Development of a Transformation System for Caldariomyces Fumago and Expression of Heterologous Proteins

154 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.Caldariomyces fumago is a filamentous fungus that secretes the enzyme chloroperoxidase in amounts greater than 1 g/l at a purity level of 90-95% on a simple fructose sugar/inorganic salts medium. Conditions were optimized for the generation of spheroplasts from C. fumago mycelium using the enzyme mixture Novozym 234. An osmotic medium was developed for maximum regeneration of the spheroplasts (40 to 50%) and sensitivity to antibiotics. Deoxycholic acid was shown to increase sensitivity while yeast extract and bacto-agar decreased it. Spheroplasts were transformed by polyethylene glycol (PEG) fusion with plasmid DNA. The DNA contained a hygromycin B resistance gene (hph), which encodes a phosphtransferase, and is flanked by promotion and termination sequences from the trpC gene of Aspergillus nidulans. Colonies which grew from the initial selection were shown to be transiently resistant as they did not grow upon reselection. Stable transformants were obtained when homologous C. fumago sequences were placed on the same plasmid with the resistance gene. Attempts to delete or disrupt the endogenous chloroperoxidase gene proved unsuccessful and provided evidence for an essential sequence in the gene area. Sequences coding for the heterologous proteins ligninase and calcitonin were cloned into the site of the chloroperoxidase structural gene and the plasmids transformed into spheroplasts. Integration of the genes into the C. fumago genome was verified by polymerase chain reaction (PCR) and/or Southern blot analysis. Small amounts of ligninase, as determined by Western blot analysis, were produced from several transformants. The reported chloroperoxidase gene sequence was discovered to have an error at the position 1055 base pairs after the transcription start site. Elimination of the error, an extra guanine residue, changed the translational phase of the last 20 base pairs of the reported gene including the assumed termination codon. A new in-phase termination codon was found 156 bp downstream resulting in a 1119 bp gene which translated into a pre-protein with 373 amino acids including the 21 amino acid signal peptide. The putative mature protein consisted of 352 amino acids. The molecular weight of unglycosylated protein was calculated to be 38,270 daltons and the glycosylated protein was determined to be approximately 42,000 daltons. The chloroperoxidase protein was reanalyzed by deglycosylation experiments, mass spectroscopy analysis, and tryptic digest/peptide sequencing.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD