BUSCADOR RECOLECTA

Background: Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification
applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available,
and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose
cultures.
Results: Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the
extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography
coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified.
Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar
cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two
lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the
poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only
slightly overproduced (14–16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes
(10–14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3
(MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins,
but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes
were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less
abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic
substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative
to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an
increase of sugar reducing ends.
Conclusions: Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic
substrates. One laccase occupied the first position in both secretomes, and three more were overproduced
together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal
of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results., This work was supported by the INDOX (KBBE-2013-613549) EU project, the BIO2014-56388-R, AGL2014-53730-R, and AGL2011-30495 projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the ProteoRed platform of the Spanish Institute of Health Carlos III (ISCIII). The work conducted by the US DOE JGI is supported by the Office of Science of the US DOE under contract number DE-AC02-05CH11231. FJR-D thanks a Ramón y Cajal contract of the Spanish MINECO, and JR thanks a contract of the CSIC project 201440E097.

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 H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 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 D3, 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., This work has been funded by the INDOX European project (KBBE-2013-7-613549), together with the BIO2014-56388-R and AGL2014-53730-R projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the BBI JU project EnzOx2 (H2020-BBI-PPP-2015-2-720297). The work conducted
by the US DOE JGI was supported by the Office of Science of the US DOE under contract number DE-AC02-05CH11231. The authors thank other members of their groups at CIB-CSIC, Novozymes, Technical University of Dresden, JenaBios, University of Naples
Federico II, Setas Kimya Sanayy, Wageningen University & Research, Anaxomics, Chiracon, BOKU, Delft University of Technology, INRABBF, Biopolis, Cheminova, CLEA, Solvay, IRNAS-CSIC and ICP-CSIC for their significant contributions to the results presented., Peer Reviewed

The recently discovered unspecific peroxygenases (UPOs) from the ascomycetes Chaetomium globosum and Humicola insolens were capable of selectively hydroxylating isophorone to 4-hydroxyisophorone (4HIP) and 4-ketoisophorone (4KIP), which are substrates of interest for the pharmaceutical and flavor-and-fragrance sectors. The model UPO from the basidiomycete Agrocybe aegerita was less regioselective, forming 7-hydroxyisophorone (and 7-formylisophorone) in addition to 4HIP. However, it was the most stereoselective UPO yielding the S-enantiomer of 4HIP with 88% ee. Moreover, using H. insolens UPO full kinetic resolution of racemic HIP was obtained within only 15 min, with >75% recovery of the R-enantiomer. Surprisingly, the UPOs from two other basidiomycetes, Marasmius rotula and Coprinopsis cinerea, failed to transform isophorone. The different UPO selectivities were rationalized by computational simulations, in which isophorone and 4HIP were diffused into the enzymes using the adaptive PELE software, and the distances from heme-bound oxygen in H2O2-activated enzyme to different substrate atoms, and the corresponding binding energies were analyzed. Interestingly, for process upscaling, full conversion of 10 mM isophorone was achieved with H. insolens UPO within nine hours, with total turnover numbers up to 5500. These biocatalysts, which only require H2O2 for activation, may represent a novel, simple and environmentally-friendly route for the production of isophorone derivatives., This work was supported by the EnzOx2 (H2020-BBI-PPP-2015-2-1-720297) EU-project, the AGL2014-53730-R (BIORENZYMERY) and CTQ2016-79138-R projects of the Spanish MINECO (co-financed by FEDER) and the CSIC (201740E071)
project. Novozymes (Bagsvaerd, Denmark) is acknowledged for providing samples of rCciUPO and rHinUPO., Peer Reviewed