Isolation and characterization of genes involved in basic metabolism of the filamentous fungus Trichoderma reesei: Dissertation

The filamentous fungus Trichoderma reesei is biotechnically interesting because of its excellent ability to secrete cellulases into its culture medium and as a potential production host for heterologous proteins. Although the cellulolytic properties of T. reesei have been thoroughly investigated, knowledge about the basic molecular biology of this fungus is still insufficient. To further develop T. reesei as a producer of cellulases as well as a host organism, it is necessary to understand the gene and promoter structure and the mechanisms of gene regulation in Trichoderma. For this purpose the glycolytic 3 phosphoglycerate kinase (pgk) gene was isolated from T. reesei by heterolo gous hybridization. The amino acid sequence deduced from the nucleotide sequence of the pgk gene is very similar to PGKs of other fungi. By analogy to other filamentous fungi, it can be postulated that the T. reesei pgk gene is interrupted by two introns of 219 and 75 nucleotides. Two transcripts, of 1.65 kb and 1.85 kb, result from two main transcription initiation sites of the pgk gene. The shorter transcript is expressed continuously, whereas the longer transcript appears at later stages of growth. The pgk gene is preceded by the regulatory elements also present in the PGK promoter of S. cerevisiae: sequences similar to the upstream activating sequence (UAS), the eukaryotic heat shock consensus (HSE) and the eukaryotic cAMP regulatory sequence CRE (ATF). The putative HSE sequence was not functional, since elevated temperatures appeared to have no effect on the total amount of pgk mRNA at elevated temperatures. A T. reesei endoglucanase I and an E. coli ß galactosidase were expressed under the pgk promoter, but the yields were very low. In order to elucidate the relationship of T. reesei to other fungi, the ribosomal RNA repeat was isolated from T. reesei. The complete 5.88 rRNA gene and parts of the small and large rRNA subunit genes were sequenced. The sequence data from the rRNA and pgk genes was used to evaluate the relationship of T. reesei to other fungi.