Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with HO as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating HO that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other HO generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.

The bacterial decarboxylase (AMDase) catalyzes the enantioselective decarboxylation of prochiral arylmalonates with high enantioselectivity. Although this reaction would provide a highly sustainable synthesis of active pharmaceutical compounds such as flurbiprofen or naproxen, competing spontaneous decarboxylation has so far prevented the catalytic application of AMDase. Here, we report on reaction engineering and an alternate protection group strategy for the synthesis of these compounds that successfully suppresses the side reaction and provides pure arylmalonic acids for subsequent enzymatic conversion. Protein engineering increased the activity of the synthesis of the (S)‐ and (R)‐enantiomers of flurbiprofen. These results demonstrated the importance of synergistic effects in the optimization of this decarboxylase. The asymmetric synthesis of both enantiomers in high optical purity (>99 %) and yield (>90 %) can be easily integrated into existing industrial syntheses of flurbiprofen, thus providing a sustainable method for the production of this important pharmaceutical ingredient.

Teaching old dogs new tricks: Alcohol dehydrogenases (ADHs) may be established redox biocatalysts but they still are good for a few surprises. ADHs can be used to oxidize aldehydes, and this was demonstrated by the oxidative dynamic kinetic resolution of profens. In the presence of a suitable cofactor regeneration system, this reaction can occur with high selectivity.

Dicarbalkoxyketenes 8a and 8b are obtained by photolysis of diazo esters 7 in cryogenic matrices or by FVT of 7 followed by matrix isolation. The photochemical Wolff rearrangement of diazomalonates 1 in Ar matrix at temperatures as low as 6.5 K produces alkoxy(alkoxycarbonyl)ketenes 3, also obtained by FVT of 1. Photolysis of 1 in CO matrix at ≥6.5 K produces the dicarbalkoxyketenes 8. Ketenes 3, 8, and 12 react with pyridine above 40 K to produce ketene−pyridine ylides (zwitterions) 9, 10, and 13, respectively.

Biocatalytic asymmetric reduction of ethyl-4,4,4-trifluoroacetoacetate under water-deficient reaction conditions using a "smart cosubstrate" 1,4-butanediol was demonstrated up to a 2 L scale. Substrate concentrations of 100 g/L were applied by using half-molar equivalent of 1,4-butanediol in methyl-tert-butylether (MTBE). Using this approach, full conversion of ethyl-4,4,4-trifluoroacetoacetate to the corresponding (S)-alcohol with an excellent enantiomeric excess (ee) of ≥99% was accomplished in 5 days. 150 g of isolated enantiopure product with high purity (94%) was obtained.