Vincenzo Lettera

Induction and transcriptional regulation of laccases in fungi.

Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet.

Transcriptional analysis of Pleurotus ostreatus laccase genes.

Fungal laccases (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) are multi-copper-containing oxidases that catalyse the oxidation of a great variety of phenolic compounds and aromatic amines through simultaneous reduction of molecular oxygen to water. Fungi generally produce several laccase isoenzymes encoded by complex multi-gene families. The Pleurotus ostreatus genome encodes 11 putative laccase coding genes, and only six different laccase isoenzymes have been isolated and characterised so far. Laccase expression was found to be regulated by culture conditions and developmental stages even if the redundancy of these genes still raises the question about their respective functions in vivo. In this context, laccase transcript profiling analysis has been used to unravel the physiological role played by the different isoforms produced by P. ostreatus. Even if reported results depict a complex picture of the transcriptional responses exhibited by the analysed laccase genes, they were allowed to speculate on the isoform role in vivo. Among the produced laccases, LACC10 (POXC) seems to play a major role during vegetative growth, since its transcription is downregulated when the fungus starts the fructification process. Furthermore, a new tessera has been added to the puzzling mosaic of the heterodimeric laccase LACC2 (POXA3). LACC2 small subunit seems to play an additional physiological role during fructification, beside that of LACC2 complex activation/stabilisation.

Identification of a new member of Pleurotus ostreatus laccase family from mature fruiting body.

Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. Most of the known laccases have fungal or plant origins, although few laccases have been also identified in bacteria and insects. Most of the fungal laccases reported thus far are extra-cellular enzymes, whereas only few enzymes from fruiting bodies have been described so far. Multiple isoforms of laccases are usually secreted by each fungus depending on species and environmental conditions. As a fact, a laccase gene family has been demonstrated in the white-rot fungus Pleurotus ostreatus. This work allowed identification and characterization of the first laccase isoenzyme from the fruiting body of P. ostreatus. Discovery through mass spectrometry of LACC12 proves the expression of a functional protein by the related deduced encoding transcript. The topology of phylogenetic tree of fungal laccases proves that LACC12 falls in cluster with the members of P. ostreatus LACC10 (=POXC) subfamily, although lacc12 deduced intron-exon structure differs from that of the subfamily members and the related locus is located in a different chromosome. Results show that the evolutionary pattern of lacc12 and that of the other laccase isozyme genes may have evolved independently, possibly through duplication-divergence events. The reported data add a new piece to the knowledge about P. ostreatus laccase multigene family and shed light on the role(s) played by individual laccase isoforms in P. ostreatus.

Efficient immobilization of a fungal laccase and its exploitation in fruit juice clarification.

The clarification step represents, in fruit juices industries, a bottleneck process because residual phenols cause severe haze formation affecting juice quality and impairing customers acceptance. An enzymatic step can be efficiently integrated in the process, and use of immobilized enzymes entails an economical advantage. In this work, covalent immobilization of recombinant POXA1b laccase from Pleurotus ostreatus on epoxy activated poly(methacrylate) beads was optimized thanks to a Response Surface Methodologies approach. Through regression analysis the process was well fitted by a quadratic polynomial equation (R(2)=0.9367, adjusted R(2)=0.8226) under which laccase activity reached 2000 ± 100 Ug(-1) of beads, with an immobilization efficiency of 98%. The immobilized biocatalyst was characterized and then tested in fruit juice clarification reaching up to 45% phenol reduction, without affecting health-effective flavanones content. Furthermore, laccase treated juice displays an improved sensory profile, due to the reduction of vinyl guaiacol, a potent off-flavor possessing a peppery/spicy aroma.

Ligand engagement on material surfaces is discriminated by cell mechanosensoring

Peptide or protein ligands can be used for molecular decoration to enhance the functionality of synthetic materials. However, some skepticism has arisen about the efficacy of such strategy in practical contexts since serum proteins largely adsorb. To address this issue, it is crucial to ascertain whether a chemically conjugated integrin-binding peptide is fully recognized by a cell even if partially covered by a physisorbed layer of serum protein; in more general terms, if competitive protein fragments physisorbed onto the surface are distinguishable from those chemically anchored to it. Here, we engraft an RGD peptide on poly-ε-caprolactone (PCL) surfaces and follow the dynamics of focal adhesion (FA) and cytoskeleton assembly at different times and culture conditions using a variety of analytical tools. Although the presence of serum protein covers the bioconjugated RGD significantly, after the first adhesion phase cells dig into the physisorbed layer and reach the submerged signal to establish a more stable adhesion structure (mature FAs). Although the spreading area index is not substantially affected by the presence of the RGD peptide, cells attached to chemically bound signals develop a stronger adhesive interaction with the materials and assemble a mechanically stable cytoskeleton. This demonstrates that cells are able to discriminate, via mechanosensoring, between adhesive motives belonging to physisorbed proteins and those firmly anchored on the material surface.

Classical breeding in Pleurotus ostreatus: a natural approach for laccase production improvement

White-rot basidiomycetes, the most common wood-rotting organisms, are characterized by their ability to produce extracellular oxidative enzymes, among which laccases are regarded as promising catalysts for many biotechnological applications. A significant obstacle to the exploitation of laccase-based bioprocesses is the large amounts of enzyme required. In this study the issue has been addressed by applying a classical breeding approach to increase laccase production yields in the white-rot fungus Pleurotus ostreatus. Starting from two different P. ostreatus varieties, three higher laccase-producing hybrids have been obtained by crossing selected compatible monokaryons. The three selected strains increased the titre of parental strains up to four fold, reaching an expression level of up to 100 000 U/L. One hybrid exhibited a more complex isoenzyme pattern, illustrating the potential of classical breeding to differentiate protein expression.

A step forward in laccase exploitation: Recombinant production and evaluation of techno-economic feasibility of the process

Protein heterologous production offers viable opportunities to tailor laccase properties to specific industrial needs. The high redox potential laccase POXA1b from Pleurotus ostreatus was chosen as case study of marketable enzyme, due to its desirable properties in terms of activity/stability profile, and already assessed applicability. POXA1b was heterologously produced in Pichia pastoris by investigating the effect of inducible and constitutive expression systems on both the yield and the cost of its production. System performances were first assessed in shaken-flasks and then scaled-up in bioreactor. The production level obtained in the inducible system is 42U/mL, while the activity value achieved with the constitutive one is 60U/mL, the highest obtained in constitutive systems so far. The economic feasibility of recombinant laccase production was simulated, describing the case of an Italian small-medium enterprise. Two scenarios were evaluated: Scenario (I) production based on methanol inducible system; Scenario (II) production based on the constitutive system, fed with glycerol. At all the scales the glycerol-based fermentation is more economic than the methanol-based one. The price forecast for rPOXA1b production is 0.34€kU-1 for glycerol-based process, and is very competitive with the current price of commercial laccase.

Cell mechanosensory recognizes ligand compliance at biomaterial interface

Cells activate signalling through ligand-receptor bonds by sensing the mechanical properties of the surrounding extracellular matrix (ECM). Ligands, indeed, have to withstand the pulling force elicited by cell receptors through focal adhesions (FAs). On this basis, we developed functional ligands to be simply adsorbed on surfaces and constituted by a two-domain peptide: one derived from ECM proteins and available to receptors to offer biochemical cues, and another adsorbed on material to withstand the tension upon receptor engagement. Tuneable compliance of the anchoring domain of the peptide ligand was verified by single peptide analysis through molecular dynamics and adsorption measurements. We showed that the highest adsorbed peptides combined with integrin cell-binding motifs allow for the cell recognition and polarization with larger mature FA areas. On the contrary, the lowest adsorbed sequences did not provide mechanical resistance to the integrin pulling action, leading to more rounded cells with smaller FA areas. This evidence demonstrates that cell mechanosensory can discriminate ligands on surfaces and should be considered as a criterion in ligand design for material bioactivation.

Laccase pretreatment for agrofood wastes valorization

Apple pomace, potato peels, and coffee silverskin are attractive agrofood wastes for the production of biofuels and chemicals, due to their abundance and carbohydrate content. As lignocellulosic biomasses, their conversion is challenged by the presence of lignin that prevents hydrolysis of polysaccharides, hence demanding a pretreatment step. In this work, the effectiveness of Pleurotus ostreatus laccases (with and without mediator) to remove lignin, improving the subsequent saccharification, was assessed. Optimized conditions for sequential protocol were set up for all agrofood wastes reaching delignification and detoxification yields correlated with high saccharification. Especially noteworthy were results for apple pomace and coffee silverskin for which 83% of and 73% saccharification yields were observed, by using laccase and laccase mediator system, respectively. The herein developed sequential protocol, saving soluble sugars and reducing the amount of wastewater, can improve the overall process for obtaining chemicals or fuels from agrofood wastes.