Thesis (MSc)- Stellenbosch University, 2001. / 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 to impart rigidity to the cells, to waterproof the vascular system, and to protect the plant against pathogens. A group of microorganisms, called white-rot fungi, are able to selectively degrade the lignin and hemicellulose from wood leaving the cellulose virtually untouched. The most widely studied fungus of this group is the basidiomycete Phanerochaete chrysosporium, which has become a model organism in studies of lignin degradation. Lignin is a large, heterogenous and water insoluble polymer and therefore the enzymes needed to degrade it have to be extracellular and non-specific. There are a number of enzymes that are involved in the degradation of lignin, including lignin peroxidases, manganese dependent peroxidases and laccases. Laecases are blue copper oxidases that require molecular oxygen to function, whereas lignin peroxidases and manganese peroxidases are heme proteins that require hydrogen peroxide. Phanerochaete chrysosporium has all three of these enzymes, as well as a system for producing the hydrogen peroxide that is necessary for peroxidases to function. For both scientific and industrial purposes, it is important to obtain linkage maps of the positions 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 genes relative to each other on the genome. Previous methods of mapping genes in P. chrysosporium involved auxotrophic mutants, radioactivity, or the use of hazardous chemicals. Here we describe an automated DNA-sequencing based mapping technique that eliminates many of the problems associated with previous techniques. Portions of the genes to be mapped were amplified from homokaryotic single basidiospore cultures using gene specific primers using the polymerase chain reaction (PCR) technique. The PCR products were sequenced to determine the segregation of alleles. Two previously mapped lignin peroxidases, lipA and lipC, were used to develop this method, and the results obtained corresponded to the known genetic linkage. A newly characterised 13-glucosidase encoding gene from P. chrysosporium was also mapped. Linkage was found between the 13-glucosidase gene and a histone (Hl) encoding gene. In P. chrysosporium the lignin peroxidase isozymes are encoded by a family of at least ten genes. Previous studies with P. chrysosporium BKM-F-1767 in defined media, wood and soil have shown differential expression of the lignin peroxidase isozymes. In this investigation the levels of expression of lignin peroxidases in P. chrysosporium ME446 cultures grown in nitrogen or carbon limited defined liquid media, as well as on aspen wood chips were determined by competitive reverse transcriptase polymerase chain reaction (RT-peR). These results were compared to those previously obtained from P. chrysosporium BKM-F-1767 to evaluate strain specific variation in the expression of lignin peroxidases. The results indicate that, although there were many similarities in the patterns of lignin peroxidase expression, there were also enough differences to conclude that there were strain specific variations in the temporal expression of the lignin peroxidases. To conclude, a fast and cost effective method for mapping genes in P. chrysosporium was developed. Also, we showed that strain specific variation in temporal expression of lignin peroxidases occurs. / AFRIKAANSE OPSOMMING: Hout bestaan uit drie hoof komponente nl. sellulose, hemisellulose en lignien. Sellulose is die hoof strukturele polimeer, terwyl die funksie van lignin in plante is om die selle te versterk, die vaskulêre sisteem waterdig te hou, en die plant teen patogene te beskerm. 'n Groep mikroërganisms, bekend as witvrotswamme, kan lignien en hemisellulose selektief uit die hout verwyder, terwyl die sellulosevesels oorbly. Vanuit hierdie groep swamme is die meeste navorsing op die basidiomiseet Phanerochaete chrysosporium gedoen Lignien is 'n groot, heterogene polimeer en is onoplosbaar in water. Die ensieme wat benodig word om lignien afte breek is daarom nie-spesifiek en kom ekstrasellulêr voor. 'n Aantal ensieme is by die afbraak van lignien betrokke, insluitend lignienperoksidase, mangaanperoksidase en lakkase. Lakkase is 'n blou koperoksidase wat suurstof benodig vir aktiwiteit. Lignienperoksidase en mangaanperoxidase is heemproteïene en benodig waterstofperoksied. Phanerochaete chrysosporium het al drie van hiedie ensieme, sowel as 'n sisteem wat waterstofperoksied produseer. Vir beide wetenskaplike en nywerheidsdoeleindes is koppelingskaarte wat die posisie van gene in die genoom van 'n organisme aandui noodsaaklik. Die meeste swamme, P. chrysosporium ingesluit, het geen fenotipiese merkers wat maklik van mekaar onderskei kan word nie, en dit is dus moeilik om 'n kaart van die ligging van gene op die genoom te bepaal. Vorige metodes om gene in P. chrysosporium te karteer het auksotrofiese mutante, radioaktiwiteit of gevaarlike chemikalieë gebruik. Ons beskryf 'n metode wat van automatiese DNA-volgordebepaling gebruik maak en wat baie van die tekortkominge van die ou metodes oorkom. Dele van die gene is met geen-spesifieke PKR-amplifikasie uit kulture van homokariotiese enkel basidiospore verkry en die DNA-volgorde is bepaal om die segregasie van die allele te ondersoek. Twee gene waarvoor 'n koppelingskaart alreeds uitgewerk is, fipA en lipt), was gebruik om hierdie metode te ontwikkel. Die resultate stem ooreen met die bekende genetiese koppeling tussen hierdie gene. 'n Geen wat onlangs in P. chrysosporium ontdek is, nl. I3-glucosidase, is ook met hierdie metode gekarteer. Koppeling is met 'n histoon (Hl) geen gevind. Die lignienperoksidase isoensieme in P. chrysosporium word deur 'n familie van ten minste tien gene gekodeer. Vorige navorsing met P. chrysosporium BKM-F-1767 in gedefineerde media, hout en grond het getoon dat 'n variasie in die uitdrukking van lignienperoxidase isoensieme voorkom. In hierdie ondersoek is 'n kultuur van P. chrysosporium ME446 in stikstof- of koolstof-beperkende vloeibare media opgegroei, as ook op aspen houtblokkies. Die vlak van uitdrukking van die lignienperoksidases is deur middel van die omgekeerde transkripsie polimerasekettingreaksie (RT-PKR) bepaal. Die resultate vir P. chrysosporium ME446 is vergelyk met vorige resultate van P. chrysosporium BKM-F-1767 om te bepaal of stamspesifieke variasies in die uitdrukking van lignienperoksidases voorkom. Daar is 'n aanduiding dat, alhoewel soortgelyke patrone in die vlakke van lignienperoksidase uitdrukking voorkom, daar ook noemenswaardige verskille is. Hieruit kan afgelui word dat stamverwante variasie van lignienperokisdase uitdrukking voorkom. Ten slotte, ons het 'n vinnige, goedkoop metode om die gene in P. chrysosporium te karteer ontwikkel. Ons het ook bewys dat stam-spesifieke variasie in die uitdrukking van die lignienperoxidase gene voorkom.

Thesis (PhD)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: White rot fungi are of interest in the paper and pulp industry because of their removal of lignin from wood. In this study over 600 Basidiomycete fungi were isolated from indigenous forests as well as from commercial Eucalyptus spp. and Pinus spp. plantations in South Africa. One hundred isolates were identified to genus level. Biochemical tests were done to screen the fungal cultures for characteristics that are favourable for biopulping, e.g. low cellulase activity with concomitant high activity of ligninolytic enzymes. Various Phanerochaete isolates with potentially high ligninolytic activity were identified. Although Phanerochaete chrysosporium Burds. has previously been isolated from the indigenous forest at Knysna in South Africa, this study showed that P. chrysosporium was a natural coloniser of wood chip piles in South Africa, indicating potential for application in industry. A possible new species of Phanerochaete, viz. Phanerochaete pseudomagnoliae nom. provo (strain PP25) from decayed wood collected in Stellenbosch, South Africa, was described and illustrated. It differs from previously described Phanerochaete species in having smaller basidiospares, and in the formation of few chlamydospores on malt extract agar but more on xylose containing media. The potential of using internal transcribed spacer DNA sequences (ITS) to infer phylogenetic relationships among species of the genus Phanerochaete was investigated. Consensus phylogenetic trees could be presented, but the presence of ambiguous aligning sequences within the ITS made inferring of phylogenetic relationships within the whole genus difficult. Fifty-five South African strains of P. chrysosporium were screened for lignin peroxidase (liP), manganese peroxidase (MnP) and glyoxal oxidase (GLOX) enzymes. Manganese peroxidase activity was quantified on agar media. The liP and GLOX activities of 13 selected strains, including control strains and P. pseudomagnoliae (PP25), were also quantified on agar media. Differences in MnP and GLOX activities existed among the strains. Preliminary biochemical characterisation of strain PP25 indicated that the most important difference was the apparent unique regulation of ligninolytic enzymes. Under low nitrogen, liP activity of the selected strains showed no significant variation, whereas strain PP25 had significantly increased liP levels under high nitrogen conditions. Restriction fragment length polymorph isms of the lignin and manganese peroxidase gene DNA fragments showed variability among strains, whereas there was probably only allelic variation for the glox gene DNA fragments. Previous research has indicated xylose oxidation activity within P. chrysosporium. To investigate whether GLOX can oxidise xylose, a purified recombinant GLOX (rGLOX) from P. chrysosporium BKM-F-1767 Burds. was used in this study. This rGLOX oxidised D-xylose and D-glucose (D-xylose > D-glucose) to produce H202. Xylose was oxidised to xylono-1 ,4-lactone with a 1:1 stoichiometric relationship between H202 produced and xylose used. Xylono-1,4-lactone was converted non-enzymatically to xylonate. This suggested that the furanose form of xylose, rather than the pyranose form, is a substrate of GLOX. The production of H202 and the removal of inhibitory compounds by GLOX could enhance ligninolytic activity. . To conclude, unique strains of P. chrysosporium have been isolated from South Africa with potential biotechnological use in paper manufacturing. The relationship of P. pseudomagnoliae nom. provo to other Phanerochaete species was evaluated and light was shed on the possible role of GLOX in lignin degradation. / AFRIKAANSE OPSOMMING: Witvrot swamme is van belang vir die papier en pulp industrie omdat hulle lignin vanaf hout kan verwyder. Meer as 600 Sasidiomiseet fungi, afkomstig vanaf inheemse woude asook kommersiële Eucalyptus spp. en Pinus spp. plantasies, IS geïsoleer. Een honderd isolate is tot op genusvlak geïdentifiseer. Die isolate is biochemies vir eienskappe wat voordelig vir "bioverpulping" kon wees, bv. die gelyktydige produksie van lae sellulosemaar hoë ligninolitiese ensiemaktiwiteit, getoets. Verskeie isolate met potensieel hoë vlakke van ligninolitiese aktiwiteit is verkry. Alhoewel Phanerochaete chrysosporium Surds. vantevore in die Knysna inheemse woud in Suid-Afrika geïsoleer is, het hierdie studie gewys dat P. chrysosporium natuurlik op hope houtblokkies voorgekom, met moontlike toepasing in die industrie. Isolaat PP25, geisoleer vannaf verrottende hout te Stellenbosch, Suid Afrika, is as 'n potensieel nuwe spesie van die genus Phanerochaete beskryf en as Phanerochaete pseudomagnoliae nom. provo benoem. Hierdie isolaat verskil van ander Phanerochaete-spesies daarin dat dit kleiner basidiospore vorm en nie klamydospore op moutekstrakagar produseer nie, maar wel op media wat xilose bevat. Die potensiaal van intern getranskribeerde spasieerder ONS opeenvolging vir die aflei van filogenetiese verhoudings tussen spesies van die genus Phanerochaete is ondersoek. Konsensus filogenetiese bome kon bepaal word, maar die teenwoordigheid van varieerbare areas het die afleiding van filogenetiese verwantskappe vir die hele genus bemoeilik. Vyf-en-vyftig Suid-Afrikaanse isolate van P. chrysosporium is vir die teenwoordigheid van lignienperoksidase- (liP), mangaanperoksidase- (MnP) en glioksaaloksidase (GLOX)-aktiwiteit getoets. Vlakke van MnP-aktiwiteit is op agarplate gekwantifiseer. Vlakke van LiP- en GLOX-ensieme op agarplate is vir 13 geselekteerde isolate, insluitend kontroles en ras PP25, gekwantifiseer. Aktiwiteit van MnP en GLOX het statisties betekenisvol tussen isolate verskil. Lignienperoksidase-aktiwiteit onder lae stikstof toestande het nie statisties betekenisvol van mekaar verskil nie. Onder hoë stikstof toestande het isolaat PP25 wel verhoogde liP-aktiwiteit getoon. Restriksie fragment polimorfismes van die lignien- en mangaanperoxidase-gene het variasie getoon, terwyl waarskynlik slegs alleliese variasie vir die glox geen waargeneem IS. Rekombinante GLOX (GLOX vanaf P. chrysosporium BKM-F-1767) het xilose en glukose (D-xilose > D-glukose) geoksideer met meegaande produksie van H202. Xilose is na xilono-1,4-laktoon geoksideer met 'n 1:1 stoigiometrie tussen H202-produksie en xilose verbruik. Xilono-1,4-laktoon is nieensiematies na xilonaat omgeskakel. Bogenoemde resultaat dui aan dat die furanose vorm van xilose die werklike substraat vir GLOX is. Deur die meegaande produksie van H202 en die verwydering van inhiberende produkte word lignoliese aangehelp. Ten slote, unieke P. chrysosporium rasse met potensiële gebruik in papiervervaardiging is in Suid-Afrika geisoleer. Die genetiese diversiteit van 'n nuwe spesie, P. pseudomagnoliae, is bepaal en nuwe lig is op die potensiële rol van GLOX in lignienafbraak gewerp.