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

Dissertation

Sirpa Vanhanen

Research output: ThesisDissertationMonograph

1 Citation (Scopus)

Abstract

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.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Penttilä, Merja, Supervisor
Award date10 May 1991
Place of PublicationEspoo
Publisher
Print ISBNs951-38-3944-3
Publication statusPublished - 1991
MoE publication typeG4 Doctoral dissertation (monograph)

Fingerprint

Trichoderma reesei
phosphoglycerate kinase
fungi
metabolism
genes
ribosomal RNA
cellulases
promoter regions
heat stress
galactosidases
regulatory sequences
consensus sequence
Trichoderma
endo-1,4-beta-glucanase
molecular biology
introns
temperature
hybridization
amino acid sequences
transcription (genetics)

Keywords

  • filamentous fungi
  • Trichoderma reesei
  • 3-phosphoglycerate kinase
  • gene and promoter structure
  • gene expression
  • ribosomal RNA
  • molecular taxonomy

Cite this

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title = "Isolation and characterization of genes involved in basic metabolism of the filamentous fungus Trichoderma reesei: Dissertation",
abstract = "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.",
keywords = "filamentous fungi, Trichoderma reesei, 3-phosphoglycerate kinase, gene and promoter structure, gene expression, ribosomal RNA, molecular taxonomy",
author = "Sirpa Vanhanen",
note = "Project code: BIO8004",
year = "1991",
language = "English",
isbn = "951-38-3944-3",
series = "Publications / Technical Research Centre of Finland",
publisher = "VTT Technical Research Centre of Finland",
number = "75",
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school = "University of Helsinki",

}

Isolation and characterization of genes involved in basic metabolism of the filamentous fungus Trichoderma reesei : Dissertation. / Vanhanen, Sirpa.

Espoo : VTT Technical Research Centre of Finland, 1991. 122 p.

Research output: ThesisDissertationMonograph

TY - THES

T1 - Isolation and characterization of genes involved in basic metabolism of the filamentous fungus Trichoderma reesei

T2 - Dissertation

AU - Vanhanen, Sirpa

N1 - Project code: BIO8004

PY - 1991

Y1 - 1991

N2 - 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.

AB - 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.

KW - filamentous fungi

KW - Trichoderma reesei

KW - 3-phosphoglycerate kinase

KW - gene and promoter structure

KW - gene expression

KW - ribosomal RNA

KW - molecular taxonomy

M3 - Dissertation

SN - 951-38-3944-3

T3 - Publications / Technical Research Centre of Finland

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -