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

RAPESEED MEAL: AN AGRO-WASTE FOR CHEAP/LOW COST LACCASE PRODUCTION

Rapeseed meal (RM) is an abundant and cheap by-product of oil extraction process. RM is a rich source of nitrogen, carbon, and minerals. Thanks to these features, RM has a striking potential for eco-friendly production of high value products in biotechnological processes. In this study, the possibility of using a low-cost medium based on the water soluble fraction of RM for laccase production by the fungus Pleurotus ostreatus has been evaluated and compared with the use of synthetic complex media. RM-based medium (RMM) led to extracellular laccase production comparable to that obtained in the presence of synthetic media. Moreover, the use of a raw material highly reduced the production costs. To further reduce cost and improve activity production the effect of the addition of nitrogen source, pure aromatic compounds and lignosulphonate, a low cost by-product of wood based industries, was evaluated. The optimized medium led to a laccase production level of more than 100,000 U/L at a cost of about 0.19* 10(-3) cent(sic)/U. The obtained results demonstrate the effective recycle of a waste to produce an industrially relevant enzyme

Low impact strategies to improve ligninolytic enzyme production in filamentous fungi: the case of laccase in Pleurotus ostreatus

The ever-increasing demand of laccases for biodelignification, industrial oxidative processes and environmental bioremediation requires the production of large quantities of enzymes at low cost. The present work was carried out to reduce laccase production costs in liquid fermentations of the white-rot fungus Pleurotus ostreatus through two different approaches. In the first, screening of fungal spent media as natural laccase inducer was performed, eliminating the presence of potentially toxic/recalcitrant and expensive exogenous inducers in the culture broth. In the latter, breeding of different strains of P. ostreatus, screened for their laccase productivity, was performed by cross-hybridisation, avoiding genetic transformation and mutagenic treatments that could produce organisms not suitable for "natural or safe processes". A laccase production level close to 80,000U/L by combining the two approaches was achieved. Autoinduction and classical breeding represent promising tools for the improvement of fungal fermentation without affecting the disposable costs that also depend on the eco-compatibility of the whole process

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 unctions 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

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

Cutaneous lichen amyloidosis associated with multiple endocrine neoplasia type 2A

The authors describe a family with multiple endocrine neoplasia type 2A and coutaneous lichen amyloidosis , by immunocitochemical study

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

The Pleurotus genome: an inventory of laccase-type genes

Many fungi produce several isoenzymes endowed with slightly different catalytic properties, being the physiological significance of this multiplicity still unknown. Likewise, this feature is shared by fungi belonging to Pleurotus genera concerning the laccase gene family (1). As a fact, four laccase gene members have already been isolated in Pleurotus sajior-caju (2), two in Pleurotus eryngii (3), and seven in Pleurotus ostreatus (4). Moreover the existence of a “laccase subfamily” consisting of three out of seven members has been postulated in the latter fungus, on the basis of sequence similarity, and intron-exon structure. Five P. ostreatus encoded isoenzymes, representing a variegated group of laccases endowed with peculiar properties, have been purified from culture broth and fully characterized (4). More recently, the identification of a new laccase isoenzyme from its fruiting body has been achieved. A more complex multicopper oxidase family has been disclosed since the release of the white-rot fungus Pleurotus ostreatus genome (http://www.jgi.doe.gov/sequencing/why/50009.html). The automatic annotation of the retrieved gene models was analyzed and improved, thanks to the information available on the structure-function of laccases. Clustering of laccase genes was evaluated along with their belonging to the already defined subfamilies (4). Genes encoding heterodimeric laccase, the large (5) and the small subunit (6), lie in the same chromosome. A cluster of seven out of twelve laccase genes in sub-telomeric region of chromosome 6 has been recently mapped by Perez and colleagues (7), persuading the authors that this location could have an evolutionary significance permitting the fungus to adapt rapidly to new lignocellulosic substrates. The presence of all the conserved residues characterizing laccases sequences (8) was checked in all the deduced proteins. These analyses allowed to understand that ten out of twelve deduced proteins correspond to laccases in sensu stricto