Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass
Abstract
Background: Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates .Results: This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks aremore »
- Authors:
- more »
- Publication Date:
- Research Org.:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE); USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE National Nuclear Security Administration (NNSA)
- OSTI Identifier:
- 2325570
- Alternate Identifier(s):
- OSTI ID: 1784623; OSTI ID: 1826711; OSTI ID: 1828023
- Report Number(s):
- PNNL-SA-162406; SAND-2021-5592J
Journal ID: ISSN 1754-6834; 101; PII: 1950
- Grant/Contract Number:
- AC02-05CH11231; AC05-76RL01830; NA0003525
- Resource Type:
- Journal Article: Published Article
- Journal Name:
- Biotechnology for Biofuels
- Additional Journal Information:
- Journal Name: Biotechnology for Biofuels Journal Volume: 14 Journal Issue: 1; Journal ID: ISSN 1754-6834
- Publisher:
- Springer Science + Business Media
- Country of Publication:
- Netherlands
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; Rhodotorula; Mevalonate pathway; Isoprenoids; Metabolic engineering; Α-bisabolene; Eucalyptol; 1,8-Cineole
Citation Formats
Kirby, James, Geiselman, Gina M., Yaegashi, Junko, Kim, Joonhoon, Zhuang, Xun, Tran-Gyamfi, Mary Bao, Prahl, Jan-Philip, Sundstrom, Eric R., Gao, Yuqian, Munoz, Nathalie, Burnum-Johnson, Kristin E., Benites, Veronica T., Baidoo, Edward E. K., Fuhrmann, Anna, Seibel, Katharina, Webb-Robertson, Bobbie-Jo M., Zucker, Jeremy, Nicora, Carrie D., Tanjore, Deepti, Magnuson, Jon K., Skerker, Jeffrey M., and Gladden, John M. Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass. Netherlands: N. p., 2021.
Web. doi:10.1186/s13068-021-01950-w.
Kirby, James, Geiselman, Gina M., Yaegashi, Junko, Kim, Joonhoon, Zhuang, Xun, Tran-Gyamfi, Mary Bao, Prahl, Jan-Philip, Sundstrom, Eric R., Gao, Yuqian, Munoz, Nathalie, Burnum-Johnson, Kristin E., Benites, Veronica T., Baidoo, Edward E. K., Fuhrmann, Anna, Seibel, Katharina, Webb-Robertson, Bobbie-Jo M., Zucker, Jeremy, Nicora, Carrie D., Tanjore, Deepti, Magnuson, Jon K., Skerker, Jeffrey M., & Gladden, John M. Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass. Netherlands. https://doi.org/10.1186/s13068-021-01950-w
Kirby, James, Geiselman, Gina M., Yaegashi, Junko, Kim, Joonhoon, Zhuang, Xun, Tran-Gyamfi, Mary Bao, Prahl, Jan-Philip, Sundstrom, Eric R., Gao, Yuqian, Munoz, Nathalie, Burnum-Johnson, Kristin E., Benites, Veronica T., Baidoo, Edward E. K., Fuhrmann, Anna, Seibel, Katharina, Webb-Robertson, Bobbie-Jo M., Zucker, Jeremy, Nicora, Carrie D., Tanjore, Deepti, Magnuson, Jon K., Skerker, Jeffrey M., and Gladden, John M. 2021.
"Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass". Netherlands. https://doi.org/10.1186/s13068-021-01950-w.
@article{osti_2325570,
title = {Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass},
author = {Kirby, James and Geiselman, Gina M. and Yaegashi, Junko and Kim, Joonhoon and Zhuang, Xun and Tran-Gyamfi, Mary Bao and Prahl, Jan-Philip and Sundstrom, Eric R. and Gao, Yuqian and Munoz, Nathalie and Burnum-Johnson, Kristin E. and Benites, Veronica T. and Baidoo, Edward E. K. and Fuhrmann, Anna and Seibel, Katharina and Webb-Robertson, Bobbie-Jo M. and Zucker, Jeremy and Nicora, Carrie D. and Tanjore, Deepti and Magnuson, Jon K. and Skerker, Jeffrey M. and Gladden, John M.},
abstractNote = {Background: Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates .Results: This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions: This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.},
doi = {10.1186/s13068-021-01950-w},
url = {https://www.osti.gov/biblio/2325570},
journal = {Biotechnology for Biofuels},
issn = {1754-6834},
number = 1,
volume = 14,
place = {Netherlands},
year = {Wed Apr 21 00:00:00 EDT 2021},
month = {Wed Apr 21 00:00:00 EDT 2021}
}
Works referenced in this record:
Toward better understanding of artifacts in variant calling from high-coverage samples
journal, June 2014
- Li, Heng
- Bioinformatics, Vol. 30, Issue 20
Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
journal, January 2014
- Garcia, David E.; Keasling, Jay D.
- PLoS ONE, Vol. 9, Issue 1
Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts
journal, October 2017
- Yaegashi, Junko; Kirby, James; Ito, Masakazu
- Biotechnology for Biofuels, Vol. 10, Issue 1
Bioprospecting—fuels from fungi
journal, February 2015
- Strobel, Gary Allan
- Biotechnology Letters, Vol. 37, Issue 5
Increasing the intracellular isoprenoid pool in Saccharomyces cerevisiae by structural fine-tuning of a bifunctional farnesyl diphosphate synthase
journal, June 2017
- Rubat, Sebastián; Varas, Ignacio; Sepúlveda, Romina
- FEMS Yeast Research, Vol. 17, Issue 4
Engineered isoprenoid pathway enhances astaxanthin production inEscherichia coli
journal, January 1999
- Wang, Chia-Wei; Oh, Min-Kyu; Liao, James C.
- Biotechnology and Bioengineering, Vol. 62, Issue 2
Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production
journal, January 2013
- Özaydın, Bilge; Burd, Helcio; Lee, Taek Soon
- Metabolic Engineering, Vol. 15
Farnesyl Diphosphate Synthase. Altering the Catalytic Site To Select for Geranyl Diphosphate Activity †
journal, December 2000
- Stanley Fernandez, Suzanne M.; Kellogg, Brenda A.; Poulter, C. Dale
- Biochemistry, Vol. 39, Issue 50
Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase
journal, June 2008
- Paradise, Eric M.; Kirby, James; Chan, Rossana
- Biotechnology and Bioengineering, Vol. 100, Issue 2
Rewriting yeast central carbon metabolism for industrial isoprenoid production
journal, September 2016
- Meadows, Adam L.; Hawkins, Kristy M.; Tsegaye, Yoseph
- Nature, Vol. 537, Issue 7622
An Evolutionarily Conserved Mechanism for Controlling the Efficiency of Protein Translation
journal, April 2010
- Tuller, Tamir; Carmi, Asaf; Vestsigian, Kalin
- Cell, Vol. 141, Issue 2
Towards synthesis of monoterpenes and derivatives using synthetic biology
journal, October 2016
- Zebec, Ziga; Wilkes, Jonathan; Jervis, Adrian J.
- Current Opinion in Chemical Biology, Vol. 34
Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase.
journal, February 1991
- Tsay, Y. H.; Robinson, G. W.
- Molecular and Cellular Biology, Vol. 11, Issue 2
Functional characterization of a geraniol synthase-encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli
journal, June 2016
- Chen, Fei; Li, Wei; Jiang, Liangzhen
- Journal of Industrial Microbiology & Biotechnology, Vol. 43, Issue 9
Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum
journal, August 2012
- Xu, Jun-Wei; Xu, Yi-Ning; Zhong, Jian-Jiang
- Applied and Environmental Microbiology, Vol. 78, Issue 22
Identification and microbial production of a terpene-based advanced biofuel
journal, September 2011
- Peralta-Yahya, Pamela P.; Ouellet, Mario; Chan, Rossana
- Nature Communications, Vol. 2, Issue 1
Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks
journal, November 2020
- Geiselman, Gina M.; Kirby, James; Landera, Alexander
- Microbial Cell Factories, Vol. 19, Issue 1
A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides
journal, June 2019
- Nora, Luísa Czamanski; Wehrs, Maren; Kim, Joonhoon
- Microbial Cell Factories, Vol. 18, Issue 1
Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production
journal, September 2013
- Alonso-Gutierrez, Jorge; Chan, Rossana; Batth, Tanveer S.
- Metabolic Engineering, Vol. 19
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
DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L −1 ) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration
journal, January 2016
- Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W.
- Energy & Environmental Science, Vol. 9, Issue 4
Chemical composition and antifungal activity of the essential oil of Zhumeria majdae , Heracleum persicum and Eucalyptus sp. against some important phytopathogenic fungi
journal, December 2017
- Davari, M.; Ezazi, R.
- Journal de Mycologie Médicale, Vol. 27, Issue 4
Production of jet fuel precursor monoterpenoids from engineered Escherichia coli : Production of Jet Fuel Precursor Monoterpenoids
journal, May 2017
- Mendez-Perez, Daniel; Alonso-Gutierrez, Jorge; Hu, Qijun
- Biotechnology and Bioengineering, Vol. 114, Issue 8
Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory
journal, September 2019
- Zhang, Chuanbo; Li, Man; Zhao, Guang-Rong
- Microbial Cell Factories, Vol. 18, Issue 1
Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides
journal, February 2020
- Geiselman, Gina M.; Zhuang, Xun; Kirby, James
- Microbial Cell Factories, Vol. 19, Issue 1
featureCounts: an efficient general purpose program for assigning sequence reads to genomic features
journal, November 2013
- Liao, Y.; Smyth, G. K.; Shi, W.
- Bioinformatics, Vol. 30, Issue 7
CNVnator: An approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing
journal, February 2011
- Abyzov, A.; Urban, A. E.; Snyder, M.
- Genome Research, Vol. 21, Issue 6
Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives
journal, July 2018
- Zada, Bakht; Wang, Chonglong; Park, Ji-Bin
- Biotechnology for Biofuels, Vol. 11, Issue 1
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
Selection on codon bias in yeast: a transcriptional hypothesis
journal, August 2013
- Trotta, Edoardo
- Nucleic Acids Research, Vol. 41, Issue 20
Strategies for strain improvement in Fusarium fujikuroi: overexpression and localization of key enzymes of the isoprenoid pathway and their impact on gibberellin biosynthesis
journal, September 2012
- Albermann, Sabine; Linnemannstöns, Pia; Tudzynski, Bettina
- Applied Microbiology and Biotechnology, Vol. 97, Issue 7
Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases
journal, September 2011
- Ma, Suzanne M.; Garcia, David E.; Redding-Johanson, Alyssa M.
- Metabolic Engineering, Vol. 13, Issue 5
Monoterpene production by the carotenogenic yeast Rhodosporidium toruloides
journal, March 2019
- Zhuang, Xun; Kilian, Oliver; Monroe, Eric
- Microbial Cell Factories, Vol. 18, Issue 1
The Sequence Alignment/Map format and SAMtools
journal, June 2009
- Li, H.; Handsaker, B.; Wysoker, A.
- Bioinformatics, Vol. 25, Issue 16
Biogenesis, molecular regulation and function of plant isoprenoids
journal, November 2005
- Bouvier, Florence; Rahier, Alain; Camara, Bilal
- Progress in Lipid Research, Vol. 44, Issue 6
Characterization of feedback-resistant mevalonate kinases from the methanogenic archaeons Methanosaeta concilii and Methanocella paludicola
journal, September 2017
- Kazieva, Ekaterina; Yamamoto, Yoko; Tajima, Yoshinori
- Microbiology, Vol. 163, Issue 9
Multiple roles of the coding sequence 5′ end in gene expression regulation
journal, December 2014
- Tuller, Tamir; Zur, Hadas
- Nucleic Acids Research, Vol. 43, Issue 1
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
Hypoxylon sp., an Endophyte of Persea indica, Producing 1,8-Cineole and Other Bioactive Volatiles with Fuel Potential
journal, October 2010
- Tomsheck, Angela R.; Strobel, Gary A.; Booth, Eric
- Microbial Ecology, Vol. 60, Issue 4