requests.

(Notes 1) Genomic DNA
It is the smallest unit of DNA information that determines a specific organism. One genomic DNA, involved in all
genetic information, is encapsulated in one cell.
(Notes 2) TAQing system
This technology was reported by Muramoto et al. in Nat. Commun. 2018 and the process simultaneously induces
multiple random DNA double-strand breaks and rejoins them in the genome by using TaqI-plasmid transfection,
resulting in large-scale genomic rearrangements. This method enables rapid improvement of phenotypes, suchas the fermentative capacity of budding yeast to accumulate bioethanol or the increased stress resistance andbiomass production for Arabidopsis thaliana.
(Notes 3) Restriction enzyme
This endonuclease recognizes specific 4-, 6- or more bases of DNA sequences and cleaves. Present in somebacteria, the enzyme functions to restrict propagation of infectious phages (viruses infect host bacteria), and so
the enzyme has come to be called a restriction enzyme. Because a restriction enzyme has the property ofcleaving DNA in a specific sequence, it has made an indispensable contribution to the development of genetic
engineering technology.
(Notes 4)
Since Germany started production of torula yeast (Cyberlindnera jadinii, Candida utilis) as a source of protein
nutrients in the 1910s, and now this yeast has been widely used as edible/fodder yeast and a dietary supplement
in many countries. The Food and Drug Administration (FDA) of the United States certified GRAS (generallyrecognized as safe) status to torula yeast.

PurifiedProtein (RE) transfection by

Double-strand breaks bycell-penetrating peptide

RE on genomic DNA

restriction enzyme(RE) protein

ゲノム再編成の誘発

Figure 1 A schematic diagram describing the procedure for TAQing2.0. In the original TAQing system, a plasmid
containing restriction enzyme (RE) gene is transfected into cells, where RE is expressed and partially activated,
resulting in the induction of double-strand breaks at the RE recognition site. In TAQing2.0, a purified RE is mixed
with cell-penetrating peptide and then delivered directly into intact cells. TAQing2.0 thus enables the induction ofgenome rearrangement. In addition, this technology is easy to apply toward species for which a generecombination assay has not been established, so we can use the TAQing2.0 method for a wider range oforganisms.

C. utilis

Mutant AG4

WTrearranged byTAQing2.0

WT

AG4

Chr I

Chr II

Chr III

Chr IV

Chr V

Chr VI

Break induced repair

Short gene conversion

Translocation

Break point

Figure 2 Microscopic images for the torula yeast (Cu) mutants obtained by TAQing2.0 (upper left; Wild Type (WT),
upper right; mutant AG4, scale bar; 5 µm) and the chromosomal structure of Cu and mutant AG4 (lower;

schematic diagram). Concerning the developed method in this study, we can introduce an active protein into yeast
cells with intact cell walls and obtain altered phenotypic Cu variants with flocculation or stress resistance. Notethat a translocation event (double-strand break and rejoin) between Chr I and Chr IV occurred in AG4, and RE
recognition sequencing TCGA at the break point in translocation is shown.

[Reference]
Magazine: Communications Biology (Online version: February 17)
Title: TAQing2.0 for genome reorganization of asexual industrial yeasts by direct protein transfection
Authors: Taishi Yasukawa, Arisa H. Oda, Takahiro Nakamura, Naohisa Masuo, Miki Tamura, Yuriko Yamasaki,
Makoto Imura, Takatomi Yamada, and Kunihiro Ohta*
DOI number: 10.1038/s42003-022-03093-6
URL: https://www.nature.com/articles/s42003-022-03093-6[Contact]
Mitsubishi Corporation Life Sciences Limited
General Affairs and Public Relations Group Human Resources and General Affairs Department
Mail: prgroup_mcls@mcls-ltd.com