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[EN] Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5- formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.

Employing the homologous DNA recombination apparatus of Saccharomyces cerevisiae as a dynamic engineering tool allows mutant libraries to be constructed in a rapid and efficient manner. Among the plethora of methods based on the yeast’s splicing apparatus, site-directed recombination (SDR) is often useful to gather information from mutations discovered in directed evolution experiments. When using SDR, the target gene is divided in segments carrying the selected mutation positions so that the resulting PCR fragments show 50% mutated and 50% wild type residues at the codons of interest. The PCR products are then assembled and cloned into yeast through one-pot transformations with the help of homologous overlapping flanking regions. By screening SDR libraries, the effect of the mutations/reversions at the different positions can be rapidly sorted out in a combinatorial manner. As such, SDR can serve as the `final polishing step´ in a laboratory evolution campaign, revealing beneficial synergies among mutations and/or overriding deleterious mutations. In practice, using SDR it is possible to discern between beneficial and negative epistasis, that is, it should be possible to collect positive synergistic mutations while discarding detrimental substitutions that affect the enzyme’s fitness.

[EN] Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.

[EN] The E-factor has become an important measure for the environmental impact of (bio)chemical reactions. However, summing up the obvious wastes generated in the laboratory neglects energy-related wastes (mostly greenhouse gases) which are generated elsewhere. To estimate these wastes, we propose to extend the E-factor by an energy-term (E-factor). At the example of a lab-scale enzyme fermentation, we demonstrate that the E-factor can constitute a multiple of the classical E-factor and therefore must not be neglected striving for a holistic estimation of the environmental impact.

[EN] By mimicking the role of human liver P450 monooxygenases, fungal unspecific peroxygenases (UPOs) can perform a range of highly selective oxyfunctionalization reactions on pharmacological compounds, including O-dealkylations and hydroxylations, thereby simulating drug metabolism. Here we have benchmarked human drug metabolite (HDM) synthesis by several evolved UPO mutants, focusing on dextromethorphan, naproxen and tolbutamide. The HDM from dextromethorphan was prepared at the semi-preparative scale as a proof of production. The structural analysis of mutations involved in the synthesis of HDMs highlights the heme access channel as the main feature on which to focus when designing evolved UPOs. These variants are becoming emergent tools for the cost-effective synthesis of HDMs from next-generation drugs.

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 tra

Enzymatic production of C–N heteropolymeric dyes at alkaline pHs is an attractive process for the textile industry. In this work, we have designed a fungal laccase by directed evolution so that it may be used at alkaline pHs for the synthesis of C–N heteropolymeric dyes (C–N polydye) from catechol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA). Firstly, several medium- and high-redox potential fungal laccases from previous laboratory evolution campaigns were benchmarked for the synthesis of the C–N polydye at pH 8.0, choosing an alkaline laccase mutant from Myceliophthora thermophila as the departure point for further engineering. Mutant libraries were then constructed, expressed in Saccharomyces cerevisiae and screened using a high-throughput colorimetric assay for the detection of the C–N polydye. By combining directed and focused molecular evolution, a novel, strongly expressed alkaline laccase variant was identified. This laccase was secreted at 37 mg/L and its catalytic efficiency for the oxidation of catechol and 2,5-DABSA at pH 8.0 was enhanced 3.5-fold relative to that of the wild-type, promoting the synthesis of the C–N polydye at basic pHs. While the improved expression was mostly the result of accumulating mutations that favor the yeast’s codon usage together with the recovery of a secretion mutation, the enhanced C–N polydye synthetic activity of the mutant laccase was dependent on the alkaline mutations it inherited. Readily secreted, this laccase mutant would appear to be a valuable platform for organic synthesis at basic pHs.

The peroxygenase from Agrocybe aegerita (AaeUPO) has been evaluated for stereoselective oxyfunctionalization chemistry under non-aqueous reaction conditions. The stereoselective hydroxylation of ethylbenzene to (R)-1-phenylethanol was performed in neat substrate as reaction medium together with the immobilized biocatalyst and tertBuOOH as oxidant. Stability and activity issues still have to be addressed. Nevertheless, gram-scale production of enantiopure (R)-1-phenylethanol was achieved with respectable 90,000 turnovers of the biocatalyst.

For more than thirty years, biotechnology has borne witness to the power of directed evolution in designing molecules of industrial relevance. While scientists all over the world discuss the future of molecular evolution, dozens of laboratory-designed products are being released with improved characteristics in terms of turnover rates, substrate scope, catalytic promiscuity or stability. In this review we aim to present the most recent advances in this fascinating research field that are allowing us to surpass the limits of nature and apply newly gained attributes to a range of applications, from gene therapy to novel green processes. The use of directed evolution in non-natural environments, the generation of catalytic promiscuity for non-natural reactions, the insertion of unnatural amino acids into proteins or the creation of unnatural DNA, is described comprehensively, together with the potential applications in bioremediation, biomedicine and in the generation of new bionanomaterials. These successful case studies show us that the limits of directed evolution will be defined by our own imagination, and in some cases, stretching beyond that.

The ligninolytic enzyme consortium is one of the most-efficient oxidative systems found in nature, playing a pivotal role during wood decay and coal formation. Typically formed by high redox-potential oxidoreductases, this array of enzymes can be used within the emerging lignocellulose biorefineries in processes that range from the production of bioenergy to that of biomaterials. To ensure that these versatile enzymes meet industry standards and needs, they have been subjected to directed evolution and hybrid approaches that surpass the limits imposed by nature. This Opinion article analyzes recent achievements in this field, including the incipient groundbreaking research into the evolution of resurrected enzymes, and the engineering of ligninolytic secretomes to create consolidated bioprocessing microbes with synthetic biology applications.

Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (~8. mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217. mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production.

[EN] The ligninolytic enzyme consortium is one of the most-efficient oxidative systems found in nature, playing a pivotal role during wood decay and coal formation. Typically formed by high redox-potential oxidoreductases, this array of enzymes can be used within the emerging lignocellulose biorefineries in processes that range from the production of bioenergy to that of biomaterials. To ensure that these versatile enzymes meet industry standards and needs, they have been subjected to directed evolution and hybrid approaches that surpass the limits imposed by nature. This Opinion article analyzes recent achievements in this field, including the incipient groundbreaking research into the evolution of resurrected enzymes, and the engineering of ligninolytic secretomes to create consolidated bioprocessing microbes with synthetic biology applications.

[EN] Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate >green catalysts>. This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts.

High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnology. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific composition of human blood (above neutral pH, high chloride concentration) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chemical syntheses, and detoxification.

A modification of the classical calcium alginate enzyme entrapment technique is described aiming to overcome some of the limitations of the former gel-based biocatalysts. Dried alginate entrapped enzymes (DALGEEs) were obtained dehydrating calcium alginate gel beads containing entrapped enzymes. A fructosyltransferase from Aspergillus aculeatus, present in Pectinex Ultra SP-L, was entrapped using this technique. The resulting DALGEEs were successfully tested both operating batchwise and in a continuous fixed-bed reactor for fructooligosaccharides (FOS) synthesis from sucrose. Interestingly, DALGEEs did not re-swell upon incubation in concentrated (600 g/L) sucrose solutions, probably due to the lowered water activity (aw) of such media. Confocal laser scanning microscopy of DALGEEs revealed that the enzyme molecules accumulated preferably in the shell of the particles. DALGEEs showed an approximately 30-fold higher volumetric activity (300 U/mL) compared with the calcium alginate gel beads. Moreover, a significant enhancement (40-fold) of the space-time-yield of fixed-bed bioreactors was observed when using DALGEEs as biocatalyst compared with gel beads (4030 g/day L of FOS vs. 103 g/day L). The operational stability of fixed-bed reactors packed with DALGEEs was extraordinary, providing a nearly constant FOS composition of the outlet during at least 700 h. It was also noticeable their resistance against microbial attack, even after long periods of storage at room temperature. The DALGEEs immobilisation strategy may also be useful for other biotransformations, in particular when they take place in low aw media. © 2013 Elsevier Ltd.

The Myceliophthora thermophila laccase was covalently immobilized on polymethacrylate-based polymers (Sepabeads EC-EP3 and Dilbeads NK) activated with epoxy groups. The enzyme immobilized on Sepabeads EC-EP3 exhibited notable activity (203 U/g) along with remarkably improved stability towards pH, temperature and storage time, but no increased resistance to organic solvents. In addition, the immobilized laccase also showed good operational stability, maintaining 84% of its initial activity after 17 cycles of oxidation of ABTS. The immobilized biocatalyst was applied to the decolorization of six synthetic dyes. Immobilized laccase retained 41% activity in the decolorization of Methyl Green in a fixed-bed reactor after five cycles. The features of these biocatalysts are very attractive for their application on the decolorization of dyes in the textile industry in batch and continuous fixed-bed bioreactors. To our knowledge, this is the first report on immobilization of laccase on Sepabeads carriers and its efficient dyes decolorization.

We have purified a fructosyltransferase from Aspergillus aculeatus present in commercial samples of Pectinex Ultra SP-L. We have also isolated and purified extracellular beta-fructofuranosidases from cultures of the yeasts Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma, Schwanniomyces occidentalis and Rhodotorula gracilis. The transferase activity of these enzymes was analysed in detail, along with that of beta-fructofuranosidase from Saccharomyces cerevisiae. Interestingly, the maximum FOS yield (expressed as weight percentage of the total amount of carbohydrates in the mixture) was significantly higher with the enzyme from A. aculeatus (61%), compared with those from X. dendrorhous, S. occidentalis and R. gracilis (17-19 %), and that from S. cerevisiae (8%). In addition, significant differences were encountered in the selectivity of the process: A. aculeatus synthesized only 1F-FOS, X. dendrorhous formed basically neo-kestose, whereas S. occidentalis and S. cerevisiae were selective towards the production of 6-kestose. R. gracilis synthesized a complex mixture of di-, tri- and tetrasaccharides.

The transglycosylation activity of a novel alpha-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with alpha-(1-2), alpha-(1-4) and alpha-(1-6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one alpha-(1-6) linkage (panose, 6-O-alpha-glucosyl-maltotriose and 6-O-alpha-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the alpha-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri- and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial alpha-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides.

The synthesis of methyl alpha-D-glucooligosaccharides, using sucrose as glucosyl donor and methyl alpha-D-glucopyranoside as acceptor, was studied with dextransucrase from Leuconostoc mesenteroides NRRL B-1299. The enzyme was immobilized by entrapment in alginate. By NMR and mass spectrometry we identified three homologous series (S1-S3) of methyl alpha-D-glucooligosaccharides. Series S2 and S3 were characterized by the presence of alpha(1-2) linkages, in combination with alpha(1-6) bonds. Two parameters, sucrose to acceptor concentration ratio (S/A) and the total sugar concentration (TSC) determined the yield of methyl alpha-D-glucooligosaccharides. The maximum concentration achieved of the first acceptor product, methyl alpha-D-isomaltoside, was 65 mM using a S/A 1:4 and a TSC of 336 g l-1. When increasing temperature, a shift of selectivity towards compounds containing alpha(1-2) bonds was observed. The formation of leucrose as a side process was very significant (reaching values of 32 g l-1) at high sucrose concentrations.