skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides

Abstract

Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile hostmore » for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.« less

Authors:
 [1];  [1];  [2];  [3];  [3];  [3];  [4];  [4]; ORCiD logo [4];  [3];  [5]; ORCiD logo [4]; ORCiD logo [4];  [3];  [3];  [1]
  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  2. Univ. of California, Berkeley, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  4. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  5. Joint BioEnergy Institute /Sandia National Laboratory
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1600027
Report Number(s):
[PNNL-SA-150034]
[Journal ID: ISSN; 1475-2859]
Grant/Contract Number:  
[AC05-76RL01830]
Resource Type:
Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
[ Journal Volume: 19]
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Rhodotorula; mevalonate pathway; diterpene; geranylgeranyl pyrophosphate synthase; mutant farnesyl pyrophosphate synthase; metabolic engineering

Citation Formats

Geiselman, Gina M., Zhuang, Xun, Kirby, James, Tran-Gyamfi, Mary, Prahl, Jan-Philip, Sundstrom, Eric R., Gao, Yuqian, Munoz Munoz, Nathalie, Nicora, Carrie D., Clay, Derek M., Papa, Gabriella, Burnum-Johnson, Kristin E., Magnuson, Jon K., Tanjore, Deepti, Skerker, Jeffrey M., and Gladden, John M. Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides. United States: N. p., 2020. Web. doi:10.1186/s12934-020-1293-8.
Geiselman, Gina M., Zhuang, Xun, Kirby, James, Tran-Gyamfi, Mary, Prahl, Jan-Philip, Sundstrom, Eric R., Gao, Yuqian, Munoz Munoz, Nathalie, Nicora, Carrie D., Clay, Derek M., Papa, Gabriella, Burnum-Johnson, Kristin E., Magnuson, Jon K., Tanjore, Deepti, Skerker, Jeffrey M., & Gladden, John M. Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides. United States. doi:10.1186/s12934-020-1293-8.
Geiselman, Gina M., Zhuang, Xun, Kirby, James, Tran-Gyamfi, Mary, Prahl, Jan-Philip, Sundstrom, Eric R., Gao, Yuqian, Munoz Munoz, Nathalie, Nicora, Carrie D., Clay, Derek M., Papa, Gabriella, Burnum-Johnson, Kristin E., Magnuson, Jon K., Tanjore, Deepti, Skerker, Jeffrey M., and Gladden, John M. Wed . "Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides". United States. doi:10.1186/s12934-020-1293-8. https://www.osti.gov/servlets/purl/1600027.
@article{osti_1600027,
title = {Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides},
author = {Geiselman, Gina M. and Zhuang, Xun and Kirby, James and Tran-Gyamfi, Mary and Prahl, Jan-Philip and Sundstrom, Eric R. and Gao, Yuqian and Munoz Munoz, Nathalie and Nicora, Carrie D. and Clay, Derek M. and Papa, Gabriella and Burnum-Johnson, Kristin E. and Magnuson, Jon K. and Tanjore, Deepti and Skerker, Jeffrey M. and Gladden, John M.},
abstractNote = {Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.},
doi = {10.1186/s12934-020-1293-8},
journal = {Microbial Cell Factories},
number = ,
volume = [19],
place = {United States},
year = {2020},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi -Isozizaene Synthase
journal, February 2014

  • Li, Ruiqiong; Chou, Wayne K. W.; Himmelberger, Julie A.
  • Biochemistry, Vol. 53, Issue 7
  • DOI: 10.1021/bi401643u

BIOCHEMISTRY: Creating Isoprenoid Diversity
journal, September 1997


CHEMISTRY: Roots of Biosynthetic Diversity
journal, April 2007


The prenyl transfer reaction. Enzymic and mechanistic studies of the 1'-4 coupling reaction in the terpene biosynthetic pathway
journal, August 1978

  • Poulter, C. Dale; Rilling, Hans C.
  • Accounts of Chemical Research, Vol. 11, Issue 8
  • DOI: 10.1021/ar50128a004

Cytotoxicity and Chemotherapeutic Potential of Natural Rosin Abietane Diterpenoids and their Synthetic Derivatives
journal, February 2019


Diterpenes as lead molecules against neglected tropical diseases: Diterpenes Acting Against Neglected Tropical Diseases
journal, November 2016

  • Barros de Alencar, Marcus Vinícius Oliveira; de Castro e. Sousa, João Marcelo; Rolim, Hercília Maria Lins
  • Phytotherapy Research, Vol. 31, Issue 2
  • DOI: 10.1002/ptr.5749

Natural Cytotoxic Diterpenoids, a Potential Source of Drug Leads for Melanoma Therapy
journal, February 2019


Biosynthesis and function of terpenoid defense compounds in maize (Zea mays)
journal, September 2018


The Phorbol Ester Fraction from Jatropha curcas Seed Oil: Potential and Limits for Crop Protection against Insect Pests
journal, November 2012

  • Ratnadass, Alain; Wink, Michael
  • International Journal of Molecular Sciences, Vol. 13, Issue 12
  • DOI: 10.3390/ijms131216157

Gibberellin Metabolism and its Regulation
journal, June 2008


Gibberellin biosynthesis and metabolism: A convergent route for plants, fungi and bacteria
journal, March 2018

  • Salazar-Cerezo, Sonia; Martínez-Montiel, Nancy; García-Sánchez, Jenny
  • Microbiological Research, Vol. 208
  • DOI: 10.1016/j.micres.2018.01.010

Current advances in gibberellic acid (GA3) production, patented technologies and potential applications
journal, August 2018

  • Camara, Marcela C.; Vandenberghe, Luciana P. S.; Rodrigues, Cristine
  • Planta, Vol. 248, Issue 5
  • DOI: 10.1007/s00425-018-2959-x

The Remarkable Structural Diversity Achieved in ent-Kaurane Diterpenes by Fungal Biotransformations
journal, February 2014


Diterpenoid biopolymers: New directions for renewable materials engineering
journal, September 2010

  • Hillwig, Matthew L.; Mann, Francis M.; Peters, Reuben J.
  • Biopolymers, Vol. 95, Issue 2
  • DOI: 10.1002/bip.21538

Engineering Aspergillus nidulans for heterologous ent-kaurene and gamma-terpinene production
journal, April 2016

  • Bromann, Kirsi; Toivari, Mervi; Viljanen, Kaarina
  • Applied Microbiology and Biotechnology, Vol. 100, Issue 14
  • DOI: 10.1007/s00253-016-7517-5

Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli
journal, November 2015


A highly efficient dilute alkali deacetylation and mechanical (disc) refining process for the conversion of renewable biomass to lower cost sugars
journal, January 2014

  • Chen, Xiaowen; Shekiro, Joseph; Pschorn, Thomas
  • Biotechnology for Biofuels, Vol. 7, Issue 1
  • DOI: 10.1186/1754-6834-7-98

Monoterpene production by the carotenogenic yeast Rhodosporidium toruloides
journal, March 2019


Concomitant Production of Lipids and Carotenoids in Rhodosporidium toruloides under Osmotic Stress Using Response Surface Methodology
journal, October 2016


Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides
journal, October 2009


Conversion of depolymerized sugars and aromatics from engineered feedstocks by two oleaginous red yeasts
journal, August 2019


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


High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture
journal, August 2007


Engineering Rhodosporidium toruloides for increased lipid production : Engineering
journal, November 2015

  • Zhang, Shuyan; Skerker, Jeffrey M.; Rutter, Charles D.
  • Biotechnology and Bioengineering, Vol. 113, Issue 5
  • DOI: 10.1002/bit.25864

Cloning of a Full-length cDNA Encoding ent -Kaurene Synthase from Gibberella fujikuroi : Functional Analysis of a Bifunctional Diterpene Cyclase
journal, January 2000

  • Toyomasu, Tomonobu; Kawaide, Hiroshi; Ishizaki, Atsuko
  • Bioscience, Biotechnology, and Biochemistry, Vol. 64, Issue 3
  • DOI: 10.1271/bbb.64.660

Regulation of product chain length by isoprenyl diphosphate synthases
journal, December 1996

  • Tarshis, L. C.; Proteau, P. J.; Kellogg, B. A.
  • Proceedings of the National Academy of Sciences, Vol. 93, Issue 26
  • DOI: 10.1073/pnas.93.26.15018

Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control
journal, July 2010

  • Leonard, E.; Ajikumar, P. K.; Thayer, K.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 31, p. 13654-13659
  • DOI: 10.1073/pnas.1006138107

Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools
journal, June 2012

  • Ham, T. S.; Dmytriv, Z.; Plahar, H.
  • Nucleic Acids Research, Vol. 40, Issue 18
  • DOI: 10.1093/nar/gks531

MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses
journal, May 2016


CONSeQuence: Prediction of Reference Peptides for Absolute Quantitative Proteomics Using Consensus Machine Learning Approaches
journal, August 2011

  • Eyers, Claire E.; Lawless, Craig; Wedge, David C.
  • Molecular & Cellular Proteomics, Vol. 10, Issue 11
  • DOI: 10.1074/mcp.M110.003384

Using Data Independent Acquisition (DIA) to Model High-responding Peptides for Targeted Proteomics Experiments
journal, June 2015

  • Searle, Brian C.; Egertson, Jarrett D.; Bollinger, James G.
  • Molecular & Cellular Proteomics, Vol. 14, Issue 9
  • DOI: 10.1074/mcp.M115.051300

Skyline: an open source document editor for creating and analyzing targeted proteomics experiments
journal, February 2010