Current Genomics - Volume 12, Issue 2, 2011

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.

Genome analyses revealed in various basidiomycetes the existence of multiple genes for blue multi-copper oxidases (MCOs). Whole genomes are now available from saprotrophs, white rot and brown rot species, plant and animal pathogens and ectomycorrhizal species. Total numbers (from 1 to 17) and types of mco genes differ between analyzed species with no easy to recognize connection of gene distribution to fungal life styles. Types of mco genes might be present in one and absent in another fungus. Distinct types of genes have been multiplied at speciation in different organisms. Phylogenetic analysis defined different subfamilies of laccases sensu stricto (specific to Agaricomycetes), classical Fe2+-oxidizing Fet3-like ferroxidases, potential ferroxidases/laccases exhibiting either one or both of these enzymatic functions, enzymes clustering with pigment MCOs and putative ascorbate oxidases. Biochemically best described are laccases sensu stricto due to their proposed roles in degradation of wood, straw and plant litter and due to the large interest in these enzymes in biotechnology. However, biological functions of laccases and other MCOs are generally little addressed. Functions in substrate degradation, symbiontic and pathogenic intercations, development, pigmentation and copper homeostasis have been put forward. Evidences for biological functions are in most instances rather circumstantial by correlations of expression. Multiple factors impede research on biological functions such as difficulties of defining suitable biological systems for molecular research, the broad and overlapping substrate spectrum multi-copper oxidases usually possess, the low existent knowledge on their natural substrates, difficulties imposed by low expression or expression of multiple enzymes, and difficulties in expressing enzymes heterologously.

Multicopper oxidases (MCO) catalyze the biological oxidation of various aromatic substrates and have been identified in plants, insects, bacteria, and wood rotting fungi. In nature, they are involved in biodegradation of biopolymers such as lignin and humic compounds, but have also been tested for various industrial applications. In fungi, MCOs have been shown to play important roles during their life cycles, such as in fruiting body formation, pigment formation and pathogenicity. Coprophilous fungi, which grow on the dung of herbivores, appear to encode an unexpectedly high number of enzymes capable of at least partly degrading lignin. This study compared the MCO-coding capacity of the coprophilous filamentous ascomycetes Podospora anserina and Sordaria macrospora with closely related non-coprophilous members of the order Sordariales. An increase of MCO genes in coprophilic members of the Sordariales most probably occurred by gene duplication and horizontal gene transfer events.

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.

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution.

Laccases (p-diphenol oxidase, EC 1.10.3.2) are blue multicopper oxidases that catalyze the reduction of dioxygen to water, with a concomitant oxidation of small organic substrates. Since the description at the end of the nineteenth century of a factor catalyzing the rapid hardening of the latex of the Japanese lacquer trees (Rhus sp.) exposed to air laccases from different origins (plants, fungi bacteria) have been continuously discovered and extensively studied. Nowadays, molecular evolution and other powerful protein modification techniques offer possibilities to develop tailored laccases for a wide array of applications including drug synthesis, biosensors or biofuel cells. Here, we give an overview on strategies and results of our laboratory in the design of new biocatalysts based on laccases.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer deaths worldwide, and the incidence of this fatal disease is still on rise. The majority of HCCs emerge in the background of a chronic liver disease, such as chronic hepatitis and liver cirrhosis. The current understanding is that majority of HCCs evolve as a consequence of chronic inflammation and due to the presence of infection with hepatitis viruses. These underlying pathogenic stimuli subsequently induce a spectrum of genetic and epigenetic alterations in several cancer-related genes, which are involved in cell-cycle regulation, cell growth and adhesion. Such widespread genomic alterations cause disruption of normal cellular signaling and finally lead to the acquisition of a malignant phenotype in HCC. In general, the type of gene alterations, such as point mutations, deletion of chromosomal regions and abnormal methylation of gene promoters differ according to the individual targeted gene. In HCC, incidence of genetic alterations is relatively rare and is limited to a subset of few cancer-specific genes, such as the tumor suppressor p53, RB genes and oncogenes such as the CTNNB1. In contrast, epigenetic changes that involve aberrant methylation of genes and other post-transcriptional histone modifications occur far more frequently, and some of these epigenetic alterations are now being exploited for the development of molecular diagnostic signatures for HCC. In addition, recent findings of unique microRNA expression profiles also provide an evidence for the existence of novel mechanisms for gene expression regulation in HCC. In this review article, we will review the current state of knowledge on the activation of various oncogenic pathways and the inactivation of tumor suppressor pathways in HCC that result in the disruption of cancer-related gene function. In addition, we will specifically emphasize the clinical implication of some of these genetic and epigenetic alterations in the management of hepatocarcinogenesis.

We have reviewed the current pig (Sus scrofa) genomic diversity within and between sites and compared them with human and other livestock. The current Porcine 60K single nucleotide polymorphism (SNP) panel has an average SNP distance in a range of 30 - 40 kb. Most of genetic variation was distributed within populations, and only a small proportion of them existed between populations. The average heterozygosity was lower in pig than in human and other livestock. Genetic inbreeding coefficient (FIS), population differentiation (FST), and Nei's genetic distance between populations were much larger in pig than in human and other livestock. Higher average genetic distance existed between European and Asian populations than between European or between Asian populations. Asian breeds harboured much larger variability and higher average heterozygosity than European breeds. The samples of wild boar that have been analyzed displayed more extensive genetic variation than domestic breeds. The average linkage disequilibrium (LD) in improved pig breeds extended to 1 - 3 cM, much larger than that in human (∼ 30 kb) and cattle (∼ 100 kb), but smaller than that in sheep (∼ 10 cM). European breeds showed greater LD that decayed more slowly than Asian breeds. We briefly discuss some processes for maintaining genomic diversity in pig, including migration, introgression, selection, and drift. We conclude that, due to the long time of domestication, the pig possesses lower heterozygosity, higher FIS, and larger LD compared with human and cattle. This implies that a smaller effective population size and less informative markers are needed in pig for genome wide association studies.

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project ‘GLIP’: Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project ‘IFCOSMO’: Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.