Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L−1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2; the Spanish projects PID2019-106166RB-100-OXYWAVE, PID2020-118968RB-100-LILI, PID2021-123332OB-C21 and PID2019-107098RJ-I00, funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/; the “Comunidad de Madrid” Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CM; the Generalitat Valenciana projects CIPROM/2021/079-PROMETEO and SEJI/2020/007; and the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. P.G.d.S. thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and the Ministry of Science and Innovation for her contract as part of the PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR. D.G.-P. thanks Juan de la Cierva Incorporación contract Ref. No.: IJC2020-043725-I, funded by MCIN/AEI/10.13039/501100011033, and the EU NextGenerationEU/PRTR program. K.Ś. thanks to Ministerio de Ciencia e Innovación and Fondo Social Europeo for a Ramón y Cajal contract (Ref. RYC2020-030596-I). We thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline

Biotransformations of industrial interest catalyzed by fungal peroxygenases

In this Thesis, the selective synthesis of different products of industrial interest such as antioxidants derived from trans-stilbene and other stilbenoids, flavour-and-fragrance additives derived from α-isophorone, and epoxides from unsaturated fatty acids (FA) and their methyl esters (FAME) catalyzed by fungal unspecific peroxygenases (UPOs, EC.1.11.2.1) was studied. With this purpose, several UPOs were tested, including those from the basidiomycetes Agrocybe aegerita (AaeUPO), Marasmius rotula (MroUPO) as well as the recombinant enzyme from Coprinopsis cinerea (rCciUPO) and those from the ascomycetes Chaetomium globosum (CglUPO) and the recombinant enzyme from Humicola insolens (rHinUPO). The first reaction studied was the oxygenation of trans-stilbene and other stilbenoids such as pinosylvin (Pin) and resveratrol (RSV). In trans-stilbene oxygenation, different reactivities were found depending on the UPO used. AaeUPO, MroUPO and rCciUPO selectively hydroxylated the aromatic rings to form the compound of interest 4,4′-dihydroxy-trans-stilbene (DHS). DHS is a RSV analogue whose preventive effects on cancer invasion and metastasis has recently been shown. The kinetic studies revealed AaeUPO as the most efficient enzyme with one and two orders of magnitude higher catalytic efficiencies (kcat/Km) for the first and second hydroxylation steps, respectively, compared to MroUPO and rCciUPO. Likewise, AaeUPO achieved the highest total turnover numbers (TTN) of up to 200 000. In addition, AaeUPO gave also the best results in Pin and RSV hydroxylation reactions with higher regioselectivity and substrate conversions. However, CglUPO failed to hydroxylate the aromatic ring and instead epoxidized the double bond of the alkenyl moiety forming trans-stilbene epoxide as the only product. True peroxygenative activity was demonstrated by incorporation of 18O from H218O2 in the oxidation products. The second reaction studied comprises the α-isophorone hydroxylation by UPOs to produce 4-hydroxyisophorone (4HIP) and 4-ketoisophorone (4KIP), which are flavor-and-fragrances molecules commonly used as additives and as intermediates in the synthesis of vitamins and carotenoids. rHinUPO and CglUPO selectively transformed the substrate (10 mM) in the compounds of interest attaining TTN of up to 5500. Interestingly, although AaeUPO was found to be less selective since this enzyme oxygenated positions 4 and 7 of α-isophorone, it was the only stereoselective enzyme producing the S-enantiomer of 4HIP with an enantiomeric excess of 88%. On the other hand, using the racemic 4HIP as substrate, a faster conversion of the S-enantiomer by rHinUPO and CglUPO was observed, leading to a kinetic resolution of the racemate with 60-75% recovery of the R-enantiomer. Surprisingly, MroUPO and rCciUPO failed to transform α-isophorone. These differences in regioselectivities could be explained by computational analysis carried out with adaptive PELE software, where differences in the distances between the substrates atoms and the oxo-heme of reactive Compound I and different binding energies were observed. The last reaction studied included the oxygenation of FAs and FAMEs to form valuable reactive epoxides. A series of mono- and poly-unsaturated FAs and FAMEs were transformed by MroUPO and CglUPO with high conversion yields, generally attaining better selectivity towards the desired epoxide with the latter enzyme. The differences were observed during oleic acid oxygenation reactions, where CglUPO showed one order of magnitude higher catalytic efficiency and higher TTN (8000) compared to MroUPO (4000). The regioselectivity observed is different from the previously described subterminal hydroxylation by the well-known rCciUPO and AaeUPO, which do not epoxidize unsaturated FAs. In conclusion, biotransformations using these novel and appealing biocatalysts would represent a potential more environmentally friendly alternative to chemical synthesis in the oxyfunctionalization reactions described during this Thesis