Linkage analysis and lignin peroxidase gene expression in Phanerochaete chrysosporium

[摘要] ENGLISH ABSTRACT:Wood is composed of three main components: cellulose, hemicellulose and lignin.Cellulose is the main structural polymer, whereas the function of lignin in plants is toimpart rigidity to the cells, to waterproof the vascular system, and to protect the plantagainst pathogens. A group of microorganisms, called white-rot fungi, are able toselectively degrade the lignin and hemicellulose from wood leaving the cellulose virtuallyuntouched. The most widely studied fungus of this group is the basidiomycetePhanerochaete chrysosporium, which has become a model organism in studies of lignindegradation.Lignin is a large, heterogenous and water insoluble polymer and therefore the enzymesneeded to degrade it have to be extracellular and non-specific. There are a number ofenzymes that are involved in the degradation of lignin, including lignin peroxidases,manganese dependent peroxidases and laccases. Laecases are blue copper oxidases thatrequire molecular oxygen to function, whereas lignin peroxidases and manganeseperoxidases are heme proteins that require hydrogen peroxide. Phanerochaetechrysosporium has all three of these enzymes, as well as a system for producing thehydrogen peroxide that is necessary for peroxidases to function.For both scientific and industrial purposes, it is important to obtain linkage maps of thepositions of genes in the genome of an organism. Most fungi, including P. chrysosporium,lack easily identifiable phenotypical markers that can be used to map the position of genesrelative to each other on the genome. Previous methods of mapping genes inP. chrysosporium involved auxotrophic mutants, radioactivity, or the use of hazardouschemicals. Here we describe an automated DNA-sequencing based mapping techniquethat eliminates many of the problems associated with previous techniques. Portions of thegenes to be mapped were amplified from homokaryotic single basidiospore cultures usinggene specific primers using the polymerase chain reaction (PCR) technique. The PCRproducts were sequenced to determine the segregation of alleles. Two previously mappedlignin peroxidases, lipA and lipC, were used to develop this method, and the resultsobtained corresponded to the known genetic linkage. A newly characterised 13-glucosidaseencoding gene from P. chrysosporium was also mapped. Linkage was found between the13-glucosidase gene and a histone (Hl) encoding gene.In P. chrysosporium the lignin peroxidase isozymes are encoded by a family of at least tengenes. Previous studies with P. chrysosporium BKM-F-1767 in defined media, wood andsoil have shown differential expression of the lignin peroxidase isozymes. In thisinvestigation the levels of expression of lignin peroxidases in P. chrysosporium ME446cultures grown in nitrogen or carbon limited defined liquid media, as well as on aspenwood chips were determined by competitive reverse transcriptase polymerase chainreaction (RT-peR). These results were compared to those previously obtained fromP. chrysosporium BKM-F-1767 to evaluate strain specific variation in the expression oflignin peroxidases. The results indicate that, although there were many similarities in thepatterns of lignin peroxidase expression, there were also enough differences to concludethat there were strain specific variations in the temporal expression of the ligninperoxidases.To conclude, a fast and cost effective method for mapping genes in P. chrysosporium wasdeveloped. Also, we showed that strain specific variation in temporal expression of ligninperoxidases occurs.

[发布机构] Stellenbosch University