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ABSTRACTA novel fungal species able to synthesize enzymes with potential synergistic actions in lignocellulose conversion was isolated from the biomass of Arundo donaxduring biodegradation under natural conditions in the Gussone Park of the Royal Palace of Portici (Naples, Italy). In this work, this species was subjected to morphological and phylogenetic analyses. Sequencing of its genome was performed, resulting in 28 scaffolds that were assembled into 27.05 Mb containing 9744 predicted genes, among which 396 belong to carbohydrate-active enzyme (CAZyme)-encoding genes. Here we describe and illustrate this previously unknown species, which was named Talaromyces borbonicus, by a polyphasic approach combining phenotypic, physiological, and sequence data.

Poly-3-hydroxybutyrate (PHB) is a biodegradable polymer produced by many bacteria. Some of the properties are similar to thermoplastics like polypropylene, hence finding application PHB can directly replace non-biodegradable polymers. But the main barrier has been the cost difference. The utilization of mixed microbial cultures facilitates the use of complex substrates and thus can reduce the cost of PHB production. In the present study, mixed culture systems, where metabolite produced by one organism may be assimilated by the other organism, were employed. Bacillus firmusNII 0830, the first organism, was used for the production of PHB since it accumulates a large amount of PHB, while the second organism, Lactobacillus delbrueckiiNII 0925, was used to provide acetic acid. Enzyme kinetic studies were performed on the PHB biosynthetic enzymes, such as β-ketothiolase, acetoacetyl CoA reductase and PHB synthase. PHB production by mixed culture was higher when compared to pure cultures. The mathematical model was then fitted to the experimental data, which can describe the dynamics of a mixed culture. The β-ketothiolase and acetoacetyl CoA reductase showed a Vmax value of 0.0093 μM/min and 0.0253 μM/min, respectively. The Kmvalues were 140.8 μM and 183.5 μM, respectively. The enzyme kinetic studies gave an idea about the action of the enzymes.

Poly-3-hydroxybutyrate (PHB) is a biodegradable polymer produced by many bacteria. Some of the properties are similar to thermoplastics like polypropylene, hence finding application PHB can directly replace non-biodegradable polymers. But the main barrier has been the cost difference. The utilization of mixed microbial cultures facilitates the use of complex substrates and thus can reduce the cost of PHB production. In the present study, mixed culture systems, where metabolite produced by one organism may be assimilated by the other organism, were employed. Bacillus firmusNII 0830, the first organism, was used for the production of PHB since it accumulates a large amount of PHB, while the second organism, Lactobacillus delbrueckiiNII 0925, was used to provide acetic acid. Enzyme kinetic studies were performed on the PHB biosynthetic enzymes, such as β-ketothiolase, acetoacetyl CoA reductase and PHB synthase. PHB production by mixed culture was higher when compared to pure cultures. The mathematical model was then fitted to the experimental data, which can describe the dynamics of a mixed culture. The β-ketothiolase and acetoacetyl CoA reductase showed a Vmax value of 0.0093 μM/min and 0.0253 μM/min, respectively. The Km values were 140.8 μMand 183.5 μM, respectively. The enzyme kinetic studies gave an idea about the action of the enzymes.

Research on regulation of cellulases and hemicellulases gene expression may be very useful for increasing the production of these enzymes in their native producers. Mechanisms of gene regulation of cellulase and hemicellulase expression in filamentous fungi have been studied, mainly in Aspergillus and Trichoderma. The production of these extracellular enzymes is an energy-consuming process, so the enzymes are produced only under conditions in which the fungus needs to use plant polymers as an energy and carbon source. Moreover, production of many of these enzymes is coordinately regulated, and induced in the presence of the substrate polymers. In addition to induction by mono- and oligo-saccharides, genes encoding hydrolytic enzymes involved in plant cell wall deconstruction in filamentous fungi can be repressed during growth in the presence of easily metabolizable carbon sources, such as glucose. Carbon catabolite repression is an important mechanism to repress the production of plant cell wall degrading enzymes during growth on preferred carbon sources. This manuscript reviews the recent advancements in elucidation of molecular mechanisms responsible for regulation of expression of cellulase and hemicellulase genes in fungi.

Laccases represent versatile catalysts being able to oxidize a wide range of aromatic substrates and are susceptible of several industrial applications based on both oxidative degradation reactions and synthetic chemistry. The range of laccase based synthetic reactions is extremely wide. Laccases are able to catalyze transformation of antibiotics based on both -lactams functionalization and phtalides functionalization. These enzymes can also catalyze derivatization of amino acids to obtain metabolically stable amino acid analogues, maximizing biological response while minimizing toxicity, thus representing an useful system for drug development. Biomolecules having antioxidative and anticancer activity can also be produced by laccase-mediated reactions of flavonoids oxidative coupling and phenoxazinones synthesis. Application of laccases to production of new derivatives of the hormones resveratrol, 17ß-estradiol, totarol and isoeugenol and oligomerization products of substituted imidazoles was also reported, with applications for pharmacological purposes due to hormonal activity of the products. The enzymatic preparation of aromatic polymeric materials by the action of laccases represents a viable and non-toxic alternative to the usual formaldehyde-based chemical production of these compounds and it has been reported for several substrates such as 2,6-dimethylphenol, 4-hydroxybenzoic acid derivatives, 3,5-dimethoxy-4-hydroxybenzoic acid and 3,5-dimethyl-4-hydroxybenzoic acid, aniline and acrylamide. Moreover, laccase-mediated biografting of phenols or certain other types of low-molecular weight compounds provides a method for tailoring the surface of lignocellulosics or for adhesion enhancement in binderless wood boards under mild conditions and usually without harmful solvents. Laccase-mediated modification of lignocellulosic materials is accomplished through two main routes: coupling of low-molecular weight compounds onto lignocellulosic materials and laccase mediated cross-linking of lignin molecules in situ. Depending on the choice of laccase substrate, properties such as improved strength properties, increased antimicrobial resistance, or hydrophilicity/ hydrophobicity can be imparted to lignocellulosic materials.

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.

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching, and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry.

A new laccase isoenzyme (POXA1b, where POX is phenol oxidase), produced by Pleurotus ostreatus in cultures supplemented with copper sulphate, has been purified and fully characterized. The main characteristics of this protein (molecular mass in native and denaturing conditions, pI and catalytic properties) are almost identical to the previously studied laccase POXA1w. However, POXA1b contains four copper atoms per molecule instead of one copper, two zinc and one iron atom per molecule of POXA1w. Furthermore, POXA1b shows an unusually high stability at alkaline pH. The gene and cDNA coding for POXA1b have been cloned and sequenced. The gene coding sequence contains 1599 bp, interrupted by 15 introns. Comparison of the structure of the poxa1b gene with the two previously studied P. ostreatus laccase genes (pox1 and poxc) suggests that these genes belong to two different subfamilies. The amino acid sequence of POXA1b deduced from the cDNA sequence has been almost completely verified by means of matrix-assisted laser desorption ionization MS. It has been demonstrated that three out of six putative glycosylation sites are post-translationally modified and the structure of the bound glycosidic moieties has been determined, whereas two other putative glycosylation sites are unmodified.

It is my great pleasure as a Guest Editor of the journal Current Genomics to present you with a ‘hot topic issue’ on fungal laccase genes. Fungi are sources of several enzymes susceptible of applications in many and diverse industrial segments such as food, beverage, textile, leather, paper and pulp, animal feed and fuel industry. These microorganisms are safe, display efficient growth under industrial production conditions and are able to secrete ample quantities of enzymes. The main fungal enzymes having industrial relevance include cellulases, xilanases, amylases, proteases, lipases, laccases, etc. Among these, laccases are blue multicopper oxidases (MCO), using the distinctive redox ability of copper ions to catalyze the oxidation of a wide range of aromatic substrates concomitantly with the reduction of molecular oxygen to water. Catalytic properties of laccases and their low substrate specificity allow their wide application in several industrial sectors such as pulp and paper, textile and cosmetic industries, for detoxification and decoloration of sewage, in organic synthesis, for degradation of xenobiotics and bioremediation, in production of wood-fiber plates, wood blocks, and cardboard without using toxic linkers, for production of detergents and in elaboration of biosensors. Much attention has been focused on isolation and recombinant expression of genes coding fungal laccases. Great interest is currently drawn by to development of tailor-made enzyme variants more appropriate for specific applications through protein engineering techniques. This issue is aimed at reporting reviews on the main results in isolation of fungal laccase genes and their recombinant expression and on the most significant advances in their protein engineering. The scope of this issue is to provide a compendium on currently available molecular tools for application of laccases from different sources. Kues and Ruhl reported an overview of laccase and other MCO genes encoded in genomes of basidiomycetes, along with a description of their phylogenetic analysis and related functions. Poggeler described MCO coding capacity of ascomycetes. Piscitelli et al. revised most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters. 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. Mate et al. described fungal laccases engineering by directed evolution to improve their functional expression or stability, using Saccharomyces cerevisiae as a heterologous host laccase. The low redox potential laccase from Myceliophthora thermophila was the first successful example and since then, specific approaches for the laboratory evolution of HRPLs that combine in vivo and in vitro tools with rational approaches were designed. The evolved fungal laccases mentioned in this review constitute platforms for further protein engineering through directed evolution, principally aimed at generating enzymes that can be used in attractive biotechnological applications. Robert et al. reported an overview on strategies and results of design of new biocatalysts based on laccases from Trametes sp. C30 by protein engineering. I would like to sincerely thank all the reviewers for their valuable suggestions to improve the quality of review articles. Special thanks to Editor-in-Chief Dr. Christian Neri for encouragement for this special issue. It was a great opportunity for me to interact with scientists from different European countries. I am convinced this issue will be useful for the scientists, academicians, industry professionals and students. I will be looking forward to editing another issue on other industrially relevant classes of fungal enzymes in future.