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Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass
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...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058980/ https://www.ncbi.nlm.nih.gov/pubmed/33883010 http://dx.doi.org/10.1186/s13068-021-01950-w |
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author | 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. |
author_facet | 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. |
author_sort | Kirby, James |
collection | PubMed |
description | 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-021-01950-w. |
format | Online Article Text |
id | pubmed-8058980 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-80589802021-04-21 Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass 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. Biotechnol Biofuels Research 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-021-01950-w. BioMed Central 2021-04-21 /pmc/articles/PMC8058980/ /pubmed/33883010 http://dx.doi.org/10.1186/s13068-021-01950-w Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research 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 |
title | Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass |
title_full | Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass |
title_fullStr | Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass |
title_full_unstemmed | Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass |
title_short | Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass |
title_sort | further engineering of r. toruloides for the production of terpenes from lignocellulosic biomass |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058980/ https://www.ncbi.nlm.nih.gov/pubmed/33883010 http://dx.doi.org/10.1186/s13068-021-01950-w |
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