Authors
Meng, H., Schwanemann, T., Lipa, M. K., Michel, C. V., Xia, J., Blank, L. M.
Abstract
3-Hydroxy fatty acids (3-HFAs) are versatile intermediates for bio-based polymers, fuels, and surfactants, and are advancing circular economy manufacturing. We built an acyl-CoA ligase-deficient chassis of Pseudomonas putida KT2440, thereby blocking 3-HFA activation and preventing its use as carbon source. This strategy decoupled synthesis from catabolism and enabled 3-HFA accumulation. We then compared two different routes for modifying the free 3-HFA composition. 1. In a direct route, overexpressing native PhaG produced C8, C10, C12, and C14 3-HFAs, achieving a total titer of 0.73 g/L in shake flasks. Functional analyses under our tested conditions support a re-assignment of PhaG: rather than acting primarily as a 3-hydroxyacyl-ACP:CoA transacylase, it functions mainly as a thioesterase, liberating free 3-HFAs from hydroxyacyl-ACP. 2. In an indirect route, overexpressing RhlA variants generated hydroxyalkanoyl-alkanoates (HAAs) that were converted to free 3-HFAs by endogenous esterase(s): RhlA from Pseudomonas aeruginosa PAO1 favored C8-C12 and yielded 0.47 g/L 3-HFAs, whereas RhlA from Burkholderia plantarii PG1 favored C10-C14 with 0.14 g/L, of which 80% was C14. Finally, we demonstrated process feasibility by up-scaling the PhaG pathway in a stirred-tank reactor. These results establish modular, stable, chassis-compatible routes for tailoring 3-HFA chain-length distributions, thereby providing a foundation for scalable, bio-based monomer supply in a circular economy.
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bioRxiv
The authors list and abstract were imported from bioRxiv on 06 Nov 2025.
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