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Title: 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 » 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.« less

Authors:
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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}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at https://doi.org/10.1186/s13068-021-01950-w

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