Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage
Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel a...
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PeerJ Inc.
2015-12-01
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| Online Access: | https://peerj.com/articles/1468.pdf |
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| author | Janice S. Chen Brendan Colón Brendon Dusel Marika Ziesack Jeffrey C. Way Joseph P. Torella |
| author_facet | Janice S. Chen Brendan Colón Brendon Dusel Marika Ziesack Jeffrey C. Way Joseph P. Torella |
| author_sort | Janice S. Chen |
| collection | DOAJ |
| description | Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into β-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous β-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha. |
| first_indexed | 2024-03-09T06:39:01Z |
| format | Article |
| id | doaj.art-3030a1ff9c234554abdb097000cdfa69 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T06:39:01Z |
| publishDate | 2015-12-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj.art-3030a1ff9c234554abdb097000cdfa692023-12-03T10:54:01ZengPeerJ Inc.PeerJ2167-83592015-12-013e146810.7717/peerj.1468Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storageJanice S. Chen0Brendan Colón1Brendon Dusel2Marika Ziesack3Jeffrey C. Way4Joseph P. Torella5Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA, United StatesDepartment of Systems Biology, Harvard Medical School, Boston, MA, United StatesWyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA, United StatesDepartment of Systems Biology, Harvard Medical School, Boston, MA, United StatesWyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA, United StatesDepartment of Systems Biology, Harvard Medical School, Boston, MA, United StatesRalstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into β-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous β-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha.https://peerj.com/articles/1468.pdf-oxidationAcyl-CoA ligaseRalstoniaBiofuelMetabolic engineering |
| spellingShingle | Janice S. Chen Brendan Colón Brendon Dusel Marika Ziesack Jeffrey C. Way Joseph P. Torella Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage PeerJ -oxidation Acyl-CoA ligase Ralstonia Biofuel Metabolic engineering |
| title | Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage |
| title_full | Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage |
| title_fullStr | Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage |
| title_full_unstemmed | Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage |
| title_short | Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage |
| title_sort | production of fatty acids in ralstonia eutropha h16 by engineering β oxidation and carbon storage |
| topic | -oxidation Acyl-CoA ligase Ralstonia Biofuel Metabolic engineering |
| url | https://peerj.com/articles/1468.pdf |
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