Multistep Biooxidation of 5-(Hydroxymethyl)furfural to 2,5-Furandicarboxylic Acid with H2O2 by Unspecific Peroxygenases.

5-(Hydroxymethyl)furfural (HMF) is a key platform chemical derived from renewable biomass sources, holding great potential as starting material for the synthesis of valuable compounds, thereby replacing petrochemical-derived counterparts. Among these valorised compounds, 2,5-furandicarboxylic acid (FDCA) has emerged as a versatile building block. Here we demonstrate the biocatalytic synthesis of FDCA from HMF via a one-pot three-step oxidative cascade performed via two operative steps under mild reaction conditions employing two unspecific peroxygenases (UPOs) using hydrogen peroxide as the only oxidant. The challenge of HMF oxidation by UPOs is the chemoselectivity of the first step, as one of the two possible oxidation products is only a poor substrate for further oxidation. The unspecific peroxygenase from Marasmius oreades (MorUPO) was found to oxidize 100 mM of HMF to 5-formyl-2-furoic acid (FFCA) with 95 % chemoselectivity. In the sequential one-pot cascade employing MorUPO (TON up to 13535) and the UPO from Agrocybe aegerita (AaeUPO, TON up to 7079), 100 mM of HMF were oxidized to FDCA reaching up to 99 % conversion and yielding 861 mg isolated pure crystalline FDCA, presenting the first example of a gram scale biocatalytic synthesis of FDCA involving UPOs.

Peroxygenase-Catalyzed Allylic Oxidation Unlocks Telescoped Synthesis of (1S,3R)-3-Hydroxycyclohexanecarbonitrile.

The unmatched chemo-, regio-, and stereoselectivity of enzymes renders them powerful catalysts in the synthesis of chiral active pharmaceutical ingredients (APIs). Inspired by the discovery route toward the LPA1-antagonist BMS-986278, access to the API building block (1S,3R)-3-hydroxycyclohexanecarbonitrile was envisaged using an ene reductase (ER) and alcohol dehydrogenase (ADH) to set both stereocenters. Starting from the commercially available cyclohexene-1-nitrile, a C-H oxyfunctionalization step was required to introduce the ketone functional group, yet several chemical allylic oxidation strategies proved unsuccessful. Enzymatic strategies for allylic oxidation are underdeveloped, with few examples on selected substrates with cytochrome P450s and unspecific peroxygenases (UPOs). In this case, UPOs were found to catalyze the desired allylic oxidation with high chemo- and regioselectivity, at substrate loadings of up to 200 mM, without the addition of organic cosolvents, thus enabling the subsequent ER and ADH steps in a three-step one-pot cascade. UPOs even displayed unreported enantioselective oxyfunctionalization and overoxidation of the substituted cyclohexene. After screening of enzyme panels, the final product was obtained at titers of 85% with 97% ee and 99% de, with a substrate loading of 50 mM, the ER being the limiting step. This synthetic approach provides the first example of a three-step, one-pot UPO-ER-ADH cascade and highlights the potential for UPOs to catalyze diverse enantioselective allylic hydroxylations and oxidations that are otherwise difficult to achieve.

Enantioselective High-Throughput Assay Showcased for the Identification of (R)- as well as (S)-Selective Unspecific Peroxygenases for C-H Oxidation.

Identifying (bio)catalysts displaying high enantio-/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Herein, a high-throughput, stereoselective screening assay is reported that gives information on enantioselectivity, stereopreference and activity as showcased for peroxygenase-catalyzed hydroxylation. The assay is based on spectrophotometric analysis of the simultaneous formation of NAD(P)H from the alcohol dehydrogenase catalyzed enantioselective oxidation of the sec-alcohol product formed in the peroxygenase reaction. The assay was applied to investigate a library comprising 44 unspecific peroxygenases (UPOs) containing 25 UPOs not reported yet. Thereby, previously non-described wild-type UPOs displaying (S)- as well as (R)-stereoselectivity for the hydroxylation of representative model substrates were identified, reaching up to 98 % ee for the (R)- and 94 % ee for the (S)-enantiomer. Homology models with concomitant docking studies indicated the structural reason for the observed complementary stereopreference.

From linoleic acid to hexanal and hexanol by whole cell catalysis with a lipoxygenase, hydroperoxide lyase and reductase cascade in Komagataella phaffii.

Green leaf volatiles (GLVs) cover a group of mainly C6-and C9-aldehydes, -alcohols and -esters. Their name refers to their characteristic herbal and fruity scent, which is similar to that of freshly cut grass or vegetables. Lipoxygenases (LOXs) catalyze the peroxidation of unsaturated fatty acids. The resulting hydroperoxy fatty acids are then cleaved into aldehydes and oxo acids by fatty acid hydroperoxide lyases (HPLs). Herein, we equipped the yeast Komagataella phaffii with recombinant genes coding for LOX and HPL, to serve as a biocatalyst for GLV production. We expressed the well-known 13S-specific LOX gene from Pleurotus sapidus and a compatible HPL gene from Medicago truncatula. In bioconversions, glycerol induced strains formed 12.9 mM hexanal using whole cells, and 8 mM hexanol was produced with whole cells induced by methanol. We applied various inducible and constitutive promoters in bidirectional systems to influence the final ratio of LOX and HPL proteins. By implementing these recombinant enzymes in Komagataella phaffii, challenges such as biocatalyst supply and lack of product specificity can finally be overcome.