A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides

Nora, Luísa Czamanski; Wehrs, Maren; Kim, Joonhoon; Cheng, Jan-Fang; Tarver, Angela; Simmons, Blake A.; Magnuson, Jon; Harmon-Smith, Miranda; Silva-Rocha, Rafael; Gladden, John M.; Mukhopadhyay, Aindrila; Skerker, Jeffrey M.; Kirby, James

doi:10.1186/s12934-019-1167-0

Title: A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides

Journal Article · 28 June 2019 · Microbial Cell Factories

DOI: https://doi.org/10.1186/s12934-019-1167-0 · OSTI ID:1545318

^[1]; Wehrs, Maren ^[2]; Kim, Joonhoon ^[3]; Cheng, Jan-Fang ^[4]; Tarver, Angela ^[4]; Simmons, Blake A. ^[5]; Magnuson, Jon ^[3]; Harmon-Smith, Miranda ^[6]; Silva-Rocha, Rafael ^[7]; Gladden, John M. ^[8];

^[5]; Skerker, Jeffrey M. ^[9]; Kirby, James ^[8]

Univ. of Sao Paulo (Brazil); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technische Univ. Braunschweig, Brunswick (Germany)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
Univ. of Sao Paulo (Brazil)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States). QB3-Berkeley

BACKGROUND:Rhodosporidium 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. RESULTS:This 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. CONCLUSIONS:A 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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1545318

Journal Information:: Microbial Cell Factories, Journal Name: Microbial Cell Factories Journal Issue: 1 Vol. 18; ISSN 1475-2859

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

09 BIOMASS FUELS
Rhodotorula
Bidirectional promoters
Metabolic engineering
Synthetic biology
Transformation
Fluorescent reporters
Promoter characterization

MOESM4 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378453 The current record is supplemented by this text	text	January 2019
MOESM4 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378453.v1 The current record is supplemented by this text	text	January 2019
A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.c.4560707 The current record is supplemented by this collection	collection	January 2019
MOESM5 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378459 The current record is supplemented by this text	text	January 2019
MOESM2 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378435 The current record is supplemented by this text	text	January 2019
A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.c.4560707.v1 The current record is supplemented by this collection	collection	January 2019
MOESM3 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378444 The current record is supplemented by this text	text	January 2019
MOESM1 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378429.v1 The current record is supplemented by this text	text	January 2019
MOESM1 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378429 The current record is supplemented by this text	text	January 2019
MOESM2 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378435.v1 The current record is supplemented by this text	text	January 2019
MOESM5 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378459.v1 The current record is supplemented by this text	text	January 2019
MOESM3 of A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides Nora, LuĂSa; Wehrs, Maren; Kim, Joonhoon https://doi.org/10.6084/m9.figshare.8378444.v1 The current record is supplemented by this text	text	January 2019