A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides

Author(s):Show less -

Nora, Luisa Czamanski ^{[1, 2, 3]} ; Wehrs, Maren ^{[2, 3, 4]} ; Kim, Joonhoon ^{[2, 5]} ; Cheng, Jan-Fang ^{[6, 7]} ; Tarver, Angela ^{[6, 7]} ; Simmons, Blake A. ^{[2, 3]} ; Magnuson, Jon ^{[2, 5]} ; Harmon-Smith, Miranda ^[6] ; Silva-Rocha, Rafael ^[1] ; Gladden, John M. ^{[2, 3, 8]} ; Mukhopadhyay, Aindrila ^{[2, 3, 7]} ; Skerker, Jeffrey M. ^{[7, 9]} ; Kirby, James ^{[2, 3, 8]}

Affiliation

^[1] Univ Sao Paulo, Ribeirao Preto Med Sch, Ave Bandeirantes 3900, BR-14049900 Sao Paulo - Brazil

^[2] US DOE, Joint BioEnergy Inst, 5885 Hollis St, Emeryville, CA 94608 - USA

^[3] Lawrence Berkeley Natl Lab, Biol Syst & Engn Div, 1 Cyclotron Rd, Berkeley, CA 94720 - USA

^[4] Tech Univ Carolo Wilhelmina Braunschweig, Inst Genet, D-38106 Braunschweig - Germany

^[5] Pacific Northwest Natl Lab, Chem & Biol Proc Dev Grp, 902 Battelle Blvd, Richland, WA 99354 - USA

^[6] US DOE, Joint Genome Inst, 2800 Mitchell Dr, Walnut Creek, CA 94598 - USA

^[7] Lawrence Berkeley Natl Lab, Environm Genom & Syst Biol Div, 1 Cyclotron Rd, Berkeley, CA 94720 - USA

^[8] Sandia Natl Labs, Dept Biomass Sci & Convers Technol, 7011 East Ave, Livermore, CA 94550 - USA

^[9] Univ Calif Berkeley, QB3 Berkeley, Berkeley, CA 94720 - USA

• Document type: Journal article
• Source: Microbial Cell Factories; v. 18, JUN 29 2019.
• Web of Science Citations: 0

Abstract

BackgroundRhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides.ResultsThis data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. A total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) were found, and are expected to be of potential value for genetic engineering of R. toruloides.ConclusionsA set of robust and constitutive promoters to facilitate genetic engineering of R. toruloides is presented here, ranging from a promoter previously used for this purpose (P7, glyceraldehyde 3-phosphate dehydrogenase, GAPDH) to stronger monodirectional (e.g., P15, mitochondrial adenine nucleotide translocator, ANT) and bidirectional (e.g., P9 and P9R, histones H3 and H4, respectively) promoters. We also identified promoters that may be useful for specific applications such as late-stage expression (e.g., P3, voltage-dependent anion channel protein 2, VDAC2). This set of characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies. (AU)

• FAPESP's process: 12/22921-8 - Synthetic biology approaches for deciphering the logic of signal integration in complex bacterial promoters
• Grantee: Rafael Silva Rocha
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 18/02227-6 - Construction of engineered yeast strains for achieving better yields of biomass degradation
• Grantee: Luísa Czamanski Nora
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree