Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway
Biosynthesis of itaconic acid occurs through decarboxylation of the TCA cycle intermediate cis-aconitate. Engineering of efficient itaconate producers often requires elimination of the highly active isocitrate dehydrogenase to conserve cis-aconitate, leading to 2-ketoglutarate auxotrophy in the prod...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-12-01
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| Series: | Metabolic Engineering Communications |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214030121000304 |
| _version_ | 1819177486185922560 |
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| author | Ken W. Lu Chris T. Wang Hengray Chang Ryan S. Wang Claire R. Shen |
| author_facet | Ken W. Lu Chris T. Wang Hengray Chang Ryan S. Wang Claire R. Shen |
| author_sort | Ken W. Lu |
| collection | DOAJ |
| description | Biosynthesis of itaconic acid occurs through decarboxylation of the TCA cycle intermediate cis-aconitate. Engineering of efficient itaconate producers often requires elimination of the highly active isocitrate dehydrogenase to conserve cis-aconitate, leading to 2-ketoglutarate auxotrophy in the producing strains. Supplementation of glutamate or complex protein hydrolysate then becomes necessary, often in large quantities, to support the high cell density desired during itaconate fermentation and adds to the production cost. Here, we present an alternative approach to overcome the glutamate auxotrophy in itaconate producers by synthetically introducing the Weimberg pathway from Burkholderia xenovorans for 2-ketoglutarate biosynthesis. Because of its independence from natural carbohydrate assimilation pathways in Escherichia coli, the Weimberg pathway is able to provide 2-ketoglutarate using xylose without compromising the carbon flux toward itaconate. With xylose concentration carefully tuned to minimize excess 2-ketoglutarate flux in the stationary phase, the final strain accumulated 20 g/L of itaconate in minimal medium from 18 g/L of xylose and 45 g/L of glycerol. Necessity of the recombinant Weimberg pathway for growth also allowed us to maintain multi-copy plasmids carrying in operon the itaconate-producing genes without addition of antibiotics. Use of the Weimberg pathway for growth restoration is applicable to other production systems with disrupted 2-ketoglutarate synthesis. |
| first_indexed | 2024-12-22T21:27:25Z |
| format | Article |
| id | doaj.art-514abe374e4641f089ea5a200a1a0ce6 |
| institution | Directory Open Access Journal |
| issn | 2214-0301 |
| language | English |
| last_indexed | 2024-12-22T21:27:25Z |
| publishDate | 2021-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Metabolic Engineering Communications |
| spelling | doaj.art-514abe374e4641f089ea5a200a1a0ce62022-12-21T18:12:00ZengElsevierMetabolic Engineering Communications2214-03012021-12-0113e00190Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathwayKen W. Lu0Chris T. Wang1Hengray Chang2Ryan S. Wang3Claire R. Shen4Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, TaiwanDepartment of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, TaiwanDepartment of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, TaiwanDepartment of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, TaiwanCorresponding author.; Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, TaiwanBiosynthesis of itaconic acid occurs through decarboxylation of the TCA cycle intermediate cis-aconitate. Engineering of efficient itaconate producers often requires elimination of the highly active isocitrate dehydrogenase to conserve cis-aconitate, leading to 2-ketoglutarate auxotrophy in the producing strains. Supplementation of glutamate or complex protein hydrolysate then becomes necessary, often in large quantities, to support the high cell density desired during itaconate fermentation and adds to the production cost. Here, we present an alternative approach to overcome the glutamate auxotrophy in itaconate producers by synthetically introducing the Weimberg pathway from Burkholderia xenovorans for 2-ketoglutarate biosynthesis. Because of its independence from natural carbohydrate assimilation pathways in Escherichia coli, the Weimberg pathway is able to provide 2-ketoglutarate using xylose without compromising the carbon flux toward itaconate. With xylose concentration carefully tuned to minimize excess 2-ketoglutarate flux in the stationary phase, the final strain accumulated 20 g/L of itaconate in minimal medium from 18 g/L of xylose and 45 g/L of glycerol. Necessity of the recombinant Weimberg pathway for growth also allowed us to maintain multi-copy plasmids carrying in operon the itaconate-producing genes without addition of antibiotics. Use of the Weimberg pathway for growth restoration is applicable to other production systems with disrupted 2-ketoglutarate synthesis.http://www.sciencedirect.com/science/article/pii/S2214030121000304Weimberg pathwayGlutamate auxotrophItaconic acid |
| spellingShingle | Ken W. Lu Chris T. Wang Hengray Chang Ryan S. Wang Claire R. Shen Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway Metabolic Engineering Communications Weimberg pathway Glutamate auxotroph Itaconic acid |
| title | Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway |
| title_full | Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway |
| title_fullStr | Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway |
| title_full_unstemmed | Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway |
| title_short | Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway |
| title_sort | overcoming glutamate auxotrophy in escherichia coli itaconate overproducer by the weimberg pathway |
| topic | Weimberg pathway Glutamate auxotroph Itaconic acid |
| url | http://www.sciencedirect.com/science/article/pii/S2214030121000304 |
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