TY - JOUR
T1 - Semi-rational Engineering of a Promiscuous Fatty Acid Hydratase for Alteration of Regioselectivity
AU - Zhang, Yan
AU - Breum, Niels Mikkel Dyrby
AU - Schubert, Sune
AU - Hashemi, Negin
AU - Kyhnau, Rikke
AU - Knauf, Marius Sandholt
AU - Mathialakan, Masuthan
AU - Takeuchi, Michiki
AU - Kishino, Shigenobu
AU - Ogawa, Jun
AU - Kristensen, Peter
AU - Guo, Zheng
AU - Eser, Bekir Engin
N1 - © 2021 Wiley-VCH GmbH.
PY - 2022
Y1 - 2022
N2 - Fatty acid hydratases (FAHs) catalyze regio- and stereo-selective hydration of unsaturated fatty acids to produce hydroxy fatty acids. Fatty acid hydratase-1 (FA-HY1) from Lactobacillus Acidophilus is the most promiscuous and regiodiverse FAH identified so far. Here, we engineered binding site residues of FA-HY1 (S393, S395, S218 and P380) by semi-rational protein engineering to alter regioselectivity. Although it was not possible to obtain a completely new type of regioselectivity with our mutant libraries, a significant shift of regioselectivity was observed towards cis-5, cis-8, cis-11, cis-14, cis-17-eicosapentaenoic acid (EPA). We identified mutants (S393/S395 mutants) with excellent regioselectivity, generating a single hydroxy fatty acid product from EPA (15-OH product), which is advantageous from application perspective. This result is impressive given that wild-type FA-HY1 produces a mixture of 12-OH and 15-OH products at 63 : 37 ratio (12-OH : 15-OH). Moreover, our results indicate that native FA-HY1 is at its limit in terms of promiscuity and regiospecificity, thus it may not be possible to diversify its product portfolio with active site engineering. This behavior of FA-HY1 is unlike its orthologue, fatty acid hydratase-2 (FA-HY2; 58 % sequence identity to FA-HY1), which has been shown earlier to exhibit significant promiscuity and regioselectivity changes by a few active site mutations. Our reverse engineering from FA-HY1 to FA-HY2 further demonstrates this conclusion.
AB - Fatty acid hydratases (FAHs) catalyze regio- and stereo-selective hydration of unsaturated fatty acids to produce hydroxy fatty acids. Fatty acid hydratase-1 (FA-HY1) from Lactobacillus Acidophilus is the most promiscuous and regiodiverse FAH identified so far. Here, we engineered binding site residues of FA-HY1 (S393, S395, S218 and P380) by semi-rational protein engineering to alter regioselectivity. Although it was not possible to obtain a completely new type of regioselectivity with our mutant libraries, a significant shift of regioselectivity was observed towards cis-5, cis-8, cis-11, cis-14, cis-17-eicosapentaenoic acid (EPA). We identified mutants (S393/S395 mutants) with excellent regioselectivity, generating a single hydroxy fatty acid product from EPA (15-OH product), which is advantageous from application perspective. This result is impressive given that wild-type FA-HY1 produces a mixture of 12-OH and 15-OH products at 63 : 37 ratio (12-OH : 15-OH). Moreover, our results indicate that native FA-HY1 is at its limit in terms of promiscuity and regiospecificity, thus it may not be possible to diversify its product portfolio with active site engineering. This behavior of FA-HY1 is unlike its orthologue, fatty acid hydratase-2 (FA-HY2; 58 % sequence identity to FA-HY1), which has been shown earlier to exhibit significant promiscuity and regioselectivity changes by a few active site mutations. Our reverse engineering from FA-HY1 to FA-HY2 further demonstrates this conclusion.
KW - Biocatalysis
KW - enzyme engineering
KW - fatty acid hydratases
KW - hydroxy fatty acids
KW - regioselectivity
UR - http://www.scopus.com/inward/record.url?scp=85122156335&partnerID=8YFLogxK
U2 - 10.1002/cbic.202100606
DO - 10.1002/cbic.202100606
M3 - Journal article
C2 - 34929055
SN - 1439-4227
VL - 23
SP - e202100606
JO - ChemBioChem
JF - ChemBioChem
IS - 4
M1 - e202100606
ER -