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Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast
Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724057/ https://www.ncbi.nlm.nih.gov/pubmed/23898325 http://dx.doi.org/10.3389/fmicb.2013.00200 |
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author | Misra, Ashish Conway, Matthew F. Johnnie, Joseph Qureshi, Tabish M. Lige, Bao Derrick, Anne M. Agbo, Eddy C. Sriram, Ganesh |
author_facet | Misra, Ashish Conway, Matthew F. Johnnie, Joseph Qureshi, Tabish M. Lige, Bao Derrick, Anne M. Agbo, Eddy C. Sriram, Ganesh |
author_sort | Misra, Ashish |
collection | PubMed |
description | Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo (13)C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. |
format | Online Article Text |
id | pubmed-3724057 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-37240572013-07-29 Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast Misra, Ashish Conway, Matthew F. Johnnie, Joseph Qureshi, Tabish M. Lige, Bao Derrick, Anne M. Agbo, Eddy C. Sriram, Ganesh Front Microbiol Microbiology Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo (13)C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. Frontiers Media S.A. 2013-07-26 /pmc/articles/PMC3724057/ /pubmed/23898325 http://dx.doi.org/10.3389/fmicb.2013.00200 Text en Copyright © Misra, Conway, Johnnie, Qureshi, Derrick, Agbo and Sriram. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. |
spellingShingle | Microbiology Misra, Ashish Conway, Matthew F. Johnnie, Joseph Qureshi, Tabish M. Lige, Bao Derrick, Anne M. Agbo, Eddy C. Sriram, Ganesh Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast |
title | Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast |
title_full | Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast |
title_fullStr | Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast |
title_full_unstemmed | Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast |
title_short | Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast |
title_sort | metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724057/ https://www.ncbi.nlm.nih.gov/pubmed/23898325 http://dx.doi.org/10.3389/fmicb.2013.00200 |
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