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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 » 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.« less

Authors:
ORCiD logo [1];  [2];  [3];  [4];  [4];  [5];  [3];  [6];  [7];  [8]; ORCiD logo [5];  [9];  [8]
+ Show Author Affiliations
  1. Univ. of Sao Paulo (Brazil); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technische Univ. Braunschweig, Brunswick (Germany)
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  4. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  5. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  6. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
  7. Univ. of Sao Paulo (Brazil)
  8. 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)
  9. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States). QB3-Berkeley
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
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. 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., and Kirby, James. Sat . "A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides". United States. doi: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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DOI:  10.1186/s12934-019-1167-0
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Works referenced in this record:

Widespread bidirectional promoters are the major source of cryptic transcripts in yeast
journal, January 2009

  • Neil, Helen; Malabat, Christophe; d’Aubenton-Carafa, Yves
  • Nature, Vol. 457, Issue 7232
  • DOI: 10.1038/nature07747

Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae
journal, January 2016

  • Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather
  • Scientific Reports, Vol. 6, Issue 1
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Bidirectional promoters generate pervasive transcription in yeast
journal, January 2009

Systems analysis of phosphate-limitation-induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides
journal, May 2018

Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts
journal, October 2017

Composability of regulatory sequences controlling transcription and translation in Escherichia coli
journal, August 2013

  • Kosuri, Sriram; Goodman, Daniel B.; Cambray, Guillaume
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 34
  • DOI: 10.1073/pnas.1301301110

5´-UTR introns enhance protein expression in the yeast Saccharomyces cerevisiae
journal, October 2016

  • Hoshida, Hisashi; Kondo, Masaki; Kobayashi, Takafumi
  • Applied Microbiology and Biotechnology, Vol. 101, Issue 1
  • DOI: 10.1007/s00253-016-7891-z

Engineering an efficient and tight d-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species
journal, October 2015

The Impact of Systems Biology on Bioprocessing
journal, December 2017

Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation
journal, September 2016

  • Zhang, Shuyan; Ito, Masakazu; Skerker, Jeffrey M.
  • Applied Microbiology and Biotechnology, Vol. 100, Issue 21
  • DOI: 10.1007/s00253-016-7815-y

Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides
journal, October 2016

Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli
journal, April 2018

  • Hwang, Hee Jin; Lee, Sang Yup; Lee, Pyung Cheon
  • Biotechnology for Biofuels, Vol. 11, Issue 1
  • DOI: 10.1186/s13068-018-1104-1

Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides
journal, February 2014

Engineering metabolism through dynamic control
journal, August 2015

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization
journal, September 2018

How introns enhance gene expression
journal, October 2017

Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides
journal, June 2012

  • Liu, Yanbin; Koh, Chong Mei John; Sun, Longhua
  • Applied Microbiology and Biotechnology, Vol. 97, Issue 2, p. 719-729
  • DOI: 10.1007/s00253-012-4223-9

The Engineering Potential of Rhodosporidium toruloides as a Workhorse for Biotechnological Applications
journal, March 2018

Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase
journal, January 2015

  • Gmeiner, Christoph; Saadati, Amirhossein; Maresch, Daniel
  • Microbial Cell Factories, Vol. 14, Issue 1
  • DOI: 10.1186/s12934-014-0183-3

The art of vector engineering: towards the construction of next-generation genetic tools
journal, September 2018

  • Nora, Luísa Czamanski; Westmann, Cauã Antunes; Martins-Santana, Leonardo
  • Microbial Biotechnology, Vol. 12, Issue 1
  • DOI: 10.1111/1751-7915.13318

featureCounts: an efficient general purpose program for assigning sequence reads to genomic features
journal, November 2013

Mitochondrial acetyl-CoA utilization pathway for terpenoid productions
journal, November 2016

Causes and Effects of N-Terminal Codon Bias in Bacterial Genes
journal, September 2013

Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides
journal, March 2019

Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora
journal, January 2016

Precise and reliable gene expression via standard transcription and translation initiation elements
journal, March 2013

  • Mutalik, Vivek K.; Guimaraes, Joao C.; Cambray, Guillaume
  • Nature Methods, Vol. 10, Issue 4
  • DOI: 10.1038/nmeth.2404

HISAT: a fast spliced aligner with low memory requirements
journal, March 2015

  • Kim, Daehwan; Langmead, Ben; Salzberg, Steven L.
  • Nature Methods, Vol. 12, Issue 4
  • DOI: 10.1038/nmeth.3317
    [ × clear filter / sort ]

    Works referencing / citing this record:

    Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides
    journal, June 2012

    • Liu, Yanbin; Koh, Chong Mei John; Sun, Longhua
    • Applied Microbiology and Biotechnology, Vol. 97, Issue 2, p. 719-729
    • DOI: 10.1007/s00253-012-4223-9

    Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation
    journal, September 2016

    • Zhang, Shuyan; Ito, Masakazu; Skerker, Jeffrey M.
    • Applied Microbiology and Biotechnology, Vol. 100, Issue 21
    • DOI: 10.1007/s00253-016-7815-y

    5´-UTR introns enhance protein expression in the yeast Saccharomyces cerevisiae
    journal, October 2016

    • Hoshida, Hisashi; Kondo, Masaki; Kobayashi, Takafumi
    • Applied Microbiology and Biotechnology, Vol. 101, Issue 1
    • DOI: 10.1007/s00253-016-7891-z

    How introns enhance gene expression
    journal, October 2017

    Engineering metabolism through dynamic control
    journal, August 2015

    The Impact of Systems Biology on Bioprocessing
    journal, December 2017

    The Engineering Potential of Rhodosporidium toruloides as a Workhorse for Biotechnological Applications
    journal, March 2018

    Mitochondrial acetyl-CoA utilization pathway for terpenoid productions
    journal, November 2016

    Bidirectional promoters generate pervasive transcription in yeast
    journal, January 2009

    Widespread bidirectional promoters are the major source of cryptic transcripts in yeast
    journal, January 2009

    • Neil, Helen; Malabat, Christophe; d’Aubenton-Carafa, Yves
    • Nature, Vol. 457, Issue 7232
    • DOI: 10.1038/nature07747

    Precise and reliable gene expression via standard transcription and translation initiation elements
    journal, March 2013

    • Mutalik, Vivek K.; Guimaraes, Joao C.; Cambray, Guillaume
    • Nature Methods, Vol. 10, Issue 4
    • DOI: 10.1038/nmeth.2404

    HISAT: a fast spliced aligner with low memory requirements
    journal, March 2015

    • Kim, Daehwan; Langmead, Ben; Salzberg, Steven L.
    • Nature Methods, Vol. 12, Issue 4
    • DOI: 10.1038/nmeth.3317

    Engineered bidirectional promoters enable rapid multi-gene co-expression optimization
    journal, September 2018

    Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae
    journal, January 2016

    • Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather
    • Scientific Reports, Vol. 6, Issue 1
    • DOI: 10.1038/srep19512

    Composability of regulatory sequences controlling transcription and translation in Escherichia coli
    journal, August 2013

    • Kosuri, Sriram; Goodman, Daniel B.; Cambray, Guillaume
    • Proceedings of the National Academy of Sciences, Vol. 110, Issue 34
    • DOI: 10.1073/pnas.1301301110

    featureCounts: an efficient general purpose program for assigning sequence reads to genomic features
    journal, November 2013

    Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides
    journal, February 2014

    The art of vector engineering: towards the construction of next-generation genetic tools
    journal, September 2018

    • Nora, Luísa Czamanski; Westmann, Cauã Antunes; Martins-Santana, Leonardo
    • Microbial Biotechnology, Vol. 12, Issue 1
    • DOI: 10.1111/1751-7915.13318

    Causes and Effects of N-Terminal Codon Bias in Bacterial Genes
    journal, September 2013

    Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase
    journal, January 2015

    • Gmeiner, Christoph; Saadati, Amirhossein; Maresch, Daniel
    • Microbial Cell Factories, Vol. 14, Issue 1
    • DOI: 10.1186/s12934-014-0183-3

    Engineering an efficient and tight d-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species
    journal, October 2015

    Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora
    journal, January 2016

    Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts
    journal, October 2017

    Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli
    journal, April 2018

    • Hwang, Hee Jin; Lee, Sang Yup; Lee, Pyung Cheon
    • Biotechnology for Biofuels, Vol. 11, Issue 1
    • DOI: 10.1186/s13068-018-1104-1

    Systems analysis of phosphate-limitation-induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides
    journal, May 2018

    Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides
    journal, October 2016

      [ × clear filter / sort ]

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