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 derivative...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-02-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-020-1293-8 |
| _version_ | 1818452778064805888 |
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| author | Gina M. Geiselman Xun Zhuang James Kirby Mary B. Tran-Gyamfi Jan-Philip Prahl Eric R. Sundstrom Yuqian Gao Nathalie Munoz Munoz Carrie D. Nicora Derek M. Clay Gabriella Papa Kristin E. Burnum-Johnson Jon K. Magnuson Deepti Tanjore Jeffrey M. Skerker John M. Gladden |
| author_facet | Gina M. Geiselman Xun Zhuang James Kirby Mary B. Tran-Gyamfi Jan-Philip Prahl Eric R. Sundstrom Yuqian Gao Nathalie Munoz Munoz Carrie D. Nicora Derek M. Clay Gabriella Papa Kristin E. Burnum-Johnson Jon K. Magnuson Deepti Tanjore Jeffrey M. Skerker John M. Gladden |
| author_sort | Gina M. Geiselman |
| collection | DOAJ |
| description | 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 for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism. |
| first_indexed | 2024-12-14T21:28:30Z |
| format | Article |
| id | doaj.art-90f1f5a0af174a618051885f0d6890d5 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-14T21:28:30Z |
| publishDate | 2020-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-90f1f5a0af174a618051885f0d6890d52022-12-21T22:46:44ZengBMCMicrobial Cell Factories1475-28592020-02-0119111210.1186/s12934-020-1293-8Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloidesGina M. Geiselman0Xun Zhuang1James Kirby2Mary B. Tran-Gyamfi3Jan-Philip Prahl4Eric R. Sundstrom5Yuqian Gao6Nathalie Munoz Munoz7Carrie D. Nicora8Derek M. Clay9Gabriella Papa10Kristin E. Burnum-Johnson11Jon K. Magnuson12Deepti Tanjore13Jeffrey M. Skerker14John M. Gladden15Department of Energy, Agile BioFoundryDepartment of Energy, Agile BioFoundryDepartment of Energy, Agile BioFoundryDepartment of Energy, Agile BioFoundryAdvanced Biofuels and Bioproducts Process Development Unit, Lawrence Berkeley National LaboratoryAdvanced Biofuels and Bioproducts Process Development Unit, Lawrence Berkeley National LaboratoryEarth and Biological Sciences Directorate, Pacific Northwest National LaboratoryEarth and Biological Sciences Directorate, Pacific Northwest National LaboratoryEarth and Biological Sciences Directorate, Pacific Northwest National LaboratoryDepartment of Energy, Agile BioFoundryAdvanced Biofuels and Bioproducts Process Development Unit, Lawrence Berkeley National LaboratoryEarth and Biological Sciences Directorate, Pacific Northwest National LaboratoryEnergy and Environment Directorate, Pacific Northwest National LaboratoryAdvanced Biofuels and Bioproducts Process Development Unit, Lawrence Berkeley National LaboratoryQB3-Berkeley, University of CaliforniaDepartment of Energy, Agile BioFoundryAbstract 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.https://doi.org/10.1186/s12934-020-1293-8RhodotorulaMevalonate pathwayDiterpeneGeranylgeranyl pyrophosphate synthaseMutant farnesyl pyrophosphate synthaseMetabolic engineering |
| spellingShingle | Gina M. Geiselman Xun Zhuang James Kirby Mary B. Tran-Gyamfi Jan-Philip Prahl Eric R. Sundstrom Yuqian Gao Nathalie Munoz Munoz Carrie D. Nicora Derek M. Clay Gabriella Papa Kristin E. Burnum-Johnson Jon K. Magnuson Deepti Tanjore Jeffrey M. Skerker John M. Gladden Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides Microbial Cell Factories Rhodotorula Mevalonate pathway Diterpene Geranylgeranyl pyrophosphate synthase Mutant farnesyl pyrophosphate synthase Metabolic engineering |
| title | Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides |
| title_full | Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides |
| title_fullStr | Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides |
| title_full_unstemmed | Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides |
| title_short | Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides |
| title_sort | production of ent kaurene from lignocellulosic hydrolysate in rhodosporidium toruloides |
| topic | Rhodotorula Mevalonate pathway Diterpene Geranylgeranyl pyrophosphate synthase Mutant farnesyl pyrophosphate synthase Metabolic engineering |
| url | https://doi.org/10.1186/s12934-020-1293-8 |
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