Data for Discovery, Characterization, and Application of Chromosomal Integration Sites for Stable Heterologous Gene Expression in Rhodotorula toruloides

Xu, Hao [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)]; Shi, Longyuan [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)]; Boob, Aashutosh [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:000000022045331X); Park, Wooyoung [School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Gwanak-gu, Seoul, 08826, Republic of Korea; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)]; Tan, Shih-I [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)]; Tran, Vinh [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000289392527); Schultz, J. Carl [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States]; Zhao, Huimin [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000290696739)

doi:10.13012/b2idb-4854964_v1

DOE OSTI · 3015300

Data for Discovery, Characterization, and Application of Chromosomal Integration Sites for Stable Heterologous Gene Expression in Rhodotorula toruloides

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Abstract

Rhodotorula toruloides is a non-model, oleaginous yeast uniquely suited to produce acetyl-CoA-derived chemicals. However, the lack of well-characterized genomic integration sites has impeded the metabolic engineering of this organism. Here we report a set of computationally predicted and experimentally validated chromosomal integration sites in R. toruloides . We first implemented an in silico platform by integrating essential gene information and transcriptomic data to identify candidate sites that meet stringent criteria. We then conducted a full experimental characterization of these sites, assessing integration efficiency, gene expression levels, impact on cell growth, and long-term expression stability. Among the identified sites, 12 exhibited integration efficiencies of 50% or higher, making them sufficient for most metabolic engineering applications. Using selected high-efficiency sites, we achieved simultaneous double and triple integrations and efficiently integrated long functional pathways (up to 14.7 kb). Additionally, we developed a new inducible marker recycling system that allows multiple rounds of integration at our characterized sites. We validated this system by performing five sequential rounds of GFP integration and three sequential rounds of MaFAR integration for fatty alcohol production, demonstrating, for the first time, precise gene copy number tuning in R. toruloides . These characterized integration sites should significantly advance metabolic engineering efforts and future genetic tool development in R. toruloides .

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Xu, Hao [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)], Shi, Longyuan [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)], Boob, Aashutosh [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:000000022045331X), Park, Wooyoung [School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Gwanak-gu, Seoul, 08826, Republic of Korea; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)], Tan, Shih-I [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)], Tran, Vinh [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000289392527), Schultz, J. Carl [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States], Zhao, Huimin [Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000290696739). 2025-02-14. Data for Discovery, Characterization, and Application of Chromosomal Integration Sites for Stable Heterologous Gene Expression in Rhodotorula toruloides. https://doi.org/10.13012/b2idb-4854964_v1

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