Rational Engineering of Hydratase from Lactobacillus Acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

Abstract

<div>Enzymatic conversion of abundant fatty acids (FAs) through fatty acid hydratases (FAHs) presents an environment-friendly and efficient route for production of high-value hydroxy fatty acids (HFAs). However, a limited diversity was achieved among HFAs to date with respect to chain length and hydroxy group position, due to high substrate- and regio-selectivity of hydratases. In this study, we compared two highly similar FAHs from <i>Lactobacillus acidophilus</i>: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilize 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 residues in FA-HY1, we observe a significant expansion of substrate scope and distinct shift and enhancement in hydration of double bonds towards omega-end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2; T391S/H393S/I378P) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA product regioisomers (10-OH:13-OH) from 99:1 to 12:88. Although kcat values are still low in comparison to wild-type FA-HY1, TM-FA-HY2 exhibited about 60-fold increase in catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) compared to wild-type FA-HY2. Important changes in regioselectivity were also observed with mutant enzymes for arachidonic acid and C18 PUFAs. In addition, TM-FA-HY2 variant exhibited high conversion rates for <i>cis</i>-5, <i>cis</i>-8, <i>cis</i>-11,<i> cis</i>-14, <i>cis</i>-17-eicosapentaenoic acid (EPA) and <i>cis</i>-8, <i>cis</i>-11, <i>cis</i>-14-eicosatrienoic acid (ETA) at preparative scale and enabled isolation of 12-hydroxy products with moderate yields. Furthermore, we demonstrated the potential of microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design and for efficient conversion of FA sources into diverse range of HFAs with high potential for various applications from polymer industry to medical field.</div><div><br></div>

Chemoenzymatic Cascade Conversion of Linoleic Acid into a Secondary Fatty Alcohol Using a Combination of 13S-Lipoxygenase, Chemical Reduction, and a Photo-Activated Decarboxylase

“…Transformation of Linoleic Acid (1) into 13-HOD (17). 13-HOD (17) was reported to serve as a bioactive lipid mediator that regulates multiple signaling processes in vivo. 55 However, this compound was very difficult to produce by using fatty acid double bond hydratases or P450 monooxygenases.…”

“…55 However, this compound was very difficult to produce by using fatty acid double bond hydratases or P450 monooxygenases. Therefore, the conversion of linoleic acid (1) into 13-HOD (17) by LOXs (Scheme 2) was examined. Although the transformation of linoleic acid (1) into 13-HPOD ( 16) by using isolated LOX enzymes was recently reported, 44,46,47,51 the whole-cell transformation of linoleic acid into 13-HPOD has rarely been investigated.…”

“…A small amount of 13-HPOD ( 16) was reduced to 13-HOD ( 17) by an intracellular reduction system of E. coli (e.g., glutathione peroxidase). The addition of TCEP or cysteine as a reducing agent in the reaction medium led to the formation of 13-HOD (17) to 85 mM (Figures 1A and S2). The biotransformation rate at t < 0.5 h reached 640 U/g dry cells.…”

“…The whole-cell biotransformation by the Cv-FAP-expressing E. coli cells was conducted as previously reported. 31 Briefly, the bioconversion was begun by adding 15 mM 13S-hydroxy-(9Z,11E)octadecadienoic acid (13-HOD) (17) under blue light into a 50 mM Tris-HCl buffer (pH 8.0) containing the recombinant cells (cell concentration: 7.2 g dry cells/L). The shaking speed and temperature were 250 rpm and 37 °C, respectively.…”

“…Several emerging ideas for the valorization of renewable oils and fatty acids were recently reported. − The unsaturated fatty acids (e.g., linoleic acid) were transformed into hydroxy fatty acids through hydration by fatty acid hydratases − or oxygenation by P450 monooxygenases , and lipoxygenases (LOXs). − The hydroxy fatty acids were further converted into keto fatty acids by long-chain secondary alcohol dehydrogenases. − For example, linoleic acid ( 1 ) was transformed into 10-keto-12­( Z )-octadecenoic acid ( 2 ) and 13-keto-9­( Z )-octadecenoic acid ( 3 ) via 10-hydroxy-12­( Z )-octadecenoic acid and 13-hydroxy-9­( Z )-octadecenoic acid, respectively ,,, (Scheme ). The keto fatty acids could be further converted into amino fatty acids ( 4 and 5 ) by transaminases , or into medium-chain fatty acids [e.g., 9-hydroxynonanoic acid ( 6 ) and non-3­( Z )-enoic acid ( 7 ) or 12-hydroxydodec-9­( Z )-enoic acid ( 8 ) and n -hexanoic acid ( 9 )], via ester formation by Baeyer–Villiger monooxygenase (BVMO). ,,,, Linoleic acid was also converted into (6 Z ,9 Z )-heptadecadiene ( 10 ) and 5,8-dihydroxy-(9 Z ,12 Z )-octadecadienoic acid ( 11 ) by the photo-activated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) − and the linoleate 5,8-diol synthases from Aspergillus nidulans, respectively (Scheme ).…”