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

Abstract

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 asmore »« less

Authors:

^[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

Publication Date:: 2019-06-29

Research Org.:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

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

OSTI Identifier:: 1545318

Grant/Contract Number:: AC02-05CH11231

Resource Type:: Accepted Manuscript

Journal Name:: Microbial Cell Factories

Additional Journal Information:: Journal Volume: 18; Journal Issue: 1; Journal ID: ISSN 1475-2859

Publisher:: BioMed Central

Country of Publication:: United States

Language:: English

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

Citation Formats


                    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., and Kirby, James. A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides.  United States: N. p., 2019. 
Web.  doi:10.1186/s12934-019-1167-0.

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                    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. A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides.  United States.  https://doi.org/10.1186/s12934-019-1167-0

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                    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., and Kirby, James. Sat .  
"A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides".  United States.  https://doi.org/10.1186/s12934-019-1167-0.  https://www.osti.gov/servlets/purl/1545318.

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@article{osti_1545318,

  title        = {A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides},

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

  abstractNote = {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.},

  doi          = {10.1186/s12934-019-1167-0},

  journal      = {Microbial Cell Factories},

  number       = 1,

  volume       = 18,

  place        = {United States},

  year         = {2019},

  month        = {6}

}

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