Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs to date with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrate various double bond positions. We reveal three active-site residues that play remarkable role in directing substrate specificity and regioselectivity of hydration. When these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, we observed a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards w-end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA regioisomers (10-OH:13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C18 PUFA substrates. In addition, TM-FA-HY2 converted EPA to its 12-hydroxy product with high conversion at preparative scale. Furthermore, we demonstrated microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design enabling production of diverse HFAs for various applications from polymer industry to medical field.
- Protein engineering
- Enzyme catalysis
- Fatty acid hydratase
- Hydroxy fatty acid
Eser, B. E., Poborsky, M., Dai, R., Kishino, S., Ljubic, A., Takeuchi, M., Jacobsen, C., Ogawa, J., Kristensen, P., & Guo, Z. (2020). Rational Engineering of Hydratase from Lactobacillus Acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity. Chembiochem, 21(4), 550-563. https://doi.org/10.1002/cbic.201900389