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- Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae
- Hye Yun Moon† , Gyu Hun Sim† , Hyeon Jin Kim , Keunpil Kim , Hyun Ah Kang
- J. Microbiol. 2022;60(1):18-30. Published online December 29, 2021
- DOI: https://doi.org/10.1007/s12275-022-1580-7
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- 7 Web of Science
- 7 Crossref
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Abstract
- We evaluated the Cre-lox and CRISPR-Cas9 systems as markerrecycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4- based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura- transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.
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Citations
Citations to this article as recorded by
- Multiple metabolic engineering of Saccharomyces cerevisiae for the production of lycopene
Jiaheng Liu, Minxia Song, Xianhao Xu, Yaokang Wu, Yanfeng Liu, Guocheng Du, Jianghua Li, Long Liu, Xueqin Lv
Food Bioengineering.2024;[Epub] CrossRef - Biochemical and Biorefinery Platform for Second-Generation Bioethanol: Fermentative Strategies and Microorganisms
Karla D. González-Gloria, Elia Tomás-Pejó, Lorena Amaya-Delgado, Rosa M. Rodríguez-Jasso, Araceli Loredo-Treviño, Anusuiya Singh, Meenu Hans, Carlos Martín, Sachin Kumar, Héctor A. Ruiz
Fermentation.2024; 10(7): 361. CrossRef - CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii
Wei Zhou, Yuanyi Li, Guosong Liu, Weichuang Qin, Dongzhi Wei, Fengqing Wang, Bei Gao
Applied Microbiology and Biotechnology.2024;[Epub] CrossRef - Establishment, optimization, and application of genetic technology in Aspergillus spp.
Jing Gao, Huiqing Liu, Zhenzhen Zhang, Zhihong Liang
Frontiers in Microbiology.2023;[Epub] CrossRef - CRISPR-Cas Technology for Bioengineering Conventional and Non-Conventional Yeasts: Progress and New Challenges
Yuanyuan Xia, Yujie Li, Wei Shen, Haiquan Yang, Xianzhong Chen
International Journal of Molecular Sciences.2023; 24(20): 15310. CrossRef - Genomic and functional features of yeast species in Korean traditional fermented alcoholic beverage and soybean products
Da Min Jeong, Hyeon Jin Kim, Min-Seung Jeon, Su Jin Yoo, Hye Yun Moon, Eun-joo Jeon, Che Ok Jeon, Seong-il Eyun, Hyun Ah Kang
FEMS Yeast Research.2023;[Epub] CrossRef - Multiplex genome editing to construct cellulase engineered Saccharomyces cerevisiae for ethanol production from cellulosic biomass
Yatika Dixit, Preeti Yadav, Arun Kumar Sharma, Poornima Pandey, Arindam Kuila
Renewable and Sustainable Energy Reviews.2023; 187: 113772. CrossRef
- Multiple metabolic engineering of Saccharomyces cerevisiae for the production of lycopene
Research Support, Non-U.S. Gov't
- NOTE] Two Novel Talaromyces Species Isolated from Medicinal Crops in Korea
- Hyunkyu Sang , Tae-Jin An , Chang Sun Kim , Gyu-Sub Shin , Gi-Ho Sung , Seung Hun Yu
- J. Microbiol. 2013;51(5):704-708. Published online October 31, 2013
- DOI: https://doi.org/10.1007/s12275-013-3361-9
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- 22 Scopus
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Abstract
- Two novel biverticillate Talaromyces species, T. angelicus and T. cnidii, were collected from the medicinal crops Angelica gigas and Cnidium officinale, respectively, in Korea. Phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region and the β-tubulin gene as well as morphological analyses revealed that the two species differ from any known Talaromyces species. Talaromyces angelicus is related to T. flavovirens in the phylogeny of the ITS region, but the new species is grouped together with Penicillium liani and T. pinophilus in terms of its β-tubulin phylogeny, and its growth rate on Czapek yeast autolysate differs from that of T. flavovirens. Talaromyces cnidii is phylogenetically similar to T. siamensis, but exhibits differences in the morphologies of the colony margin, metulae, and conidia.
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