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Optimized oxidoreductases for medium and large scale industrial biotransformations

Final Report Summary - INDOX (Optimized oxidoreductases for medium and large scale industrial biotransformations)

Executive Summary:
Lignin-degrading basidiomycetes and related fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different coppercontaining oxidoreductases. Heme peroxidases comprise ligninolytic peroxidases and dyedecolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate peroxidases. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological potential due to their ability to catalyze a variety of regio- and stereoselective monooxygenase reactions with H2O2 as source of oxygen and final electron acceptor. Flavooxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical, respectively. The group of copper oxidoreductases also includes other H2O2 generating enzymes (copper-radical oxidases), together with classical laccases (multicopper oxidases) that are the oxidoreductases with the largest number of reported applications. However, the recently described lytic polysaccharide monooxygenases currently attract the highest interest, since they are able to break down (oxidatively) crystalline cellulose, a major bottleneck in lignocellulose biorefineries (together with lignin degradation). Interestingly, some flavin-containing dehydrogenases also play a key role "fueling" (directly or indirectly) electrons for polysaccharide monooxygenase activation.

Many of the above oxidoreductases have been engineered in INDOX to attain the required selectivity and catalytic efficiency and operational properties (stability included) using both rational design and directed evolution. Using ad hoc software and current computational capacities, it is possible to predict the substrate access to the active site (biophysical simulations) and the efficiency of the electron transfer (biochemical simulations) in enzyme screening and engineering, reducing in orders of magnitude the time of experimental work. The above is illustrated by a series of oxyfunctionalization and oxidation reactions (e.g. enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, chiral alcohols, furandicarboxylic acid, indigo dyes and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation) to be presented to the chemical sectors as successful case stories of the potential of different oxidoreductases in medium and large-scale industrial biotransformations.

Project Context and Objectives:
The INDOX Project is a collaborative research initiative funded by the European Commission 7th Framework Programme (FP7) and gathering together the expertise of sixteen participants from seven EU countries plus one partner from an EU candidate country. The final aim of INDOX is to provide relevant industrial case stories to demonstrate the efficacy of optimized oxidative biocatalysts on targeted reactions, compared to chemical conversion processes, and to establish the processes scalability, sustainability and cost-efficiency versus chemical conversions. The chemical industry (some specialties excluded) is not yet embracing enzymatic oxidation reactions to a significant extent primarily due to lack of biocatalysts with the required selectivity, availability and compatibility with the rigorous process conditions (high substrate concentrations, use of solvents, and strongly oxidative conditions). The above bottlenecks have been addressed in INDOX by enzyme engineering and process optimization using state-of-the-art technologies, as will be summarized in the next section.

Enzymes catalyze a huge amount of chemical reactions in living organisms, which take place under mild conditions compatible with life, and with exquisite substrate specificity. Nowadays, the availability of genetic engineering tools permits large-scale production of enzymes and other proteins at low cost by isolating (or synthesizing) the corresponding genes and introducing them into adequate expression hosts after their cloning in expression vectors. Simultaneously, protein engineering using rational and non-rational designs permits to adapt these enzymes to the industrial application conditions, and to increase their expression yields. This situation represented a real breakthrough for industrial biotechnology enabling the production of commercial enzymes at very low costs. However, most industrial enzymes commercialized by Novozymes and other biotechnology companies belong to the group of hydrolases, together with other enzymes occupying quantitatively less important industrial niches. Among them, enzymes catalyzing oxidation/reduction reactions - oxidoreductases - represent an environmentally-friendly alternative to harsh chemicals in different industrial processes that include oxidative transformations. Oxidoreductases still have a modest penetration in the chemical markets but their potential is very noteworthy, especially when considering the best known enzymes (such as horseradish peroxidase or laccase) together with the new oxidoreductase types recently described (such as peroxygenases and dye-decolorizing peroxidases, DyPs). Among these oxidoreductase types, those being "self-sufficient", in the sense that they are activated by easily available extracellular oxidizers (such as oxygen or peroxide) without the need for auxiliary proteins and expensive cosubstrates, have the highest potential as industrial biocatalysts. The latter is a characteristic of extracellular microbial oxidoreductases, and especially of those produced by fungi involved in the natural decay of lignocellulosic biomass, and humus turnover. Three types of secreted fungal oxidoreductases with great potential to catalyze reactions of industrial interest have been explored in INDOX, namely: heme-peroxidases/peroxygenases, flavo-oxidases, and copper-oxidoreductases. Multi-enzymatic and double-oxidation cascade reactions have also been considered to reduce the costs and increase the efficiency of the enzymatic processes, together with several other strategies.

Due to the characteristics described above, the members of the oxidoreductase groups explored in INDOX seemed especially well suited to perform two types of industrially-relevant reactions, namely oxidation of target aromatic and other substrates of interest, and oxyfunctionalization of target aliphatic and aromatic molecules with very low chemical reactivity. In this context, the following selected industrial target reactions formed the basis for the screening and optimization of new biocatalysts within the INDOX project: i) Intermediates for agrochemicals and APIs (active pharmaceutical ingredients) and flavors/fragrances, including benzylic, aromatic and terpene oxyfunctionalizations, chiral chemicals, ionones and epoxidation products; ii) Precursors for specialty polymers including 2,5-hydroxymethylfurfural (HMF) products, and di-alcohols and hydroxy/di-acids from alkanes and fatty acids, and functionalized plant polymers (including oxidized cellulose and lignin); and iii) Intermediates for dye-stuff production including phenolic and amine derivatives, indole and indole derivatives, and aniline polymers. The selection of the aforementioned reactions has been based on proof of reaction at laboratory scale, industrial interest and possibility for broadening the application field to similar substrate classes.

The project flow has comprised the following steps: i) Recovery of selective oxidative biocatalysts for target reactions from the groups of heme-peroxidases/ peroxygenases, flavo-oxidases and copper-oxidoreductases showing high substrate conversion under laboratory conditions, from fungal genomes and other sources; ii) Improvement of their oxidation activity and stability by protein engineering using rational design (based on structural-functional information), directed evolution and hybrid approaches, combined with computer simulations, to fulfill the operational and catalytic conditions required by the chemical industry; and iii) Optimization of reaction conditions (including several immobilization technologies and novel enzymatic cascade reactions). Finally, several processes were selected for evaluation of cost efficiency compared to chemical processing.

According to the general concept outlined above, the following specific S&T objectives were defined, to be developed in the different work packages of the INDOX work plan:

1. To identify oxidoreductases of interest for target biotransformations by genome, transcriptome and/or culture mining, and characterize them, together with already available enzymes, to establish relationships between the catalytic properties and the molecular structure characteristics.

2. To tailor the selected oxidoreductases to obtain ad hoc biocatalysts with optimized properties for oxidation, oxyfunctionalization, chiral synthesis and polymerization reactions, using protein engineering and computational tools.

3. To produce the oxidoreductases isolated in the previous genome (or transcriptome) screening using both laboratory and other expression hosts, yielding enzymes for laboratory engineering and bioprocess development.

4. To optimize enzymatic or chemo-enzymatic systems based on the new and/or engineered oxidoreductases by: i) Enzyme immobilization; ii) In situ generation of cofactors; iii) Development of preparations acting in "water-free" media; and iv) Multi-enzymatic and double-oxidation cascade reactions.

5. To develop oxidation/oxyfunctionalization bioprocesses of interest using the new and/or engineered oxidoreductases acting in optimized systems for: i) Intermediates of agrochemicals/APIs; ii) Precursors for specialty polymers; iii) Dye-stuff intermediates; iv) Functionalized polymers for the pulp and paper; and v) Fragrances.

6. To provide relevant industrial case stories by up-scaling at least three of the previously developed bioprocesses for the production of agrochemicals, chiral compounds, dye stuffs and/or precursors for specialty polymers, based on the use of new and/or engineered oxidoreductase biocatalysts.

Project Results:
Oxidoreductases for medium and large-scale industrial biotransformations

Angel T. Martínez*, Francisco J. Ruiz-Dueñas, Susana Camarero (CIB, CSIC, Madrid, Spain)
Henrik Lund#, Jesper Vind, Morten Tovborg (Novozymes A/S, Bagsvaerd, Denmark)
Martin Hofrichter, Christiane Liers, René Ullrich (TU Dresden, Zittau, Germany)
Katrin Scheibner (JenaBios GmBH, Jena, Germany)
Giovanni Sannia, Alessandra Piscitelli, Cinzia Pezzella (University Federico II, Naples, Italy)
Mehmet E. Sener (Setas Kimya Sanayi AS, Tekirdag, Turkey)
Willem J.H. van Berkel (Wageningen University & Research, the Netherlands)
Victor Guallar, Fátima Lucas (Anaxomics, Barcelona, Spain)
Ralf Zuhse (Chiracon GmBH, Luckenwalde, Germany)
Roland Ludwig (BOKU, Vienna, Austria)
Frank Hollmann (University of Technology, Delft, the Netherlands)
Eric Record, Craig Faulds (INRA, Marseille, France)
Marta Tortajada (Biopolis, Valencia, Spain)
Ib Winckelmann (Cheminova A/S Lemvig, Denmark)
Jo-Anne Rasmussen (CLEA Technologies BV Delft, the Netherlands)
Mirjana Gelo-Pujic (Rhodia, Paris, France)
Ana Gutiérrez, José C del Río (IRNAS, CSIC, Seville, Spain)
Miguel Alcalde (Institute of Catalysis, CSIC, Madrid, Spain)


# In memoriam
* Corresponding author (
¶ Abbreviations: AAD, aryl-alcohol dehydrogenase; AAO, aryl-alcohol oxidase; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); API, active pharmaceutical ingredients; CDH, cellobiose dehydrogenase; CPK, Corey/Pauling/Koltun (atom coloring convention); CRO, copper-radical oxidase; DFF, 2,5-diformylfuran; DyP, dye-decolorizing peroxidase; FDCA, 2,5-furandicarboxylic acid; FFCA, 2,5-formylfurancarboxylic acid; GDH, glucose dehydrogenase; GMC glucose-methanol-choline oxidase/dehydrogenase superfamily; GOX, glucose oxidase; HMF, 5-hydroxymethylfurfural; HSQC, heteronuclear single quantum correlation (NMR experiment); HTP, hemethiolate peroxidases; LiP, lignin peroxidase; LPMO, lytic polysaccharide monooxygenase; LRET, long-range electron transfer; MCO, multicopper oxidase; MnP, manganese peroxidase; MOX, methanol oxidase; NMR, nuclear magnetic resonance; P2O, pyranose 2-oxidase; PELE, protein energy landscape exploration (software); QM/MM, mixed quantum mechanics/molecular mechanics; UPO, unspecific peroxygenase; VAO, vanillyl-alcohol oxidase; VP, versatile peroxidase


Lignin-degrading basidiomycetes and related fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases. Heme peroxidases comprise classical ligninolytic peroxidases and new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate peroxidases. 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 source of oxygen and final electron acceptor. Flavooxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical, respectively. 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. However, the recently described lytic polysaccharide monooxygenases attract the highest attention among copper oxidoreductases, since they are able to break down (oxidatively) crystalline cellulose, a major bottleneck in lignocellulose biorefineries (together with lignin degradation by peroxidases and laccases). Interestingly, some flavin-containing dehydrogenases also play a key role "fueling" (directly or indirectly) electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, using both rational design and directed evolution, to attain the selectivity, catalytic efficiency and operational properties (stability included) required for their industrial utilization. Using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site (biophysical simulations) and electron transfer efficiency (biochemical simulations), reducing in orders of magnitude the time of experimental work in enzyme screening and engineering. The above is illustrated by a series of oxyfunctionalization and oxidation reactions (e.g. enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, chiral alcohols, furandicarboxylic acid, indigo dyes and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation) as successful case stories demonstrating the potential of oxidoreductases in medium and large-scale industrial biotransformations.

• Heme peroxidases/peroxygenases
• Oxidases and dehydrogenases
• Laccases
• Lytic polysaccharide monooxygenases
• Biophysical and biochemical computational modeling
• Rational design
• Directed evolution
• Enzyme cascades
• Selective oxyfunctionalizations
• Lignocellulose biorefinery

1. Fungal oxidoreductases

Oxidoreductases take advantage from the incorporation of different cofactors (such as heme, flavin and metal ions) to catalyze redox reactions, using a variety of electron acceptors and a great number of electron-donating substrates, and yielding many products of industrial interest (Gygli and van Berkel, 2015). Fungi, including wood-rotting basidiomycetes, are involved in the oxidative degradation of lignocellulosic biomass, recycling the carbon fixed by plant photosynthesis through a battery of secreted (and robust) high redox-potential oxidoreductases (Martínez et al., 2016). Fungal oxidoreductases of biotechnological interest typically include: i) heme-containing peroxidases and peroxygenases, being activated by H2O2 as electron acceptor; ii) flavin-containing oxidases and dehydrogenases, being activated by O2 and other oxidizers (such as Fe3+ and quinones), respectively; and iii) copper-containing oxidases and monooxygenases, being activated by O2 (the latter with a more complicated catalytic mechanism) (Fig. 1).

Classical fungal oxidoreductases include basidiomycete ligninolytic peroxidases, and ascomycete and basidiomycete multicopper oxidases (MCO¶, mainly laccases) with different redox potentials and abilities to act on lignin-derived products (optionally using redox mediators). Moreover, new heme- and copper-containing oxidoreductases of high biotechnological interest have been recently discovered including: i) unspecific peroxygenases (UPOs) catalyzing a variety of regio- stereo-selective oxyfunctionalizations with H2O2 working as the oxygen source and final electron acceptor; and ii) copper-containing lytic polysaccharide monooxygenases (LPMOs), which turned out to be the “missing” enzymes in the microbial attack to crystalline cellulose and other polysaccharides.

Enzymes of the glucose-methanol-choline oxidase/dehydrogenase (GMC) and copper-radical oxidase (CRO) superfamilies have been classically investigated as the source of H2O2 for: i) ligninolytic peroxidase in white-rot (lignin-degrading) basidiomycetes; or ii) hydroxyl radical generation via Fenton reaction in brown-rot (cellulose-degrading) basidiomycetes. However, the preferential or optional use of other electron acceptors by some of them (dehydrogenase activity) suggests additional functions, e.g. preventing lignin repolymerization or fueling electrons to LPMOs. These and other fungal flavooxidases are also emerging industrial biocatalysts.

2. Oxidoreductases as industrial biocatalysts

The above oxidoreductases are biocatalysts of interest for establishing a circular economy with the highest potential in: i) the production of renewable building blocks from plant biomass; and ii) their use for the manufacture of sustainable chemicals and materials in lignocellulose biorefineries (Fig. 2). However, the chemical industry (some specialties excluded) is not yet embracing enzymatic oxidation reactions to a significant extent primarily due to lack of biocatalysts with the required selectivity, availability and compatibility with the rigorous process conditions (high substrate concentrations, use of solvents, and strongly oxidative conditions).

The above bottleneck has been addressed through enzyme engineering and process optimization using state-of-the-art technologies, and some recent (2014-2017 period) representative results are reviewed here. The following oxidation and oxyfunctionalization target reactions formed the basis for the screening and optimization of new biocatalysts in this study, whose selection was based on proof of reaction at laboratory scale, industrial interest of the corresponding products and possibility for broadening the application field to similar substrate classes: i) Intermediates for agrochemicals and active pharmaceutical ingredients (APIs), flavors and fragrances, chiral alcohols, epoxidation products and drug metabolites; ii) Precursors for specialty polymers including 5-hydroxymethylfurfural (HMF) products such as 2,5-furandicarboxylic acid (FDCA), diols, hydroxyacids and diacids (from alkanes and fatty acids), and functionalized plant polymers including oxidized cellulosic fibers and lignin; and iii) Intermediates for dye-stuff production including phenolic and amine derivatives, indole and indole derivatives, and aniline polymers (Fig. 3).

The work performed comprises: i) Recovery of selective oxidative biocatalysts from fungal genomes and other sources for target reactions from the groups of heme-peroxidases/ peroxygenases, flavo-oxidases and copper-oxidoreductases; ii) Improvement of their oxidation activity and stability by protein engineering using rational design (based on structural-functional information), directed evolution and hybrid approaches (in yeast and other expression systems), combined with computer simulations speeding the engineering process, to better fulfill the operational and catalytic conditions required by the chemical industry; and iii) Optimization of reaction conditions including enzyme cascade reactions.

The above multidisciplinary approach was possible by creating a highly-specialized consortium of SMEs, large companies and research/academic institutions in the frame of the INDOX project “Optimized oxidoreductases for medium and large scale industrial biotransformations” ( Production of the new optimized biocatalysts, and their introduction into the chemical market will take advantage from the participation of the world-leading company in the sector of industrial enzymes (Novozymes) together with several chemical companies (such as Cheminova, Rhodia, Setas and Chiracon) willing to implement new medium- and large-scale enzymatic biotransformation processes. Moreover, these successful case stories demonstrate the potential of oxidative biotransformations in related chemical sectors.

3. Heme-containing peroxidases/peroxygenases

Classical peroxidases and heme-containing peroxygenases are members of the peroxidase-catalase and hemethiolate peroxidase (HTP) superfamilies, respectively. Although these enzymes share a heme cofactor, the phylogenetic connection between them would be remote or null. Classical ligninolytic peroxidases are known for near forty years, and representatives for their three families – lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) – have been extensively characterized due to biotechnological interest (Fig. 1A) (Martínez et al., 2016). Distribution of the corresponding genes in white-rot and brown-rot fungal genomes sequenced at JGI ( provides strong evidence on their involvement in lignin degradation (Barrasa et al., 2016). In this way, genes of the ligninolytic peroxidase families are found in all the white-rot (ligninolytic) basidiomycete genomes sequenced to date, but absent from all the brown-rot (cellulolytic) sequenced genomes. Recently, additional information on these enzymes has been obtained in post-genomic studies where the complete inventory of peroxidase genes from different basidiomycete genomes was heterologously expressed and the different LiP, MnP and VP products, among others, were analyzed providing the first demonstration of: i) VP ability to degrade lignin; and ii) C-terminal tail contribution to long MnP stability, among other relevant information (Fernández-Fueyo et al., 2014d; 2014a).

In contrast to well-known ligninolytic peroxidases, the first basidiomycete peroxygenase (currently known as unspecific peroxygenase, UPO) was reported only twelve years ago from Agrocybe aegerita (Fig. 1C). Ascomycete chloroperoxidase also belongs to the HTP superfamily but it has low oxygenase activity. Unrelated vanadium-containing (and H2O2 resistant) chloroperoxidase also catalyzes reactions of biotechnological interest (Fernández-Fueyo et al., 2015b; 2016b). Interestingly, HTP genes are well represented in most basidiomycete genomes sequenced at JGI, as well as in additional genomes sequenced in the search for HTP genes (Kellner et al., 2016). Despite this wide genomic presence and the biotechnological interest of mono(per)oxygenation reactions, which resulted in several recent patents (Landvick et al., 2016a; 2016b), just a few UPOs have been purified and characterized to date, and central aspects of their catalytic mechanism have been only recently solved (Wang et al., 2015).

UPOs share the same active site and reaction chemistry of cytochrome P450 monooxygenases (P450s) resulting in highly versatile oxidation/oxygenation reactions, which can be classified as follows: a) two-electron oxidations with O-transfer; b) two-electron oxidations with O-transfer and bond cleavage; c) two-electron oxidations with O-transfer to heteroatoms; and d) one electron oxidations (as typical peroxidases) (Fig. 4A) (Hofrichter et al., 2015). However, compared with P450s that need a flavin-containing reductase or protein domain and a source of reducing power, UPOs can be considered as “self-sufficient” monooxygenases only requiring a source of H2O2 to be activated (Fig. 4B). Nevertheless, UPOs exhibit some catalase activity, along with an oxidative instability, that must be taken into account for correct H2O2 dosage (Karich et al., 2016). In addition, UPOs are secreted enzymes and, therefore, more stable than P450s that are generally intracellular membrane-bound proteins.

Also recently, the so-called dye-decolorizing peroxidases (DyPs) have been described in basidiomycetes (Strittmatter et al., 2015) (Fig. 1B). Their classification in the CDE superfamily (including chlorite dismutase, DyP and EfeB protein from Escherichia coli) reveals a different phylogenetic origin (Linde et al., 2015b). In this case, the convergence with ligninolytic peroxidases not only includes a histidine residue as heme iron ligand (while a cysteine occupies this position in HTPs) but also a long-range electron transfer (LRET) mechanism for oxidation of bulky lignin-derived and dye substrates. Although some surface tyrosines have been suggested as the beginning of LRET pathways in fungal DyP (Strittmatter et al., 2015), combination of electron paramagnetic resonance and directed mutagenesis studies revealed that such pathway starts at a radical-forming catalytic tryptophan, as previously described in LiP and VP (Baratto et al., 2015; Linde et al., 2015a). Simultaneously, the first fungal DyP oxidizing Mn2+ to Mn3+, as MnP and VP do, has been described (Fernández-Fueyo et al., 2015a), providing another example of evolutionary convergence between unrelated enzymes oxidizing lignin products.

In the search for new peroxidases/peroxygenases (and enzymes of the other oxidoreductase superfamilies discussed below) the genomic inventories have been complemented with enzyme screening in fungi with different lifestyles and transcriptomic and secretomic studies using natural substrates under laboratory conditions (Barrasa et al., 2014; Fernández-Fueyo et al., 2014b; 2016a; Hori et al., 2014).

4. Flavin-containing oxidases/dehydrogenases

The GMC superfamily includes flavin-containing (i) oxidases, such as aryl-alcohol oxidase (AAO), methanol oxidase (MOX), pyranose 2-oxidase (P2O) and glucose oxidase (GOX), and (ii) dehydrogenases, such as cellobiose dehydrogenase (CDH, which contains flavin and heme domains) and glucose dehydrogenase (GDH). Their distribution, phylogenetic relationships and potential role in lignocellulose degradation has been recently reviewed based on the analysis of 10 sequenced Polyporales genomes (Ferreira et al., 2015a).

Opposite to that described above for ligninolytic peroxidases, a strict distribution of genes of different H2O2-generating oxidases was not observed in wood-rotting fungi. However, AAO appears as the most frequent GMC oxidase in the white-rot species, where H2O2 activates ligninolytic peroxidases. In contrast, MOX genes are more abundant in the brown-rot species, where H2O2 is reduced by Fe2+ yielding hydroxyl radical involved in the initial attack on cellulose by these fungi. AAO and P2O are secreted proteins and, therefore, can be more easily involved in the extracellular degradation of lignocellulosic materials. However, several pieces of evidence indicate that oxidases lacking such secretion mechanism, such as GOX and especially MOX, can be also involved in lignocellulose decay thanks to an alternative secretion process or simply by hyphal lysis. AAO can be considered as the model GMC oxidase in lignocellulose decay, with recent studies on the catalytic mechanism of the best known Pleurotus eryngii enzyme (Ferreira et al., 2015b) (Fig. 1F), and isolation of new AAOs of interest (Couturier et al., 2016).

Among GMC dehydrogenases, CDH is involved in cellulose decay by white-rot fungi, in agreement with gene distribution in sequenced genomes (Kracher et al., 2016). The effect of CDH is related to its synergistic action with LPMO, which is described in the next section. Such reaction implies electron transfer from the flavin domain (Fig. 1E), where cellobiose is oxidized to cellobionolactone, to the heme (or cytochrome) domain, in an intramolecular reaction that has been characterized from a structural and mechanistic point of view (Kracher et al., 2015; Tan et al., 2015). As will be explained below, other dehydrogenases can also promote the action of LPMO by quinone redox cycling, as in the case of the first basidiomycete GDH and the first aryl-alcohol dehydrogenase (AAD) reported to date (both identified in the Pycnoporus cinnabarinus genome) (Mathieu et al., 2016; Piumi et al., 2014). The structural basis for the quinone-reducing ability of these AADs seems related to a wider active-site access channel, compared with that of AAO that only enables the access of O2 (Fig. 5).

Another two microbial flavooxidases of biotechnological interest in organic synthesis are vanillyl-alcohol oxidase (VAO; Fig. 1D) and eugenol oxidase, which belong to a different protein superfamily, have ascomycete and bacterial origins, respectively, and differ in oligomerization degree due to a single loop identified in the dimer interface (Ewing et al., 2016).

5. Copper-containing oxidoreductases

Three different copper-containing oxidoreductases are considered in this section: i) CRO; ii) MCO (laccases); and iii) LPMOs. The CRO family is characterized by the presence of a copper ion and a protein radical involved in catalysis, and includes glyoxal oxidase, the first H2O2-producing enzyme described in the model white-rot fungus Phanerochaete chrysosporium, and galactose oxidase that, in addition to oxidize monosaccharides, has some activity on benzylic alcohols (Kalum et al., 2014b). CROs are present in most basidiomycete genomes, and some of them showed new catalytic properties of interest after their heterologous expression (Daou et al., 2016). Among MCOs, laccases are characterized by the presence of four copper ions (Fig. 1G) and, together with peroxidases, are the most thoroughly studied oxidoreductases in wood rotting fungi and, by far, the largest number of biotechnological applications have been reported for these multicopper enzymes (Mate and Alcalde, 2016; Pezzella et al., 2015). Due to the high number of well characterized laccases with different redox potentials and other properties, recent work on these enzymes focused on tailoring the catalytic properties of commercially available, and other laccases of interest, for target reactions by rational design, often guided by computational simulations, and directed evolution, as described in the next sections.

In contrast with the above copper-containing enzymes that are known for many years (fungal laccase was for the first time reported in the early 1960's), LPMOs (Fig. 1H) were recognized as a new oxidoreductase family, playing a crucial role in cellulose degradation, only six years ago (Martínez, 2016). Recently, LPMOs with different activities have been reported from several fungi (Isaksen et al., 2014; Patel et al., 2016). Paradoxically, the first sequences of this new oxidoreductase family (from genomes and other sources) were stored for years in databases (such as CAZY, as corresponding to family GH61. This striking confusion originated from: i) whole sequence similarities with glycoside hydrolases, including the presence of carbohydrate binding domains in some of them; ii) weak hydrolase activity of some LPMOs or their contaminating proteins; and iii) requirement of a reducing (electron providing) substrate for enzyme activation. Such activation, initially obtained with artificial reductants such as ascorbic acid, reduces Cu2+ to Cu+, which reacts with O2 forming a reactive copper-superoxide complex. The resulting monooxygenase activity causes the oxidative breakdown of crystalline polysaccharide chains (lytic oxygenase activity). The search for the natural LPMO reductants described below is a hot topic in lignocellulose degradation with an enormous importance for lignocellulose biorefineries.

As summarized in Fig. 6, different mechanisms can operate fueling electrons for LPMO activation in wood-rotting fungi. Among them, CDH is able to transfer electrons from cellulose products to LPMO using its heme domain, after intramolecular electron transfer from the flavin domain where the reaction with cellobiose takes place (Kracher et al., 2015; Loose et al., 2016; Tan et al., 2015). Other GMCs provide alternative routes for LPMO activation, as shown for GDH (Garajova et al., 2016) that can redox cycle quinones (from lignin degradation or fungal metabolism) for a continuous supply of easily oxidizable hydroquinones to LPMO (Kracher et al., 2016). Another LPMO activating routes could involve photosynthetic pigments and lignin-derived phenols whose radicals after LPMO oxidation would be reduced back by some lignin fractions/domains (Martínez, 2016)

Due to the relatively recent reports on LPMO structure and activity, several important aspects of its reaction mechanism are still to be fully understood: from the reactive oxygenation species to the interaction with substrates. Concerning the latter aspect, recent evidence from 2D nuclear magnetic resonance (NMR) spectroscopy suggests that reductants, such as CDH, and cellulose bind the same region of the LPMO molecule (Fig. 7), in contrast with the alternative hypothesis that suggested the existence of a LRET pathway for enzyme activation (Cu2+ reduction) starting at a different region of the protein surface (Courtade et al., 2016).

6. Biophysical and biochemical computational modeling

Oxidoreductase engineering has benefited from different computational simulations, where the target reaction to be achieved (or the enzyme property to be improved) was explored with in silico biophysical and biochemical tools, enabling significant reduction of time dedicated to experimental engineering work.

Biophysical modeling typically included dynamic simulations of substrate diffusion to the enzyme active site, optimized docking and, if it can represent a limiting step, analysis of product diffusion to the solvent region. The state-of-the-art technology for modeling substrate and product diffusion in enzymes is PELE (an acronym for Protein Energy Landscape Exploration) being capable of accurately reproducing long time scale processes in only few hours of CPU (

The PELE algorithm, which combines a steered stochastic approach with protein structure prediction methods capable of projecting the migration dynamics of ligands in proteins, is based on three main steps: i) ligand and protein local perturbation, including translation and rotation of the ligand, and protein α-carbon displacement following an anisotropic network model approach; ii) side-chain sampling, by placing all side chains local to the ligand; and iii) energy minimization of a region including, at least, all residues local to the atoms involved in (i) and (ii). Typically, a simulation involves several processors running multiple steps and sharing information towards addressing a common task.

As shown in different studies described in next sections, PELE modeling properly informs about substrate access and positioning at the enzyme active site (Babot et al., 2015a) including distances and angles between the redox centers (Lucas et al., 2016; Molina-Espeja et al., 2016a) but, in oxidoreductase reactions, this is often followed by electron transfer estimation by quantum calculations, as described below. Furthermore, a computational methodology accumulating beneficial interactions between a laccase and a target substrate has resulted, through a repurposing strategy, useful to design a novel polar binding scaffold to anchor negatively charged groups (Giacobelli et al., 2017).

Following the biophysical study (PELE simulation of substrate diffusion into the active site) it is possible to perform the quantum biochemical characterization of the electronic coupling involved in the oxidation process. In a model study with a basidiomycete peroxidase, the values obtained showed a striking correlation with the oxidation turnover values determined experimentally validating the computational approach (Fig. 8) (Acebes et al., 2016). Quantum calculations have been also used to estimate stacking interaction energies of different alcohol substrates at the active site of AAO, being able to successfully predict changes in the enzyme reaction mechanism (from ping-pong to ternary complex interactions with reducing and oxidizing substrates) (Ferreira et al., 2015b)

In other cases, e.g. when the substrate can adopt different oxidation poses at the active site of the native enzyme or in silico mutated variants, mixed quantum mechanics/molecular mechanics (QM/MM) calculations can be performed to predict the best active site mutations from the estimated average spin density on the substrate molecule. This methodology has been applied for engineering laccase for aniline oxidation, as described below, and the calculations validated by experimental determination of the kinetic constants of the best predicted variant (Santiago et al., 2016). In this way, a binding focused general strategy based on QM/MM reactivity scoring has been proposed for laccase engineering (Monza et al., 2015).

A similar approach has been used to rationalize the improvements observed in: i) laccase oxidation of phenols (sinapic acid) after saturation mutagenesis at the substrate binding site (Pardo et al., 2016b); and ii) stereoselective sulfoxidation after directed mutagenesis at the DyP active site (Linde et al., 2016). Mixed QM/MM calculations were also used to identify the LRET pathways that characterize peroxidase oxidation of bulky substrates, using the “e-pathway” approach (where relevant residues are successively included in the QM region, while the rest of the protein is in the MM region) as shown for anthraquinoid dye oxidation by DyP (Linde et al., 2015a).

7. Engineering oxidative enzymes

Oxidoreductase engineering for industrial application considers both directed mutagenesis (rational design) and directed molecular evolution, as well as combinations of both (semi-rational approaches) on the whole protein or focused on target regions (Maté et al., 2016; Molina-Espeja et al., 2016b; Pardo and Camarero, 2015b; Viña-González et al., 2016), including the development of new directed evolution methods (González-Pérez et al., 2014b). A prerequisite for all protein engineering methodologies is the availability of an expression system to generate improved variants. Therefore, the heterologous expression (in Saccharomyces cerevisiae, Escherichia coli or other systems) of oxidoreductase genes was first optimized for UPO (Alcalde et al., 2014; Molina-Espeja et al., 2014), DyP (Linde et al., 2014), AAO (Viña-González et al., 2015), ligninolytic peroxidases (García-Ruiz et al., 2014) and VAO (Gygli and van Berkel, 2017) engineering.

In rational engineering studies, VP has been used as a model peroxidase and both oxidative and alkaline inactivation have been addressed to develop better variants for industrial application. Two different strategies have been successfully combined to improve the VP oxidative stability against H2O2: i) substitution of easily oxidizable residues (methionines located between the cofactor and the surface catalytic tryptophan); and ii) mutation of distal heme pocket residues for reducing the efficiency of peroxidase reaction with H2O2 and, consequently, the accumulation of compound I, whose reaction with H2O2 excess inactivates the enzyme via non-catalytic compound III (Sáez-Jiménez et al., 2015a).

While the improvement of oxidative stability was based on our knowledge on VP structure-function relationships, a different strategy was successfully applied for rational improvement of alkaline stability, based on: i) selection of a naturally-stable peroxidase (MnP) from genome screening (and heterologous expression); ii) identification of the structural determinants for this stability (such as H-bonds, salt bridges and basic residues exposed to the solvent in the crystal structure); and iii) introducing them in the target enzyme (VP) by directed mutagenesis (Sáez-Jiménez et al., 2015d).

Rational design has been also used to create a peroxidase (VP) with strong ligninolytic activity due to its ability to act at extremely acidic pH (that increases the redox potential of the heme iron). With this purpose, the catalytic tryptophan of VP (and LiP) was introduced in a peroxidase (MnP) scaffold from genome screening that was stable under these acidic conditions (Fernández-Fueyo et al., 2014c). In another example of rational design, the active site of DyP has been broadened resulting in stereoselective sulfoxidation reactions due to efficient substrate docking near the reactive oxygen of enzyme compound I, as shown by computational modeling using the crystal structure of the engineered variant (Linde et al., 2016).

Paralleling the rational design described above, directed evolution and hybrid strategies have been applied to improve the H2O2 (González-Pérez et al., 2014a) and alkaline (González-Pérez et al., 2016) stability of VP. On the other hand, polyvalent UPO was submitted to directed evolution to enhance its mono(per)oxygenase activity (and reduce competing one-electron oxidation peroxidase activity) for oxyfunctionalizations of biotechnological interest, such as naphthalene hydroxylation to 1-naphthol (Fig. 9) (Gómez de Santos et al., 2016; Molina-Espeja et al., 2015b; 2016a). Moreover, directed evolution and other engineering approaches (such as domain swapping) have also been applied to laccase variants expressed in yeast to improve properties of interest, such as oxidation of different phenolic substrates (Maté and Alcalde, 2016; 2015; Pardo et al., 2016a; Pardo and Camarero, 2015a; 2015b; Vicente et al., 2016) and ad hoc high-throughput screening strategies have been developed (Pardo and Camarero, 2017).

The engineered enzymes were expressed using a Saccharomyces cerevisiae-Pichia pastoris tandem system (Molina-Espeja et al., 2015a) and the industrial expression technology of Novozymes (Vind et al., 2015) for the applications described below (together with Escherichia coli expression and in vitro activation for some laboratory-scale reactions). The industrial applicability often improved by enzyme immobilization (Lettera et al., 2016; Poraj-Kobielska et al., 2015b).

8. Enzymatic oxyfunctionalization

Basidiomycete UPOs appear as a highly promising biocatalysts for a variety of aromatic and aliphatic oxyfunctionalization reactions of industrial interest (Bormann et al., 2015; Hofrichter and Ullrich, 2014). Some selective oxygenation reactions have also been developed with engineered variants of other peroxidases, such as DyP sulfoxidation mentioned above (Linde et al., 2016). Several oxyfunctionalization reactions of biotechnological interest are described below.

1-Naphthol - a large market chemical (40,000 tons/year) used in the production of herbicides, insecticides, pharmaceuticals and dye precursors - can be obtained by naphthalene hydroxylation using an UPO variant evolved with this purpose through an ad hoc screening protocol (Fig. 9) (Molina-Espeja et al., 2016a). Epoxidation of non-cyclic alkenes and terpenes of interest for the chemical sector using different UPOs has also been obtained (Lund et al., 2016).

25-Hydroxyvitamin D3 - of interest in feeding chickens and other farm animals to reduce skeleton problems caused by rapid growth and reduced mobility - can be produced using the Coprinopsis cinerea UPO due to the ad hoc active-site architecture for vitamin D3 regioselective hydroxylation (Fig. 10) (Babot et al., 2015b; Lucas et al., 2016). Related hydroxylations are those catalyzed by different basidiomycete peroxygenases on a variety of steroidal substrates (mainly at their 25 position) yielding products with antimicrobial properties, among others (Babot et al., 2015a).

Desmethylation in the synthesis of human metabolites of the bile acid reabsorption inhibitor SAR548304, a seven-step chemical reaction using palladium catalysis and laborious chromatographic purification with an overall yield of only 27%, can be obtained by one-pot selective (and high yield) N-desmethylation using the Marasmius rotula UPO (Fig. 11) (Kiebist et al., 2015). Also of pharmaceutical interest is the use of the same UPO for the enzymatic removal of corticoid side-chain, involving hydroxylation and C-C bond cleavage (Poraj-Kobielska et al., 2015a).

Stereoselective hydroxylation of ethylbenzene into (R)-1-phenylethanol, an example of enzymatic production of chiral secondary alcohols, can be achieved in a methanol supported and by-product free multi-enzymatic cascade using: a) Agrocybe aegerita UPO; b) methanol oxidase for H2O2 supply; c) formaldehyde dismutase, converting each two formaldehyde molecules into one molecule of methanol (reacting again with methanol oxidase) and one molecule of formic acid; d) formate dehydrogenase converting formic acid into CO2 and H2O at expenses of NAD(P)+; and e) 3-hydroxybenzoate-6-hydroxylase regenerating NAD(P)+ from the NAD(P)H formed in the previous dehydrogenase reaction (Fig. 12) (Hollmann and Ni, 2016; Ni et al., 2016). Concerning the last step in the enzyme cascade, other NAD(P)H regeneration systems have also been reported (Pham et al., 2015).

Last but not least, enzymatic oxygenation of long-chain n-alkanes at their unreactive terminal position has been very recently reported using the Marasmius rotula UPO, yielding dicarboxylic acids and other oxygenated products from these largely inert compound family (Olmedo et al., 2016). Note that all other basidiomycete peroxygenases and wild-type P450s mostly produce subterminal diols, among other products from alkane oxygenation (Lund et al., 2014).

9. Enzymatic oxidations

A series of oxidation reactions of biotechnological interest are catalyzed by different oxidoreductases, UPOs included, for the production of polymer building blocks, dyeing molecules, and other compounds of interest, as described below.

According to the US Department of Energy, HMF is one of the top-ten renewable chemicals for a sustainable bioeconomy, as the precursor of FDCA and other platform chemicals, such as 2,5-diformylfuran (DFF). HMF-derived FDCA is the bio-based alternative to fossil-based terephthalic acid for the production of polyester-type plastics. HMF is an aromatic (benzylic) alcohol and, therefore, a substrate of AAO, and this oxidase also acts on hydrated aldehydes (gem-diols). Therefore, an enzyme cascade has been developed where the limiting step in HMF oxidation by AAO, i.e. the 2,5-formylfurancarboxylic acid (FFCA) oxidation to FDCA, is catalyzed by UPO at expenses of the H2O2 generated by AAO in the two initial oxidation steps (Fig. 13) (Carro et al., 2015). On the other hand, a galactose oxidase variant has been claimed for the conversion of HMF into DFF, since it selectively catalyzes the first oxidation step (Kalum et al., 2014b).

A variety of indigo dyes, differing in the benzene ring substituents, can be obtained by oxidative coupling of the 3-hydroxyindol precursors obtained by indole hydroxylation by UPO (Fig. 14A) (Herold-Majumdar et al., 2016; Kalum et al., 2014a). In this way enzymatic in situ dyeing of fabrics is possible, with the indigo dye becoming insoluble once oxidized, and the color remaining after fabric washing (Fig. 14B-D). Additionally, green routes for the production of polymeric dyes can be developed by the enzymatic copolymerization of aniline-type and phenolic precursors, such as 2,5-diaminebenzenesulfonic acid and resorcinol, by laccase (Pezzella et al., 2016) and ad hoc evolved variants (Vicente et al., 2016). Such reactions are milder and more environmentally-friendly than the chemical counterparts, and dyeing of natural and synthetic fabrics is comparable to that obtained with commercial dyes from chemical synthesis (such as Nyloset Brown N2R) (Fig. 15).

Synthesis of conductive polyaniline has been widely investigated during the last decade for a range of applications (sensor devices, rechargeable batteries, etc). However, the conducting polyaniline polymer is industrially produced under strongly acidic conditions and using strong oxidants. Recently, a laccase variant that exhibits improved aniline oxidation has been obtained by a combination of computational simulations and directed evolution tools (Santiago et al., 2016) as a green alternative for polyaniline synthesis under industrially relevant conditions (de Salas et al., 2016) (Fig. 16).

Lignin removal is a bottleneck for biomass conversion into fuels and chemicals in lignocellulose biorefineries that is overcome using strong physicochemical treatments. Mimicking those changes caused by lignin-degrading white-rot fungi (van Kuijk et al., 2016), isolated oxidoreductases (especially laccases in the presence/absence of redox mediators) have been applied as milder and more environmentally-friendly pretreatments of woody and nonwoody lignocellulosic feedstocks (Rencoret et al., 2016). In these studies, changes in lignin structure during the enzymatic treatment have been followed using 2D-NMR, the state-of-the-art technology for structural characterization of lignin (Rico et al., 2015).

Concerning technical lignins from lignocellulose biorefineries (with variable phenolic content), treatment with fungal oxidoreductases (as laccases and peroxidases) can be followed by growth of selected bacteria acting as a microbial sink for the synthesis of products of interest through metabolic engineering (Salvachúa et al., 2016). Interestingly, using a water soluble lignin (lignosulfonate) as substrate, and a combination of 2D-NMR and stopped-flow rapid spectrophotometry, it has been possible to demonstrate that: i) ligninolytic peroxidases are able to abstract electrons directly from the lignin polymer; ii) the minor phenolic moiety of lignin is preferentially oxidized by these enzymes; and iii) the catalytic tryptophan of VP and LiP is strictly required to oxidize the nonphenolic lignin (Sáez-Jiménez et al., 2015b; Sáez-Jiménez et al., 2015c; Sáez-Jiménez et al., 2016).

In the near future, advanced processing of biomass in lignocellulose biorefineries could take advantage from synthetic biology tendencies and tools to combine the extracellular lignin and cellulose degrading machineries (ligninosome and cellulosome) and the intracellular fermentative capabilities of microorganisms to develop biofuel-producing "white-rot yeasts" and other tailor-made microorganisms (Fig. 17) (Alcalde, 2015; González-Pérez, 2016; González-Pérez and Alcalde, 2014).


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 the groups of 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, INRA-Marseille, Biopolis, Cheminova, CLEA, Rhodia, IRNAS-CSIC, and ICP-CSIC for their significant contributions to the results presented. FJR-D thanks a MINECO Ramon y Cajal contract.


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Martínez, A. T., 2016. How to break down crystalline cellulose. Science 352:1050-1051.

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Maté, D., Alcalde, M., 2016. Directed evolution of fungal laccases: An update, p. 91-112. In A. Rahman (ed.), Advances in Genome Science vol 4. Genes in Health and Disease. Bentham Books.

Maté, D., González-Pérez, D., Mateljak, I., Gómez de Santos, P., Vicente, A. I., Alcalde, M., 2016. The pocket manual of directed evolution: Tips and tricks, p. 185-214. In G. Brahmachari, A. Demain, and J. L. Adrio (eds.), Biotechnology of Microbial Enzymes: Production, Biocatalysis and Industrial Applications. Elsevier, Amsterdam.

Maté, D. M., Alcalde, M., 2015. Laccase engineering: From rational design to directed evolution. Biotechnol. Adv. 33:25-40.

Mate, D. M., Alcalde, M., 2016. Laccase: a multi-purpose biocatalyst at the forefront of biotechnology. Microbial Biotechnol. online.

Mathieu, Y., Piumi, F., Valli, R., Aramburu, J. C., Ferreira, P., Faulds, C. B. et al., 2016. Activities of secreted aryl alcohol quinone oxidoreductases from Pycnoporus cinnabarinus provide insights into fungal degradation of plant biomass. Appl. Environ. Microbiol. 82:2411-2423.

Molina-Espeja, P., Garcia-Ruiz, E., Gonzalez-Perez, D., Ullrich, R., Hofrichter, M., Alcalde, M., 2014. Directed evolution of unspecific peroxygenase from Agrocybe aegerita. Appl. Environ. Microbiol. 80:3496-3507.

Molina-Espeja, P., Ma, S., Maté, D. M., Ludwig, R., Alcalde, M., 2015a. Tandem-yeast expression system for engineering and producing unspecific peroxygenase. Enzyme Microb. Technol. 73-74:29-33.

Molina-Espeja, P., Plou, F., Alcalde, M., 2015b. Unspecific peroxygenase mutants with high monooxygenase activity and their applications. Patent (Spain) P201531641.

Molina-Espeja, P., Canellas, M., Plou, F. J., Hofrichter, M., Lucas, F., Guallar, V. et al., 2016a. Synthesis of 1-naphthol by a natural peroxygenase engineered by directed evolution. ChemBioChem 17:341-349.

Molina-Espeja, P., Viña-Gonzalez, J., Gomez-Fernandez, B. J., Martin-Diaz, J., Garcia-Ruiz, E., Alcalde, M., 2016b. Beyond the outer limits of nature by directed evolution. Biotechnol. Adv. 34:754-767.

Monza, E., Lucas, F., Camarero, S., Alejaldre, L. C., Martínez, A. T., Guallar, V., 2015. Insights on laccase engineering from molecular simulations: towards a binding focused strategy. J. Phys. Chem. Lett. 6:1447-1453.

Ni, Y., Fernández-Fueyo, E., Baraibar, A. G., Ullrich, R., Hofrichter, M., Yanase, H. et al., 2016. Peroxygenase-catalyzed oxyfunctionalization reactions promoted by the complete oxidation of methanol. Angew. Chem. 55:798-801.

Olmedo, A., Aranda, C., del Río, J. C., Kiebist, J., Scheibner, K., Martínez, A. T. et al., 2016. From alkanes to carboxylic acids: Terminal oxygenation by a fungal peroxygenase. Angewandte Chemie 55:12248-12251.

Pardo, I., Camarero, S., 2015a. Exploring the oxidation of lignin-derived phenols by a library of laccase mutants. Molecules 20:15929-15943.

Pardo, I., Camarero, S., 2015b. Laccase engineering by rational and evolutionary design. Cell Mol. Life Sci. 72:897-910.

Pardo, I., Camarero, S., 2017. Colorimetric high-troughput screening assays for the directed evolution of fungal laccases. Methods Mol. Biol. (in press).

Pardo, I., Rodríguez-Escribano, D., Camarero, S., 2016a. A highly stable hybrid laccase obtained by domain swapping. Appl. Environ. Microbiol. (in press).

Pardo, I., Santiago, G., Gentili, P., Lucas, F., Monza, E., Medrano, F. J. et al., 2016b. Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid. Catal. Sci. Technol. 6:3900-3910.

Patel, I., Kracher, D., Ma, S., Garajova, S., Haon, M., Faulds, C. B. et al., 2016. Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6. Biotechnol. Biofuels 9:108.

Pezzella, C., Giacobbe, S., Giacobelli, V. G., Guarino, L., Kylic, S., Sener, M. et al., 2016. Green routes towards industrial textile dyeing: A laccase based approach. J. Mol. Catal. B-Enzym.

Pezzella, C., Guarino, L., Piscitelli, A., 2015. How to enjoy laccases. Cell Mol. Life Sci. 72:923-940.

Pham, N. H., Hollmann, F., Kracher, D., Preims, M., Haltrich, D., Ludwig, R., 2015. Engineering an enzymatic regeneration system for NAD(P)H oxidation. J. Mol. Catal. B-Enzym. 120:38-46.

Piumi, F., Levasseur, A., Navarro, D., Zhou, S. M., Mathieu, Y., Ropartz, D. et al., 2014. A novel glucose dehydrogenase from the white-rot fungus Pycnoporus cinnabarinus: production in Aspergillus niger and physicochemical characterization of the recombinant enzyme. Appl. Microbiol. Biotechnol. 98:10105-10118.

Poraj-Kobielska, M., Gröbe, G., Kiebist, J., Grün, M., Ullrich, R., Scheibner, K. et al., 2015a. Verfahren zur Deacylierung von Corticoiden. Patent (Germany) DE102014005371.

Poraj-Kobielska, M., Peter, S., Leonhardt, S., Ullrich, R., Scheibner, K., Hofrichter, M., 2015b. Immobilization of unspecific peroxygenases (EC in PVA/PEG gel and hollow fiber modules. Biochem. Eng. J. 98:144-150.

Rencoret, J., Pereira, A., del Río, J. C., Martínez, A. T., Gutiérrez, A., 2016. Laccase-mediator pretreatment of wheat straw degrades lignin and improves saccharification. Bioenerg. Res. 9:917-930.

Rico, A., Rencoret, J., del Río, J. C., Martínez, A. T., Gutiérrez, A., 2015. In-depth 2D NMR study of lignin modification during pretreatment of Eucalyptus wood with laccase and mediators. Bioenerg. Res. 8:211-230.

Sáez-Jiménez, V., Acebes, S., Guallar, V., Martínez, A. T., Ruiz-Dueñas, F. J., 2015a. Improving the oxidative stability of a high redox potential fungal peroxidase by rational design. PLoS ONE10(4):e0124750. doi:10.1371/journal.pone.0124750.

Sáez-Jiménez, V., Baratto, M. C., Pogni, R., Rencoret, J., Gutiérrez, A., Santos, J. I. et al., 2015b. Demonstration of lignin-to-peroxidase direct electron transfer. A transient-state kinetics, directed mutagenesis, EPR and NMR study (vol 290, pag 23201, 2015). J. Biol. Chem. 290:30268.

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Wang, X., Ullrich, R., Hofrichter, M., Groves, J. T., 2015. Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive. Proc. Natl. Acad. Sci. USA 112:3686-3691

Fig. 1. Classical and new oxidoreductase families involved in lignocellulose degradation and other biotransformations of interest: A. Pleurotus eryngii VP (3FJW); B. Auricularia auricula-judae DyP (4W7J); C. Agrocybe aegerita UPO (2YP1); D. Penicillium simplicissimum VAO monomer (1VAO); E. Neurospora crassa CDH with flavin and heme domains (4QI7); F Pleurotus eryngii AAO (3FIM); G. Pycnoporus cinnabarinus laccase (2XYB); and H. Thermoascus aurantiacus LPMO (2YET). Cofactors (heme/FAD and copper ions as red and yellow sticks and orange spheres, respectively) and residues relevant for catalysis (Corey/Pauling/Koltun, CPK, colored sticks) such as: i) His, Cys and Met/His ligands of heme iron in A/B, C and E, respectively; ii) His/Arg, Asp/Arg and Glu/Arg involved in activation by H2O2 in A, B and C, respectively; iii) 2 Glu and 1 Asp forming the Mn binding site in A; iv) catalytic Trp in A and B; v) active site residues in D-F, including His linked to FAD in D; vi) 4 copper ions in G; and vii) 2 His and 1 Tyr copper ligands in H.

Fig. 2. Oxidative biocatalysts for circular economy. Production of renewable building blocks and manufacture of sustainable chemicals and materials are two steps where oxidative biocatalysts (OxiCats) can exert the most positive impact enabling greener and more efficient biotransformation routes. Adapted from

Fig. 3. Some model target chemicals whose production using oxidative enzymes has been evaluated.

Fig. 4. Basidiomycete UPO catalyzing a variety of monooxygenation reactions (and others) with advantages over P450s. A. UPO oxidation and oxyfunctionalization reactions, at expenses of H2O2, include: a) two-electron oxidations with O-transfer; b) two-electron oxidations with O-transfer and cleavage reaction; c) two-electron oxidations with O-transfer to heteroatoms; and d) one-electron oxidations. B. While intracellular P450s (top) require a source of reducing power (NAD[P]H) and an auxiliary flavin-containing reductase or protein domain (often wasting a significant part of the reducing power in unproductive H2O2 formation), secreted UPO just needs a source of H2O2 to be activated (being also more robust due to its extracellular nature). Partially adapted from Hofrichter et al. (2015).

Fig. 5. Wider FAD-access channels in LPMO-activating AADs compared with AAO. The channels connecting the active-site cavity to solvent in Pleurotus eryngii AAO (PDB 3FIM) (A) and three AADs (AAQO1-AAQO3) from the Pycnoporus cinnabarinus genome and secretome (B-D, homology models) are shown. Channels were depicted by CAVER, with FAD as spheres and several active-site residues as sticks (CPK colored), including two catalytic histidines (green carbons) and other residues (cyan carbons) affecting the size and shape of the FAD access channel (see bottleneck in A). Adapted from Mathieu et al. (2016).

Fig. 6. Different LPMO activation routes. After initial studies, where LPMO activity was detected using artificial reductants, several mechanisms have been shown to operate “fueling” electrons to the LPMO copper cofactor for the oxidative breakdown (red units) of crystalline cellulose. These alternative mechanisms involve other enzymes, such as CDH (being directly oxidized by LPMO) and GDH (acting through redox cycling of quinones derived from lignin decay or fungal metabolism), as well as lignin-derived phenoxy radicals (being reduced by lignin) and light-activated photosynthetic pigments (Chl). Adapted from Kracher et al. (2016) and Martínez (2016).

Fig. 7. 2D-NMR study on LPMO binding to substrates. His83, one of the copper ligands together with His155 and His1 side-chain and terminal amine (N) (A), participates in cellulose binding on LPMO (C) as shown by strong signal displacement in the 1H-15N heteronuclear single quantum (HSQC) spectra acquired in the absence (black contour) and the presence (cyan contour) of a cellulose hexasaccharide (Glc6) (the unaffected signal of Val42 is shown as reference) (B). More interestingly, a smaller but significant displacement of the above NMR signal in the presence of CDH suggests that the same site, near the catalytic copper, is also involved in binding of CDH, or other copper-reducing molecules, for LPMO activation. The strongest interactions (NMR displacements) correspond to red surface in A, followed by blue surface. Adapted from Courtade et al. (2016).

Fig. 8. Simulations guiding introduction of a new peroxidase activity. A highly-stable peroxidase was engineered for oxidation of a new substrate (2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid], ABTS) using PELE simulations for substrate diffusion in wild and mutant enzyme (left) and electron transfer estimation by quantum calculations after proper docking at the active site (right). The substrate binding residues to be introduced were identified by previous substrate diffusion on an active enzyme, and the new activity of the engineered enzyme was experimentally confirmed by PCR mutagenesis, heterologous expression and estimation of kinetic constants. ABTS-heme distances vs interaction energies in the model active enzyme (red dots) and the wild and mutated target enzyme (blue dots) during PELE simulations are shown in left. Detail of ribbon-type structure with docked ABTS and heme as CPK-colored sticks (other ABTS molecules during PELE diffusion as blue lines) and solvent access surface in grayish blue are shown in right. Adapted from Acebes et al. (2016).

Fig. 9. Directed evolution and enzymatic production of 1-naphthol. A. Fast red-based screening resulting in several UPO variants hydroxylating naphthalene. B. Mutations in the best UPO variant (JaWa) located at the heme channel (top) and the surface (bottom) of the UPO crystal structure. C. HPLC profile of 1-naphtol production by JaWa compared with a second variant (PaDa-I). Adapted from Molina-Espeja et al. (2016a).

Fig. 10. Regioselective hydroxylation by UPO. Regioselective conversion of vitamin D3 (1) into 25-hydroxyvitamin D3 (2) by Coprinopsis cinerea UPO (A) was shown by GC-MS profiles of reactions (B, black line) compared with controls (B, red line), and explained by PELE simulations revealing an optimal minimum for oxygen transfer from heme compound-I to substrate C25 after its diffusion at the heme access channel (C). Double peaks in B are pyro-isomers formed during GC-MS analysis. Substrate and cofactor in C are shown as CPK-colored sticks, while relevant amino acid residues are shown as CPK spheres (part of the solvent access surface is also shown). Adapted from Lucas et al. (2016).

Fig. 11. Synthesis of human metabolites by basidiomycete peroxygenase. A. The bile acid reabsorption inhibitor SAR548304 is N-desmethylated by Marasmius rotula UPO, as shown by HPLC analysis at different reaction times. B. The postulated N-desmethylation mechanism (from 1 to 2) may proceed via an unstable hemiaminal intermediate and formation of N-formyl derivatives (14 + 15) arising from a geminal alcohol intermediate. Adapted from Kiebist et al. (2015).

Fig. 12. Stereoselective hydroxylation in a byproduct-free enzyme cascade. Ethylbenzene conversion into (R)-1-phenyethanol by UPO, at expenses of methanol oxidation to CO2 and H2O, was obtained by increasing the number of cascade steps (A) resulting in higher product yield (B) as shown by green vs blue and red lines and reactions. AaeUPO, Agrocybe aegerita UPO; PpAOx, Pichia pastoris MOX; PpFDM, formaldehyde dismutase from Pseudomonas putida; CbFDH, formate dehydrogenase from Candida boidinii; Rj3HB6H, 3-hydroxybenzoate-6-hydroxylase from Rhodococcus jostii. Adapted from Ni et al. (2016).

Fig. 13. Enzymatic production of FDCA from renewable HMF. A. Scheme for a cosubstrate-free cascade including: i) HMF oxidation by AAO to DFF, whose hydrated (gem-diol) form undergoes a second AAO oxidation yielding FFCA that is not substrate of AAO due to its low hydration degree; and ii) Conversion of unhydrated FFCA into FDCA by UPO at expenses of the H2O2 previously formed by AAO, with H2O as the only by-product. B. Time course of HMF products by the above cascade (GC-MS analysis) with AAO and UPO additions at times 0 and 4 h, respectively (DFF was not detected due to its rapid conversion by AAO). Adapted from Carro et al. (2015).

Fig. 14. Enzymatic indigo dyes for fabric dyeing. A. Proposed reaction mechanism for the UPO-catalyzed formation of different indigo dyes. (1) 1H-indoles; (2) 2,3-epoxy-1H-indoles; (3), 3,3-dihydro-1H-indol-2-ones; (4) 3-hydroxy-1H-indoles; (5) indigos: R= H (indigo), R=Br (6,6'-dibromoindigo) or R= Cl (6,6'-dichloroindigo). B. Oxidized and reduced (soluble) forms of Tyrian purple (6,6'-bromoindigo) during the dyeing process. C. Enzymatic in situ dyeing of cotton fabrics. D. Dyeing persistence after washing one of the above fabrics dyed with enzymatic Tyrian purple. In part (A and B) adapted from Kalum et al. (2014a) and Herold-Majumdar et al. (2016).

Fig. 15. Multifiber test of enzymatic biodyes. Two dyes obtained by laccase copolymerization of 2,5-diaminobenzenesulfonic acid and resorcinol in different ratios (A,B) yielded results on natural and synthetic fibers comparable to a commercial dye (C). Adapted from Pezzella et al. (2016).

Fig. 16. Polyaniline production with a laccase double mutant (DM). A. Formula of conductive polyaniline. B. Two mutations predicted by PELE simulations enabled efficient electron transfer (estimated by QM/MM calculations) from docked aniline (CPK-colored spheres) to laccase copper-1 (cyan spheres) in DM (right) compared with parental laccase (left). C. Experimental demonstration of faster polyaniline production (left) and darker color (right) by DM compared with parental laccase. Adapted from Santiago et al. (2016).

Fig. 17. Scheme of proposed Saccharomyces cerevisiae engineering for its use in lignocellulose biorefinery as a “white-rot yeast”. Adapted from González-Perez (2016).

Potential Impact:
A) Potential impact of industrial biotransformations

i) Impact on the European Chemical sector

In a broader perspective the INDOX project is envisioned to change the competitive basis and future outlook of the European Chemical Industry at large by show-casing a number of scalable industrial bio-processes that can allow chemical companies to compete effectively without having to constantly strive for the lowest possible labor-cost and, at the same time, looking for more sustainable chemical processing. The introduction of innovative and more efficient technologies, as the new enzymatic biocatalysts developed in INDOX, could be a key factor to increase the competitiveness of the European Chemical sector, whose position in the world markets is declining in favor of Asian countries (with pessimistic forecasts for the near future). Given the large dimension of this sector, any competitiveness increase should have a very important economic and social (employment) impact in Europe.

The INDOX choice of oxidation reactions as focus area is important as oxidations can be very difficult to manage with traditional synthesis tools, hence enzymes may not only offer a more sustainable route to produce industrial chemicals, but even be able to offer more cost-effective routes for their manufacture. Depending on the target molecule the development of a new enzymatic route may provide multiple advantages, such as shortening synthesis routes, providing high selectivity and formation of fewer by-products, as well as higher yields and less energy consumption, among others. The much milder reaction conditions usually offered by enzymatic synthesis routes also allow for establishing production plants with much lower capital expense (CAPEX) thus enabling a much more flexible production platform.

Given that the chemical industry is not yet embracing enzymatic oxidation reactions to a significant extent, an important objective of INDOX was to show the efficacy of such reactions for relevant industrial processes, trying to demonstrate that these are scalable as well as to assess the sustainability and cost-efficiency versus existing conversion processes. An important fact is that most of the proposed target reactions/products were known by the partners to be fundamentally feasible from a technical point of view so that the individual work packages served the objective of trying to mitigate the factors limiting the commercial viability (yields, selectivity, total turnover numbers, enzyme-stability, peroxide-cost, etc.).

During the course of the project many reactions and process configurations were tested at the laboratory scale leading to a selection of those giving the most promising results to be further optimized and assessed at a larger scale by the companies involved. This resulted in a preliminary techno-economic analysis of three selected biotransformations (for the enzymatic production of a bulk chemical and a dye-stuff as well as fibre enzymatic treatment for wood panel applications) plus a number of other enzymatic reactions that have being investigated to a significant extent. The results obtained so far, although not yet enabling a direct commercialization at the current stage, are a good example of the potential applicability of enzymatic technologies in industrial processes and have opened the door to a further optimization of the biotransformations. In fact, given the range of potential implementation, the work is already continuing in a new project beyond INDOX funded by the Bio-based Industries Joint Undertaking initiative, focusing on the enzymatic production of bio-based plastic building blocks and polymer precursors as well as flavor and fragrance ingredients and APIs.

ii) Impact on the European Biotechnology sector

The European Biotechnology companies already represent the majority of the global production of industrial enzymes. However, in the context of a greater world, and particularly with the expansion of the Asian markets, it will be necessary to initiate the development of further enzyme-based innovation to ensure a future extension of these companies. In INDOX, the European key-player in enzyme production, Novozymes producing 45% of the industrial enzymes applied worldwide with a company turnover over 1.5 billion €/year, was involved as leading industrial partner. Therefore, the project and the joint development of new oxidoreductases will have a strong impact on this sector and a realistic chance to develop and launch innovative new products to the market in the near future. In addition, a complementary SME, BIOS, dealing with enzyme production for specialty markets (wild-type enzymes for fine chemicals and for research purpose), is an integral part of the consortium.

As the exploitation of the enzymes will be driven not only by the identification of suitable industrial applications, but also by the availability of the commercial biocatalysts required, the INDOX strategy has been directed to develop and optimize these new biocatalysts, their larger scale production and the optimization of the enzyme-based bioprocesses.

Current oxidoreductase enzymes in the market are mainly laccases, phenol-oxidizing peroxidases, sugar oxidases and catalases. Applications are diverse with several enzymes being used in the textile industry as well as in the food industry. However, none of them catalyze oxyfunctionalizations of poorly activated organics, a challenging oxidation type that is catalyzed by cytochrome P450 monooxygenases and the so-called peroxygenases investigated in INDOX, which present several key advantages over P450s (enabling their use in medium/large scale biotransformations) since they are more stable, do neither need auxiliary proteins nor expensive electron donors and can be applied as isolated proteins rather than in whole-cell biotransformations.

Peroxygenases are just an example of the potential of fungal oxidoreductases as oxidative industrial biocatalysts. Other new oxidoreductases (such as lytic-polysaccharide monooxygenases and dye-decolourizing peroxidases), together with tailor-made well-known oxidoreductases (from the groups of laccases, peroxidases and oxidases, among others) also have high potential to catalyze other industrially-relevant oxidative biotransformations, as those investigated in the INDOX project. This will result in very significant growth opportunities for industrial oxidoreductases that today only represent a very small single-digit percentage of the approximately 2.7 billion € market of industrial enzymes (largely dominated by hydrolytic enzymes). In this way, the INDOX project can help to develop several new industrial markets for oxidative enzymes, in particular as large-scale oxidation catalysts for sustainable production of bulk and large-volume specialty chemicals as well as for research use and for production of high-value specialty chemicals and chiral intermediates.

iii) Reducing the environmental impact of the chemical industry

The potential applications of the enzymes and biocatalytic oxidative processes considered in INDOX are directly linked to the concept of Green Chemistry as a carrier for effective environmental sustainability. It is widely accepted that biocatalytic processes offer extensive advantages over traditional chemistry in terms of environmental impact, as a result of milder reactions conditions (physiological pH and temperature), biodegradable enzymatic catalysts that generate less waste, and substitution of organic/toxic solvents by water.

It must also be stressed that the targeted processes and manufacturing sectors for application (e.g. agrochemical production, specialty polymer synthesis, dyes and pulp and paper industries) are some of the most polluting among chemical industries, attending to their air and water emissions (e.g. 36% of all volatile organic compounds released in industrial chemical production correspond to synthetic resin manufacturing, 90% of biological oxygen demand in water is due to pulp and paper industries).

It has been shown that enzyme-catalyzed oxidations result in:
- Reduction of toxic reagents, solvents and metal catalysts
- Reduced consumption of energy and water
- Reduction of waste and CO2 production

The results of the INDOX project open the door to a broader use of optimized oxidoreductase based biocatalysts that will help to attain the above described objectives in many oxidative industrial biotransformations.


The results obtained during the INDOX project have been efficiently disseminated though Scientific publications, Congress presentations, dissemination articles, and the Website of the project.

Eighty-eight scientific publications plus six book chapters have been produced reporting results from studies funded by INDOX. These publications include high impact journals where general aspects of the results obtained are described (such as Science, J. Amer. Chem. Soc., Proc. Natl. Aca. Sci. USA and Nature Commun.), multidisciplinary journals (such as PLoS ONE, Sci. Rep., Cell. Mol. Life Sci. and J. Vis. Exp.) and specialized journals in the areas of Biotechnology (such as Biotechnol. Biofuels, Biofpr, Enzyme Microb. Technol., Trends Biotechnol., Curr. Biotechnol., Biotechnol. Adv. and J. Biotechnol.), Chemistry (such as Green Chem., J. Phys. Chem. B, ChemBioChem., Curr. Opin. Chem. Biol. and Angew. Chem.), Biochemistry (such as J. Biol. Chem., Biochem. J., Biochemistry, FEBS J., Arch. Biochem. Biophys., Biochem. Eng. J., Acta Crystallogr. D. Biol. Crystallogr. and Anal. Biochem.), Genetics (such as PLoS Genetics, Genome Announcements and Fungal Genet. Biol.), Catalysis (such as ACS Catal., ChemCatChem, Catal. Sci. Technol. and J. Mol. Catal. B-Enzym.), Microbiology (such as Appl. Environ. Microbiol., FEMS Microbiol. Ecol., Appl. Microbiol. Biotechnol., AMB Express, and Mycologia) and other specialized journals (such as Ind. Crops Prod., Protein Express. Purif., Protein Sci., Molecules, Adv. Exp. Med. Biol., Food Chem., J. Animal Sci.Biotechnol. Frontiers in Plant Science, Planta, J. Anal. Appl. Pyrolysis, Bioengineered, Biomass Bioenerg. and Bioenerg. Res.). The complete list of publications is included in section 4.2 of this report.

The national and international Congress presentations where INDOX results were disseminated by the different project partners attained a total of two hundred and twenty-five (such as 17th and 18th Meetings of the Spanish Lignocellulose Network in Madrid and Jaen, respectively, Annual Conf. for General and Applied Microbiology in Dresden, DSM Nutritional Products in Basel, VIII Meeting of the Spanish Protein Network in Madrid, MECP14 in Madrid, 10th Int. Conf. on Protein Stabilisation in Stresa, Protein Multiverse Workshop in Madrid, 7th ICEP Conf. in Vitória, JGI User Meeting 2014 and 2015 in Walnut Creek, EWLP-2014 in Seville and EWLP-2016 in Autrans, Lignobiotech-III in Concepción and Lignobiotech-IV in Madrid, OxiZymes-2014 in Vienna and OxiZymes-2016 in Wageningen, SEBBM-2014 in Granada and SEBBM-2015 in Valencia, CMIBM'14 in Oviedo, 18th Int. Symp. on Flavins and Flavoproteins in Phetchaburi, Biotrans-2015 in Vienna, ECFG-2014 in Seville and ECFG-2016 in Paris, JEB 2015 in Madrid, XII BIONNALE meeting in Berlin, 4th Visegrad Symp. on Structural Systems Biology in Nové Hrady, 8th International Conference on Polymer and Fiber Biotechnology in Braga, ICPP8 International Conference on Porphyrins and Phtalocyanines in Istanbul, XXX Annual Meeting of Reference Network on Theoretical and Computational Chemistry in Barcelona, ANQUE-ICC-BIOTEC 2014 in Madrid, 16th European Congress on Biotechnology in Edinburgh, EMBO practical course in Paris, 10th International Mycological Congress in Bangkok, 2014 Int. Conf. “Lignin biosynthesis and utilization”in Umeå, 7th Int. Congress on Biocatalysis, Biotech Meeting in Kookmin University, Seoul, 2nd Transnational Workshop on Genomics and Transcriptomics in Geobiotech and White Biotech in Freiberg, 8th Int. Conf. Mushroom Biology and Mushroom Products in New Delhi, Int. Conf. on Natural Resources, Green Technology and Sustainable Development in Zagreb, Agrocybe-aegerita-Meeting in Goethe-Universität, Frankfurt, Carnival Conference Session in Cologne, Biodiversity Exploratories Conference in Wernigerode, Frühjahrstagung der DECHEMA-Biotechnologen in Frankfurt, XVI Netherlands' Catalysis and Chemistry Conference in Noordwijkerhout, Pavia Workshop on Protein Expression and Crystallization in Brallo di Pregola, 8th Conference on Recombinant Protein in Palma de Maiorca, CBM11 in Espoo, Cell Factories & Biosustainability Conference in Copenhagen, CALTECH and Occidental City Colleage in Pasadena, 29th Annual Symposium of The Protein Society in Barcelona, 2015 Meeting of the Mycological Society of America in Edmonton, Gordon Research Conference on Cellulosomes, Cellulases & Other Carbohydrate Modifying Enzymes in Andover, Gordon Research Conference on Biocatalysis in Biddeford, Biocatalysis & Bioprocessing in Shanghai, 18th International Symposium on Wood, Fibre and Pulping Chemistry in Vienna, IV New trends in Computational Chemistry for Industry Applications in Barcelona, 2nd Lund Symposium on lignin and hemicellulose valorization, CHAINS 2014 and 2015 in Veldhoven, Ecology of soil Microorganisms 2015 in Prague, 3rd Encontro de Jovens Investigadores de Biologia Computacional Estrutura in Coimbra, Mycology Symposium in Zürich, Int. Conf. Molecular Recognition (BiFi) 2015 and 2016 in Zaragoza, 18th and 20th Austrian Carbohydrate Workshop in Vienna, Industrial Biotechnology Forum (IBB) 2016 in Munich, 26th Colloque du CBSO, Evian-les-Bains, EMBO Conference 2016 in Oulu, 2nd Protein Engineering Canada Conference in Ottawa, Enzyme Engineering: Bright Strategies from Theory and Experiments, CECAM-HQ-EPFL in Lausanne, 17th European Congress on Biotechnology (ECB 2016) in Krakow, 7th Int. Symp. Advanced Energy Science in Kyoto, 8th ÖGMBT Annual Meeting in Graz, VI Congr. Microb. Indus. & Biotec Microb. in Leon, Clusterkonferenz Chemie und Kunststoffe in Schwarzheide, 5th Int. Conf. Novel Enzymes in Groningen, BiRT Seminar Series in Tulln and ESIB-2016 in Graz). In the above congresses, ninety-six oral presentations and one hundred and twenty-nine poster presentations disseminated the project results. The complete list of congress presentations is included in section 4.2 of this report.

Moreover several partners have been involved in the organization of the following international conferences where a high number of communications showing INDOX results were presented (as shown in Section 4.2 below):

- 13th European Workshop on Lignocellulosics and Pulp, held in Seville (Spain) on 24-27 June 2014, organized by CSIC (IRNAS + CIB)

- OxiZymes 2014, held in Vienna (Austria) on 1-4 July 2014 organized by BOKU.

- OxiZymes 2016, held in Wageningen (The Netherlands) on 1-3 July 2016 organized by WUR and TUDelft

- Lignobiotech IV Symposium, held in Madrid (Spain) on 19-22 June 2016 organized by CSIC (CIB).

In addition to the above dissemination to the scientific community, some of the main INDOX results were also disseminated to broader audiences by means of the following articles and presentations describing the project scope and main results emphasizing the discovery and engineering of new industrial biocatalysts for a more environmentally-sound and cost-efficient development of the industrial sector:

- “Mushroom enzymes could work cost saving magic for API makers say researchers” published in the portal on 9April 2014.

- “Biocatalysts to improve the chemical industry” presented in the CommBeBiz webinar on “Research projects review developments with bioeconomy industry” held on 10 July 2015.

- “The INDOX Project” presented at the workshop on “Maximising the Impact of KET Biotechnology” held in Brussels on 22 September 2015.

- “Nature to improve chemical industry” published in 2016 CommBeBiz Magazine on Bioeconomy Innovation.

- “Going back to nature for greener production” published in CORDIS Results in brief on 7-Dec-2016

- “Catalysing the EU chemicals sector” (article requested by the European Commission’s DG Research to be published during the coming weeks behind its Infocentre “success stories” tab)

A brochure including a brief description of the project concept, objectives and members was produced at the beginning of the project and is available since then to be downloaded from the INDOX website (see below). In addition, a number of copies were printed and distributed. At the end of the project, a second document summarizing the main results and achievements of the project has been prepared, in the form of a scientific publication submitted to an Open Access journal. The publication will also be available from the INDOX website once it has been published.

Finally, The INDOX website ( merits a special mention since, from the beginning of the project, provided to the scientific community, and the interested general audience, updated information on the specific project advances and resulting publications, together with general information on the pursued objectives, the composition of the consortium and expertise of the different partners, and different related events and relevant international news, providing links to the corresponding internet pages.


As explained above, the multidisciplinary approach undertaken in INDOX has been supported by a highly-specialized consortium of SMEs, large companies and research/academic institutions. Production of the new optimized biocatalysts and their introduction into the chemical market takes advantage from the participation of the world-leading company in the sector of industrial enzymes, together with some chemical companies willing to implement new medium- and large-scale biotransformation processes. The following patents covering specific findings and/or applications of the oxidoreductases investigated in INDOX were submitted in the course of the project:

1. Alcalde, M., Molina-Espeja, P., García-Ruiz, E., González-Pérez, D., Ullrich, R. y Hofrichter, M. (2014) Unspecific peroxygenase with high monooxygenase activity. Patent (Spain) P201430595-1 (application submitted)

2. Gómez de Santos, P., Molina-Espeja, P., Plou, F. y Alcalde, M. (2016) Unspecific peroxygenase mutants with high monooxygenase activity and their uses. PCT/ES2016/070809 (Patent application submitted)

3. Herold-Majumdar, O.M. Tovborg, M., Hofrichter, M., Lund, H. y Poraj-Kobielska, M. (2016) Enzymatic production of indigo. Patent application submitted

4. Hollmann, F. y Ni, Y. (2016) Enzymatic conversion using hydrogen peroxide. Patent (NL) NL2013351A

5. Kalum, L., Morant, M.D. Lund, H., Jensen, J., Lapainaite, I., Soerensen, N.H. Pedersen, S., Østergaard, L. y Xu, F. (2014) Enzymatic oxidation of 5-hydroxymethylfurfural and derivatives thereof. Patent (International) WO2014-015256A2, US20150152452A1

6. Kalum, L., Lund, H., Hofrichter, M. y Ullrich, R. (2014) Enzymatic preparation of indigo dyes and intermediates. Patent (International) WO2014122109A1:

7. Landvick, S., Ostergaard, L.H. y Kalum, L. (2016) Polypeptides Having Peroxygenase Activity and Polynucleotides Encoding Same. Patent (USA) US20160244731A1:

8. Landvick, S., Ostergaard, L.H. y Kalum, L. (2016) Polypeptides having peroxygenase activity. Patent (International) WO2014056916A3:

9. Lund, H., Brask, J., Kalum, L., Gutiérrez, A., Babot, E.D. Ullrich, R., Hofrichter, M., Martínez, A.T. y del Rio, J.C. (2014) Enzymatic preparation of diols. Patent (USA) US20140234917A1:

10. Molina-Espeja, P., Plou, F. y Alcalde, M. (2015) Unspecific peroxygenase mutants with high monooxygenase activity and their applications. Patent application submitted (Spain) P201531641

11. Poraj-Kobielska, M., Gröbe, G., Kiebist, J., Grün, M., Ullrich, R., Scheibner, K. y Hofrichter, M. (2015) Verfahren zur Deacylierung von Corticoiden. Patent (Germany) DE102014005371:

12. Vind, J., Ostergaard, L.H. de Leonardo, M., Kalum, L. y Amougi, E. (2015) Peroxygenase variants. Patent (International) WO2015079064A3:

Additional details on the above list are described in Section 4.2 B below.

List of Websites:
Project website:

Project Coordinator:
Prof Angel T. Martínez
Mobile phone: 34 659960911

Project Secretariat:
Dr Marta Pérez-Boada
Mobile phone: 34 650080476

Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC)
Biological Research Centre (CIB)
Calle Ramiro de Maeztu 9, E-28040 Madrid, Spain
Phone: 34 918373112; Fax: 34 915360432