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

Abstract

BACKGROUND: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. RESULTS: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. CONCLUSION:This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host formore » 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:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1618899
Alternate Identifier(s):
OSTI ID: 1600027; OSTI ID: 1604735
Report Number(s):
PNNL-SA-150034
Journal ID: ISSN 1475-2859; 24; PII: 1293
Grant/Contract Number:  
AC02-05CH11231; AC05-76RL01830
Resource Type:
Published Article
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Name: Microbial Cell Factories Journal Volume: 19 Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
Springer Science + Business Media
Country of Publication:
United Kingdom
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 B., 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 Kingdom: N. p., 2020. Web. https://doi.org/10.1186/s12934-020-1293-8.
Geiselman, Gina M., Zhuang, Xun, Kirby, James, Tran-Gyamfi, Mary B., 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 Kingdom. https://doi.org/10.1186/s12934-020-1293-8
Geiselman, Gina M., Zhuang, Xun, Kirby, James, Tran-Gyamfi, Mary B., 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 Kingdom. https://doi.org/10.1186/s12934-020-1293-8.
@article{osti_1618899,
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 B. 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 = {BACKGROUND: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. RESULTS: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. CONCLUSION: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 = 1,
volume = 19,
place = {United Kingdom},
year = {2020},
month = {2}
}

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https://doi.org/10.1186/s12934-020-1293-8

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