RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides

Koh, Hyun Gi; Tohidifar, Payman; Oh, Hyunjoon; Ye, Quanhui; Jung, Suk-Chae; Rao, Christopher V.; Jin, Yong-Su

doi:10.1021/acssynbio.4c00848

RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides

Journal Article · Tue Apr 15 00:00:00 EDT 2025 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.4c00848· OSTI ID:2561408

Koh, Hyun Gi ^[1]; Tohidifar, Payman ^[2]; ^[2]; ^[2]; ^[2]; ^[2]; ^[2]

Hongik University, Sejong (Korea, Republic of)
University of Illinois at Urbana−Champaign, IL (United States)

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ (R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. Furthermore, this result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides, providing a streamlined method for addressing genetic engineering challenges in the yeast.

View Accepted Manuscript (DOE)

Research Organization:: Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0018420

OSTI ID:: 2561408

Journal Information:: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Journal Issue: 5 Vol. 14; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (40)

Analysis of carotenoid profile changes and carotenogenic genes transcript levels inRhodosporidium toruloidesmutants from an optimizedAgrobacterium tumefaciens‐mediated transformation method Sun, Zeping; Lv, Jing; Ji, Chaofan Biotechnology and Applied Biochemistry, Vol. 68, Issue 1 https://doi.org/10.1002/bab.1895	journal	May 2020
Application of adaptive laboratory evolution to improve the tolerance of Rhodotorula strain to methanol in crude glycerol and development of an effective method for cell lysis Wei, Shiyu; Wang, Hongyang; Fan, Meixi Biotechnology Journal, Vol. 19, Issue 1 https://doi.org/10.1002/biot.202300483	journal	December 2023
Engineering Rhodosporidium toruloides for increased lipid production : Engineering Zhang, Shuyan; Skerker, Jeffrey M.; Rutter, Charles D. Biotechnology and Bioengineering, Vol. 113, Issue 5 https://doi.org/10.1002/bit.25864	journal	November 2015
Characterization the carotenoid productions and profiles of three Rhodosporidiumtoruloides mutants from Agrobacterium tumefaciens‐mediated transformation Lin, Xinping; Gao, Ning; Liu, Sasa Yeast, Vol. 34, Issue 8 https://doi.org/10.1002/yea.3236	journal	May 2017
Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts Abbott, Erika P.; Ianiri, Giuseppe; Castoria, Raffaello Applied Microbiology and Biotechnology, Vol. 97, Issue 1 https://doi.org/10.1007/s00253-012-4561-7	journal	November 2012
Integrating transcriptomic and metabolomic analysis of the oleaginous yeast Rhodosporidium toruloides IFO0880 during growth under different carbon sources Jagtap, Sujit Sadashiv; Deewan, Anshu; Liu, Jing-Jing Applied Microbiology and Biotechnology, Vol. 105, Issue 19 https://doi.org/10.1007/s00253-021-11549-8	journal	September 2021
Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16 Tsai, Yung-Yu; Ohashi, Takao; Kanazawa, Takenori Current Genetics, Vol. 63, Issue 2 https://doi.org/10.1007/s00294-016-0629-8	journal	July 2016
Ascomycota has a faster evolutionary rate and higher species diversity than Basidiomycota Wang, HaiYing; Guo, ShouYu; Huang, ManRong Science China Life Sciences, Vol. 53, Issue 10 https://doi.org/10.1007/s11427-010-4063-8	journal	October 2010
The enzyme patterns of Ascomycota and Basidiomycota fungi reveal their different functions in soil Manici, Luisa M.; Caputo, Francesco; De Sabata, Davide Applied Soil Ecology, Vol. 196 https://doi.org/10.1016/j.apsoil.2024.105323	journal	April 2024
Engineering of non-model eukaryotes for bioenergy and biochemical production Ploessl, Deon; Zhao, Yuxin; Shao, Zengyi Current Opinion in Biotechnology, Vol. 79 https://doi.org/10.1016/j.copbio.2022.102869	journal	February 2023
Toward rapid and efficient utilization of nonconventional substrates by nonconventional yeast strains Koh, Hyun Gi; Yook, Sangdo; Oh, Hyunjoon Current Opinion in Biotechnology, Vol. 85 https://doi.org/10.1016/j.copbio.2023.103059	journal	February 2024
Developing Rhodotorula as microbial cell factories for the production of lipids and carotenoids Zhang, Ting-Ting; Wu, Ai-Hua; Aslam, Muhammad Green Carbon, Vol. 2, Issue 4 https://doi.org/10.1016/j.greenca.2024.09.004	journal	December 2024
Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides Koh, Hyun Gi; Kang, Nam Kyu; Kim, Eun Kyung Journal of Biotechnology, Vol. 278 https://doi.org/10.1016/j.jbiotec.2018.04.017	journal	July 2018
Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides Bonturi, Nemailla; Pinheiro, Marina Julio; de Oliveira, Paola Monteiro Metabolic Engineering Communications, Vol. 15 https://doi.org/10.1016/j.mec.2022.e00200	journal	December 2022
The Engineering Potential of Rhodosporidium toruloides as a Workhorse for Biotechnological Applications Park, Young-Kyoung; Nicaud, Jean-Marc; Ledesma-Amaro, Rodrigo Trends in Biotechnology, Vol. 36, Issue 3 https://doi.org/10.1016/j.tibtech.2017.10.013	journal	March 2018
Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in Yarrowia lipolytica Liu, Hu-Hu; Wang, Chong; Lu, Xiang-Yang Journal of Agricultural and Food Chemistry, Vol. 67, Issue 35 https://doi.org/10.1021/acs.jafc.9b03727	journal	August 2019
Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides Koh, Hyun Gi; Kim, Jinhong; Rao, Christopher V. ACS Synthetic Biology, Vol. 12, Issue 11 https://doi.org/10.1021/acssynbio.3c00443	journal	October 2023
A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae O’Riordan, Nicola M.; Jurić, Vanja; O’Neill, Sarah K. ACS Synthetic Biology, Vol. 13, Issue 4 https://doi.org/10.1021/acssynbio.3c00743	journal	March 2024
Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii Crozet, Pierre; Navarro, Francisco J.; Willmund, Felix ACS Synthetic Biology, Vol. 7, Issue 9 https://doi.org/10.1021/acssynbio.8b00251	journal	August 2018
Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector Szymczak, Andrea L.; Workman, Creg J.; Wang, Yao Nature Biotechnology, Vol. 22, Issue 5 https://doi.org/10.1038/nbt957	journal	April 2004
PCR-amplification of GC-rich regions: 'slowdown PCR' Frey, Ulrich H.; Bachmann, Hagen S.; Peters, Jürgen Nature Protocols, Vol. 3, Issue 8 https://doi.org/10.1038/nprot.2008.112	journal	July 2008
Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector Liu, Ziqing; Chen, Olivia; Wall, J. Blake Joseph Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-02460-2	journal	May 2017
A unified multi-kingdom Golden Gate cloning platform Chiasson, David; Giménez-Oya, Victor; Bircheneder, Martin Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-019-46171-2	journal	July 2019
Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber) Basu, Saumik; Pereira, Adriano E.; Pinheiro, Daniele H. Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-019-47020-y	journal	July 2019
Fast and efficient genetic transformation of oleaginous yeast Rhodosporidium toruloides by using electroporation Liu, Hongdi; Jiao, Xiang; Wang, Yanan FEMS Yeast Research, Vol. 17, Issue 2 https://doi.org/10.1093/femsyr/fox017	journal	March 2017
Codon usage tabulated from international DNA sequence databases: status for the year 2000 Nakamura, Yasukazu; Gojobori, Takashi; Ikemura, Toshimichi Nucleic Acids Research, Vol. 28, Issue 1, p. 292-292 https://doi.org/10.1093/nar/28.1.292	journal	January 2000
Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides Lin, Xinping; Wang, Yanan; Zhang, Sufang FEMS Yeast Research, Vol. 14, Issue 4 https://doi.org/10.1111/1567-1364.12140	journal	February 2014
A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology Larroude, Macarena; Park, Young‐Kyoung; Soudier, Paul Microbial Biotechnology, Vol. 12, Issue 6 https://doi.org/10.1111/1751-7915.13427	journal	May 2019
Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii Plucinak, Thomas M.; Horken, Kempton M.; Jiang, Wenzhi The Plant Journal, Vol. 82, Issue 4 https://doi.org/10.1111/tpj.12844	journal	May 2015
Draft Genome Sequences of Rhodosporidium toruloides Strains ATCC 10788 and ATCC 10657 with Compatible Mating Types Hu, Jie; Ji, Lianghui Genome Announcements, Vol. 4, Issue 2 https://doi.org/10.1128/genomeA.00098-16	journal	March 2016
Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides Koh, Chong Mei; Liu, Yanbin BMC Microbiology, Vol. 14, Issue 1 https://doi.org/10.1186/1471-2180-14-50	journal	January 2014
GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris Prielhofer, Roland; Barrero, Juan J.; Steuer, Stefanie BMC Systems Biology, Vol. 11, Issue 1 https://doi.org/10.1186/s12918-017-0492-3	journal	December 2017
A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, Luísa Czamanski; Wehrs, Maren; Kim, Joonhoon Microbial Cell Factories, Vol. 18, Issue 1 https://doi.org/10.1186/s12934-019-1167-0	journal	June 2019
Heterologous synthesis of chlorophyll b in Nannochloropsis salina enhances growth and lipid production by increasing photosynthetic efficiency Koh, Hyun Gi; Kang, Nam Kyu; Jeon, Seungjib Biotechnology for Biofuels, Vol. 12, Issue 1 https://doi.org/10.1186/s13068-019-1462-3	journal	May 2019
Engineering non-conventional yeast Rhodotorula toruloides for ergothioneine production Liu, Ke; Xiang, Gedan; Li, Lekai Biotechnology for Biofuels and Bioproducts, Vol. 17, Issue 1 https://doi.org/10.1186/s13068-024-02516-2	journal	May 2024
A Modular Cloning System for Standardized Assembly of Multigene Constructs Weber, Ernst; Engler, Carola; Gruetzner, Ramona PLoS ONE, Vol. 6, Issue 2 https://doi.org/10.1371/journal.pone.0016765	journal	February 2011
High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice Kim, Jin Hee; Lee, Sang-Rok; Li, Li-Hua PLoS ONE, Vol. 6, Issue 4, Article No. e18556 https://doi.org/10.1371/journal.pone.0018556	journal	April 2011
Dna base Composition of some Species of Yeasts and Yeast-Like Fungi Nakase, Takashi; Komagata, Kazuo The Journal of General and Applied Microbiology, Vol. 17, Issue 5 https://doi.org/10.2323/jgam.17.363	journal	January 1971
Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides Johns, Alexander M. B.; Love, John; Aves, Stephen J. Frontiers in Microbiology, Vol. 7 https://doi.org/10.3389/fmicb.2016.01666	journal	October 2016
An Evolved Strain of the Oleaginous Yeast Rhodotorula toruloides, Multi-Tolerant to the Major Inhibitors Present in Lignocellulosic Hydrolysates, Exhibits an Altered Cell Envelope Fernandes, Mónica A.; Mota, Marta N.; Faria, Nuno T. Journal of Fungi, Vol. 9, Issue 11 https://doi.org/10.3390/jof9111073	journal	November 2023

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
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