Journal of Microbiology

Journal Article

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

265 View
0 Download
7 Web of Science
7 Crossref

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.

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; 3(4): 397. 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

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

124 View
0 Download
20 Crossref

Abstract PDF

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.

Citations

Citations to this article as recorded by

Modern taxonomy and approaches to the identification of the genus Talaromyces (Trichocomaceae, Eurotiales)
Е. А. Antonov, A. V. Aleksandrova, I. I. Antonova
Микология и фитопатология.2024;[Epub] CrossRef
Talaromyces sedimenticola sp. nov., isolated from the Mariana Trench
Hongbo Zhou, Liting Xu, Wenxian Liu, Kaiwen Ta, Xincun Wang, Jianwei Guo, Wenxi Luo, Zhiyuan Peng, Qiaoni Huang, Yuguang Wang
Antonie van Leeuwenhoek.2024;[Epub] CrossRef
New Fungal Strains from Peat Soil in Malaysia: Morphological and Molecular Characteristics
Efaq Ali Noman, Adel Ali Al-Gheethi, Baliks A. Talip, Radin Maya Saphira Radin Mohamed, Reyad Almoheer, Fairoz Ali Al-Wrafy, Najeeb Al-Shorgani, Hesham Ali El Enshasy
Sustainability.2023; 15(7): 5902. CrossRef
A New Species and Five New Records of Talaromyces ( Eurotiales , Aspergillaceae ) Belonging to Section Talaromyces in Korea
Thuong T. T. Nguyen, Hyang Burm Lee
Mycobiology.2023; 51(5): 320. CrossRef
Fine Identification and Classification of a Novel Beneficial Talaromyces Fungal Species from Masson Pine Rhizosphere Soil
Xiao-Rui Sun, Ming-Ye Xu, Wei-Liang Kong, Fei Wu, Yu Zhang, Xing-Li Xie, De-Wei Li, Xiao-Qin Wu
Journal of Fungi.2022; 8(2): 155. CrossRef
Isolation, Identification and Insecticidal Activity of the Secondary Metabolites of Talaromyces purpureogenus BS5
Ying Yue, Mingfang Jiang, Hanying Hu, Jinghui Wu, Haoran Sun, Hong Jin, Taiping Hou, Ke Tao
Journal of Fungi.2022; 8(3): 288. CrossRef
New Species of Talaromyces (Fungi) Isolated from Soil in Southwestern China
Zhi-Kang Zhang, Xin-Cun Wang, Wen-Ying Zhuang, Xian-Hao Cheng, Peng Zhao
Biology.2021; 10(8): 745. CrossRef
Six Newly Recorded Fungal Taxa from Freshwater Niche in Korea
Hyo Jin Lim, Thuong T. T Nguyen, Hyang Burm Lee
Mycobiology.2021; 49(2): 105. CrossRef
Four new species of Talaromyces section Talaromyces discovered in China
Shangzhu Wei, Xiuli Xu, Long Wang
Mycologia.2021; 113(2): 492. CrossRef
Discovery and Extrolite Production of Three New Species of Talaromyces Belonging to Sections Helici and Purpurei from Freshwater in Korea
Thuong T. T. Nguyen, Jens Christian Frisvad, Paul M. Kirk, Hyo Jin Lim, Hyang Burm Lee
Journal of Fungi.2021; 7(9): 722. CrossRef
New section and species in Talaromyces
Bing-Da Sun, Amanda J. Chen, Jos Houbraken, Jens C. Frisvad, Wen-Ping Wu, Hai-Lei Wei, Yu-Guang Zhou, Xian-Zhi Jiang, Robert A. Samson
MycoKeys.2020; 68: 75. CrossRef
Mycoflora assessment, growth and toxigenic features of patulin‐producers in kiwifruit in China
Yuan Wang, Kewei Feng, Bin Liu, Zhiwei Zhang, Jianping Wei, Yahong Yuan, Tianli Yue
Journal of the Science of Food and Agriculture.2018; 98(7): 2573. CrossRef
Three new species of Talaromyces sect. Talaromyces discovered from soil in China
Xian-Zhi Jiang, Zhong-Dong Yu, Yong-Ming Ruan, Long Wang
Scientific Reports.2018;[Epub] CrossRef
Notes for genera: Ascomycota
Nalin N. Wijayawardene, Kevin D. Hyde, Kunhiraman C. Rajeshkumar, David L. Hawksworth, Hugo Madrid, Paul M. Kirk, Uwe Braun, Rajshree V. Singh, Pedro W. Crous, Martin Kukwa, Robert Lücking, Cletus P. Kurtzman, Andrey Yurkov, Danny Haelewaters, André Aptro
Fungal Diversity.2017; 86(1): 1. CrossRef
NewTalaromycesspecies from indoor environments in China
A.J. Chen, B.D. Sun, J. Houbraken, J.C. Frisvad, N. Yilmaz, Y.G. Zhou, R.A. Samson
Studies in Mycology.2016; 84(1): 119. CrossRef
Species List of Aspergillus, Penicillium and Talaromyces in Korea, Based on ‘One Fungus One Name’ System

The Korean Journal of Mycology.2016;[Epub] CrossRef
Cellulase and Xylanase Production by the Mexican Strain Talaromyces stollii LV186 and Its Application in the Saccharification of Pretreated Corn and Sorghum Stover
Montserrat Orencio-Trejo, Jessica Torres-Granados, Arelis Rangel-Lara, Esmeralda Beltrán-Guerrero, Santos García-Aguilar, Cessna Moss-Acosta, Humberto Valenzuela-Soto, Susana De la Torre-Zavala, Argel Gastelum-Arellanez, Alfredo Martinez, Axel Tiessen, Ed
BioEnergy Research.2016; 9(4): 1034. CrossRef
Talaromyces neofusisporus and T. qii, two new species of section Talaromyces isolated from plant leaves in Tibet, China
Qi-Ming Wang, Yong-Hong Zhang, Bo Wang, Long Wang
Scientific Reports.2016;[Epub] CrossRef
Five new Talaromyces species with ampulliform-like phialides and globose rough walled conidia resembling T. verruculosus
Cobus M. Visagie, Neriman Yilmaz, Jens C. Frisvad, Jos Houbraken, Keith A. Seifert, Robert A. Samson, Karin Jacobs
Mycoscience.2015; 56(5): 486. CrossRef
Aspergillus, Penicillium and Talaromyces isolated from house dust samples collected around the world
C.M. Visagie, Y. Hirooka, J.B. Tanney, E. Whitfield, K. Mwange, M. Meijer, A.S. Amend, K.A. Seifert, R.A. Samson
Studies in Mycology.2014; 78(1): 63. CrossRef

First
Prev
Page of 1
Next
Last

Search