Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei:Dissertation

The biosynthesis of cellobiohydrolase (CBH I, EC 3.2.1.91) of Trichoderma reesei was studied in order to obtain more information about the basic biochemistry and secretion processes of filamentous fungi.The induction and regulation of cellulase biosynthesis were studied using different inducers.The information obtained provided a basis for choosing a method of cloning the cellulase genes.Poly(A) mRNA was isolated from T reesei cultures actively synthesizing cellulases and translated in vitro.In vitro synthesized polypeptides corresponding to the cellulase proteins cellobiohydrolase I, cellobiohydrolase II and endoglucanase I were immunoprecipitated using antisera against the natural enzymes and characterised.When mRNA from glucosegrown cells was added to the in vitro protein synthesis system only negligible amounts of cellulases were immunoprecipitated, indicating that regulation of the expression of these proteins is at the transcriptional level.Sophorose was found to be the best inducer for cellobiohydrolase I and it was used as inducer in in vivo labeling experiments.In order to analyze the glycans of cellobiohydrolase I the extracellular glycoproteins of T. reesei were metabolically labeled with ³H mannose after sophorose induction.The labeled cellobiohydrolase I was purified by irnmunoprecipitation and SDS polyacrylamide gel electrophoresis.The glycans of cellobiohydrolase I were analyzed by chemical degradation procedures and by specific glycosidase digestions.The results indicated that cellobiohydrolase I contained both O- and N glycosidic glycans.The O-linked glycans consisted of one to four hexose residues, which were composed mainly of mannose.The N linked glycans were of high mannose type, with the structures (Man)5(GlcNAc)2 and (Man)9(GlcNAc)2. Analysis of the in vivo synthesized cellobiohydrolase I revealed that the N linked glycans are most probably added co-translationally.The first residues of the O linked glycans may also be added co-translationally translationally, but the chains are completed later during the secretion process, which takes about 20 minutes.The light and transmission electron microscopy of the morphological changes in a T. reesei mutant strain induced to produce cellulases revealed that the induced mycelium had a higher content of vacuoles and membraneous structures than the repressed mycelium.The glycan analysis showed that the N linked glycans of cellobiohydrolase I were of high mannose-type, resembling the glycans in animal cells, whereas the O-linked glycans resembled those produced by yeast.Because many problems have been encountered in the production of recombinant products in prokaryotes T. reesei may have considerable potential as a host for the production of a range of foreign glyco proteins, including those from animal sources