Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae
Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engi...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Springer-Verlag
2017
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| Online Access: | http://hdl.handle.net/1721.1/107177 https://orcid.org/0000-0001-6909-4568 |
| _version_ | 1811075067540406272 |
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| author | Formenti, Luca Riccardo Phon, Too Heng Nielsen, Michael Lynge Lantz, Anna Eliasson Kielland-Brandt, Morten C. Carlsen, Simon Zhou, Kang Stephanopoulos, Gregory Parayil Kumaran, Ajikumar |
| author2 | Massachusetts Institute of Technology. Department of Chemical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Chemical Engineering Formenti, Luca Riccardo Phon, Too Heng Nielsen, Michael Lynge Lantz, Anna Eliasson Kielland-Brandt, Morten C. Carlsen, Simon Zhou, Kang Stephanopoulos, Gregory Parayil Kumaran, Ajikumar |
| author_sort | Formenti, Luca Riccardo |
| collection | MIT |
| description | Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe–4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U–[superscript 13]C[subscript 6] glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron–sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron–sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron–sulfur cluster proteins in its cytosol. |
| first_indexed | 2024-09-23T10:00:07Z |
| format | Article |
| id | mit-1721.1/107177 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T10:00:07Z |
| publishDate | 2017 |
| publisher | Springer-Verlag |
| record_format | dspace |
| spelling | mit-1721.1/1071772022-09-30T18:14:45Z Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae Formenti, Luca Riccardo Phon, Too Heng Nielsen, Michael Lynge Lantz, Anna Eliasson Kielland-Brandt, Morten C. Carlsen, Simon Zhou, Kang Stephanopoulos, Gregory Parayil Kumaran, Ajikumar Massachusetts Institute of Technology. Department of Chemical Engineering Carlsen, Simon Zhou, Kang Stephanopoulos, Gregory Parayil Kumaran, Ajikumar Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe–4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U–[superscript 13]C[subscript 6] glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron–sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron–sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron–sulfur cluster proteins in its cytosol. National Institutes of Health (grant no. 1-R01-GM085323-01A1) Denmark. Technical University Singapore-MIT Alliance 2017-03-03T22:49:46Z 2017-03-03T22:49:46Z 2013-05 2013-03 2016-05-23T12:09:37Z Article http://purl.org/eprint/type/JournalArticle 0175-7598 1432-0614 http://hdl.handle.net/1721.1/107177 Carlsen, Simon, Parayil Kumaran Ajikumar, Luca Riccardo Formenti, Kang Zhou, Too Heng Phon, Michael Lynge Nielsen, Anna Eliasson Lantz, Morten C. Kielland-Brandt, and Gregory Stephanopoulos. “Heterologous Expression and Characterization of Bacterial 2-C-Methyl-d-Erythritol-4-Phosphate Pathway in Saccharomyces Cerevisiae.” Applied Microbiology and Biotechnology 97, no. 13 (May 1, 2013): 5753–5769. https://orcid.org/0000-0001-6909-4568 en http://dx.doi.org/10.1007/s00253-013-4877-y Applied Microbiology and Biotechnology Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ Springer-Verlag Berlin Heidelberg application/pdf Springer-Verlag Springer-Verlag |
| spellingShingle | Formenti, Luca Riccardo Phon, Too Heng Nielsen, Michael Lynge Lantz, Anna Eliasson Kielland-Brandt, Morten C. Carlsen, Simon Zhou, Kang Stephanopoulos, Gregory Parayil Kumaran, Ajikumar Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae |
| title | Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae |
| title_full | Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae |
| title_fullStr | Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae |
| title_full_unstemmed | Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae |
| title_short | Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae |
| title_sort | heterologous expression and characterization of bacterial 2 c methyl d erythritol 4 phosphate pathway in saccharomyces cerevisiae |
| url | http://hdl.handle.net/1721.1/107177 https://orcid.org/0000-0001-6909-4568 |
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