Genetic improvement of enzyme production in industrially important fungal strains: Dissertation

Helena Nevalainen

Research output: ThesisDissertation

2 Citations (Scopus)

Abstract

Filamentous fungi have a number of significant advantages for production of enzymes for industrial applications.In this study, enzyme producing filamentous fungal trains used in biotechnology have been improved genetically.Strains producing up to four times more enzyme than the initial parent, were obtained.The work is divided in two parts.The first part deals with mutagenesis and screening for induction and isolation of fungal strains with enhanced enzyme production and the second part concerns the application of recombinant DNA in developing new industrial strains.Different fungi have been selected for production of particular enzymes.Through mutagenesis and screening, improved strains of Aspergillus awamori and A. niger well suited to industrial scale production of amyloglucosidase and ß-galactosidase, respectively, have been isolated and characterised.The mesophilic cellulolytic filamentous fungus Trichoderma reesei is an important organism in studies of enzymatic hydrolysis of cellulose for production of fuels and chemicals.In this work, a series of hypercellulolytic T. reesei strains capable of production of cellulases on a relatively cheap fermentation medium has been induced and isolated.The genetic regulation of cellulase biosynthesis in T. reesei was studied through induction and isolation of mutant strains unable to hydrolyze Walseth cellulose.Cellulase negative mutants were enriched on plates using the antibiotic nystatin.Results suggest a separate regulation of actual cellulases to that of the ß-glucosidase enzyme in this fungus. Recombinant DNA methods have been introduced to study the expression of filamentous fungal genes in yeast and to gain important basic information of the structure and function of fungal cellulase genes.For further investigations in Trichoderma, a transformation system is needed and work towards this goal has been started by isolation of auxotrophic mutants of T. reesei.Cloning of genes from filamentous fungi by expression in the yeast Saccharomyces cerevisiae, as a general method, was studied.Yeast transformants carrying A. niger DNA were screened for functional expression of genes coding for the secreted enzymes ß-galactosidase, ß-glucosidase and amyloglucosidase and for the expression of Aspergillus genes complementing the yeast leu 2 and ura 3 mutations.Only the gene for ß-glucosidase was obtained in this way its expression being detected as blue coloured transformant colonies on plates containing the Xglu reagent.In addition to A. niger ß-glucosidase, the T. reesei CBH I gene coding for cellobiohydrolase I, the most abundant enzyme present in culture fluids of the fungus, is shown to express in yeast.CBH I enzyme produced by recombinant yeast shows activity comparable to that of T. reesei enzyme.However, the expression of genomic filamentous fungal genes in yeast seems to be rare and when it occurs the level of expression is very low. For efficient production of filamentous fungal enzymes in yeast, yeast regulatory signals and cDNAs of the genes to be expressed, need to be used.In future, both approaches, mutagenesis and screening and recombinant DNA, will be needed in development of new fungal strains for industry.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Award date29 Nov 1985
Place of PublicationEspoo
Publisher
Print ISBNs951-38-2436-5
Publication statusPublished - 1985
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

genetic improvement
Trichoderma reesei
yeasts
enzymes
glucosidases
recombinant DNA
fungi
endo-1,4-beta-glucanase
mutagenesis
genes
galactosidases
cellulases
screening
cellulose
Aspergillus awamori
cellulose 1,4-beta-cellobiosidase
nystatin
auxotrophs
fuel production
mutants

Keywords

  • genetic engineering
  • genetics
  • improvement
  • enzymes
  • production
  • fungi
  • theses

Cite this

Nevalainen, H. (1985). Genetic improvement of enzyme production in industrially important fungal strains: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Nevalainen, Helena. / Genetic improvement of enzyme production in industrially important fungal strains : Dissertation. Espoo : VTT Technical Research Centre of Finland, 1985. 98 p.
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title = "Genetic improvement of enzyme production in industrially important fungal strains: Dissertation",
abstract = "Filamentous fungi have a number of significant advantages for production of enzymes for industrial applications.In this study, enzyme producing filamentous fungal trains used in biotechnology have been improved genetically.Strains producing up to four times more enzyme than the initial parent, were obtained.The work is divided in two parts.The first part deals with mutagenesis and screening for induction and isolation of fungal strains with enhanced enzyme production and the second part concerns the application of recombinant DNA in developing new industrial strains.Different fungi have been selected for production of particular enzymes.Through mutagenesis and screening, improved strains of Aspergillus awamori and A. niger well suited to industrial scale production of amyloglucosidase and {\ss}-galactosidase, respectively, have been isolated and characterised.The mesophilic cellulolytic filamentous fungus Trichoderma reesei is an important organism in studies of enzymatic hydrolysis of cellulose for production of fuels and chemicals.In this work, a series of hypercellulolytic T. reesei strains capable of production of cellulases on a relatively cheap fermentation medium has been induced and isolated.The genetic regulation of cellulase biosynthesis in T. reesei was studied through induction and isolation of mutant strains unable to hydrolyze Walseth cellulose.Cellulase negative mutants were enriched on plates using the antibiotic nystatin.Results suggest a separate regulation of actual cellulases to that of the {\ss}-glucosidase enzyme in this fungus. Recombinant DNA methods have been introduced to study the expression of filamentous fungal genes in yeast and to gain important basic information of the structure and function of fungal cellulase genes.For further investigations in Trichoderma, a transformation system is needed and work towards this goal has been started by isolation of auxotrophic mutants of T. reesei.Cloning of genes from filamentous fungi by expression in the yeast Saccharomyces cerevisiae, as a general method, was studied.Yeast transformants carrying A. niger DNA were screened for functional expression of genes coding for the secreted enzymes {\ss}-galactosidase, {\ss}-glucosidase and amyloglucosidase and for the expression of Aspergillus genes complementing the yeast leu 2 and ura 3 mutations.Only the gene for {\ss}-glucosidase was obtained in this way its expression being detected as blue coloured transformant colonies on plates containing the Xglu reagent.In addition to A. niger {\ss}-glucosidase, the T. reesei CBH I gene coding for cellobiohydrolase I, the most abundant enzyme present in culture fluids of the fungus, is shown to express in yeast.CBH I enzyme produced by recombinant yeast shows activity comparable to that of T. reesei enzyme.However, the expression of genomic filamentous fungal genes in yeast seems to be rare and when it occurs the level of expression is very low. For efficient production of filamentous fungal enzymes in yeast, yeast regulatory signals and cDNAs of the genes to be expressed, need to be used.In future, both approaches, mutagenesis and screening and recombinant DNA, will be needed in development of new fungal strains for industry.",
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author = "Helena Nevalainen",
year = "1985",
language = "English",
isbn = "951-38-2436-5",
series = "Technical Research Centre of Finland. Publications",
publisher = "VTT Technical Research Centre of Finland",
number = "26",
address = "Finland",
school = "University of Helsinki",

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Nevalainen, H 1985, 'Genetic improvement of enzyme production in industrially important fungal strains: Dissertation', Doctor Degree, University of Helsinki, Espoo.

Genetic improvement of enzyme production in industrially important fungal strains : Dissertation. / Nevalainen, Helena.

Espoo : VTT Technical Research Centre of Finland, 1985. 98 p.

Research output: ThesisDissertation

TY - THES

T1 - Genetic improvement of enzyme production in industrially important fungal strains

T2 - Dissertation

AU - Nevalainen, Helena

PY - 1985

Y1 - 1985

N2 - Filamentous fungi have a number of significant advantages for production of enzymes for industrial applications.In this study, enzyme producing filamentous fungal trains used in biotechnology have been improved genetically.Strains producing up to four times more enzyme than the initial parent, were obtained.The work is divided in two parts.The first part deals with mutagenesis and screening for induction and isolation of fungal strains with enhanced enzyme production and the second part concerns the application of recombinant DNA in developing new industrial strains.Different fungi have been selected for production of particular enzymes.Through mutagenesis and screening, improved strains of Aspergillus awamori and A. niger well suited to industrial scale production of amyloglucosidase and ß-galactosidase, respectively, have been isolated and characterised.The mesophilic cellulolytic filamentous fungus Trichoderma reesei is an important organism in studies of enzymatic hydrolysis of cellulose for production of fuels and chemicals.In this work, a series of hypercellulolytic T. reesei strains capable of production of cellulases on a relatively cheap fermentation medium has been induced and isolated.The genetic regulation of cellulase biosynthesis in T. reesei was studied through induction and isolation of mutant strains unable to hydrolyze Walseth cellulose.Cellulase negative mutants were enriched on plates using the antibiotic nystatin.Results suggest a separate regulation of actual cellulases to that of the ß-glucosidase enzyme in this fungus. Recombinant DNA methods have been introduced to study the expression of filamentous fungal genes in yeast and to gain important basic information of the structure and function of fungal cellulase genes.For further investigations in Trichoderma, a transformation system is needed and work towards this goal has been started by isolation of auxotrophic mutants of T. reesei.Cloning of genes from filamentous fungi by expression in the yeast Saccharomyces cerevisiae, as a general method, was studied.Yeast transformants carrying A. niger DNA were screened for functional expression of genes coding for the secreted enzymes ß-galactosidase, ß-glucosidase and amyloglucosidase and for the expression of Aspergillus genes complementing the yeast leu 2 and ura 3 mutations.Only the gene for ß-glucosidase was obtained in this way its expression being detected as blue coloured transformant colonies on plates containing the Xglu reagent.In addition to A. niger ß-glucosidase, the T. reesei CBH I gene coding for cellobiohydrolase I, the most abundant enzyme present in culture fluids of the fungus, is shown to express in yeast.CBH I enzyme produced by recombinant yeast shows activity comparable to that of T. reesei enzyme.However, the expression of genomic filamentous fungal genes in yeast seems to be rare and when it occurs the level of expression is very low. For efficient production of filamentous fungal enzymes in yeast, yeast regulatory signals and cDNAs of the genes to be expressed, need to be used.In future, both approaches, mutagenesis and screening and recombinant DNA, will be needed in development of new fungal strains for industry.

AB - Filamentous fungi have a number of significant advantages for production of enzymes for industrial applications.In this study, enzyme producing filamentous fungal trains used in biotechnology have been improved genetically.Strains producing up to four times more enzyme than the initial parent, were obtained.The work is divided in two parts.The first part deals with mutagenesis and screening for induction and isolation of fungal strains with enhanced enzyme production and the second part concerns the application of recombinant DNA in developing new industrial strains.Different fungi have been selected for production of particular enzymes.Through mutagenesis and screening, improved strains of Aspergillus awamori and A. niger well suited to industrial scale production of amyloglucosidase and ß-galactosidase, respectively, have been isolated and characterised.The mesophilic cellulolytic filamentous fungus Trichoderma reesei is an important organism in studies of enzymatic hydrolysis of cellulose for production of fuels and chemicals.In this work, a series of hypercellulolytic T. reesei strains capable of production of cellulases on a relatively cheap fermentation medium has been induced and isolated.The genetic regulation of cellulase biosynthesis in T. reesei was studied through induction and isolation of mutant strains unable to hydrolyze Walseth cellulose.Cellulase negative mutants were enriched on plates using the antibiotic nystatin.Results suggest a separate regulation of actual cellulases to that of the ß-glucosidase enzyme in this fungus. Recombinant DNA methods have been introduced to study the expression of filamentous fungal genes in yeast and to gain important basic information of the structure and function of fungal cellulase genes.For further investigations in Trichoderma, a transformation system is needed and work towards this goal has been started by isolation of auxotrophic mutants of T. reesei.Cloning of genes from filamentous fungi by expression in the yeast Saccharomyces cerevisiae, as a general method, was studied.Yeast transformants carrying A. niger DNA were screened for functional expression of genes coding for the secreted enzymes ß-galactosidase, ß-glucosidase and amyloglucosidase and for the expression of Aspergillus genes complementing the yeast leu 2 and ura 3 mutations.Only the gene for ß-glucosidase was obtained in this way its expression being detected as blue coloured transformant colonies on plates containing the Xglu reagent.In addition to A. niger ß-glucosidase, the T. reesei CBH I gene coding for cellobiohydrolase I, the most abundant enzyme present in culture fluids of the fungus, is shown to express in yeast.CBH I enzyme produced by recombinant yeast shows activity comparable to that of T. reesei enzyme.However, the expression of genomic filamentous fungal genes in yeast seems to be rare and when it occurs the level of expression is very low. For efficient production of filamentous fungal enzymes in yeast, yeast regulatory signals and cDNAs of the genes to be expressed, need to be used.In future, both approaches, mutagenesis and screening and recombinant DNA, will be needed in development of new fungal strains for industry.

KW - genetic engineering

KW - genetics

KW - improvement

KW - enzymes

KW - production

KW - fungi

KW - theses

M3 - Dissertation

SN - 951-38-2436-5

T3 - Technical Research Centre of Finland. Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Nevalainen H. Genetic improvement of enzyme production in industrially important fungal strains: Dissertation. Espoo: VTT Technical Research Centre of Finland, 1985. 98 p.