Investigation and optimization of the filamentous fungus Caldariomyces fumago as enzyme production host

In this work, investigations and optimizations of the filamentous fungus Caldariomyces fumago as enzyme production host were performed. For UV mutagenesis experiments with C. fumago, UV irradiation parameters were determined. A survival curve was recorded and an irradiation time of 4 min was chosen for all following UV experiments leading to a survival rate of 21 %. By colony screening of randomly mutagenized C. fumago cells 6 albino (white) mutants displaying a slight growth reduction on solid media were isolated. In production media these strains were red-colored due to the production of the heme-containing chloroperoxidase (CPO). Their culture supernatant samples lacked the intense black fungal pigment thereby enabling to follow the formation of CPO by visual inspection of the medium color and simplifying downstream processing. Surprisingly, elimination of the undesirable pigmentation was accompanied by improved CPO secretion. Namely, the CPO content in the culture supernatant samples of the white C. fumago mutants increased significantly faster compared to the wild type. To examine the nature of the black pigment of the C. fumago wild-type strain several inhibitors commonly used to block the synthesis of different pigment in fungi were tested. The addition of 50 µg ml-1 tricyclazole, a specific inhibitor of the 1,8-dihydroxynaphthalene (D2HN) melanin synthesis pathway, induced a significant color change of the C. fumago wild type mycelium proposing the black pigment of C. fumago to be D2HN melanin. A further C. fumago mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant sample lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity could be detected. SDS-PAGE analysis of mutant culture supernatant sample showed production of a 38 kDa protein while wild type samples contain the 42 kDa CPO protein. Protein identification using nanoLC-ESI-MS/MS was performed and based on 3 peptides the protein was identified as CPO. In addition, the CPO gene was sequenced. However, no differences in the CPO gene sequence of wild type and mutant were found indicating a post-translational defect in protein maturation. Deglycosylation experiments using CPO from wild type and cpo1 were carried out. After removing N-linked oligosaccharides from wild type CPO a protein band at 38 kDa was detected. These results reveal that the mutant protein lacks the heme group as well as the N-glycans. Addition of different amounts of tunicamycin, an antibiotic that blocks N-linked glycosylation of proteins, to C. fumago wild type cultures did either lead to a strong reduced growth behavior when a too high concentration was applied or had no influence on CPO activity and no visible change in protein size on SDS-PAGE gels was obtained. To select transformants of C. fumago for the development of more efficient transformation systems based on auxotrophic markers the URA3 gene encoding 5’-orotidine monophosphate decarboxylase (OMPD) was chosen. Mutants lacking OMPD activity could be easily selected after UV irradiation since they are able to grow in the presence of 5-FOA but require the addition of uracil or uridine to the medium. Fifty-three strains were isolated and their growth properties were examined by plating on different media. Three strains showed the growth properties which are dedicated to uracil or uridine auxotrophic mutants having a defective URA3 gene. Those cells were transformed with a plasmid containing the URA3 gene either of A. nidulans or C. fumago. However, no complementation of the auxotrophy of any of the 3 strains was achieved. Since mutants with a defective URA5 gene also show the determined growth properties it was assumed that the URA5 gene is affected. Experiments of expressing horseradish peroxidase (HRP) and the carotenoid cleaving peroxidase MsP2 from Marasmius scorodonius using the highly efficient and specific secretion apparatus of C. fumago were performed. Due to the lack of CPO knock-outs strains, the C. fumago wild type strain had to be used in the present work. The construct for the expression of HRP consisted of the N-terminal signal peptide of CPO and the C-terminal propeptide of CPO. The constructs for MsP2 consisted either of the N-terminal secretion signal of CPO or of MsP2 and the C-terminal propeptide of CPO was absent or present. The constructs were transformed into the rDNA region of the C. fumago genome. For all constructs of both enzymes no visible protein bands were detected on SDS-PAGE gels. Enzyme assays specific for HRP and MsP2 were developed. However, no enzyme activity was detected for exemplarily chosen transformants. Several limits of heterlogous gene expression using C. fumago are discussed.