Yeast Saccharomyces cerevisiae as a tool in cloning and analysis of fungal genes: Applications for biomass hydrolysis and utilisation: Dissertation

Research output: ThesisDissertationCollection of Articles

Abstract

The baker s yeast, Saccharomyces cerevisiae has been employed by man for centuries in manufacturing of bread, beer, and wine. In science, it has become a useful tool as well. In this work, methods were developed in order to study the molecular biology of the cellulolytic filamentous fungus Trichoderma reesei with the aid of yeast. Cellulose is the most abundant carbon source in nature, and its enzymatic degradation is essential for carbon turnover. In addition, cellulose is used as a raw material in microbial processes. In this work, a previously unknown cellulase-encoding gene was cloned by expression in yeast and detection of hydrolysis halos on substrate plates. This EGV enzyme consists of an exceptionally small core domain, a cellulose-binding domain, and a linker region connecting the two. EGV belongs to family GH45 of glycosyl hydrolases. Additionally, a gene encoding a beta -1,3-1,4-glucanase enzyme was cloned and studied. The enzyme was produced in insect cells, and analysis of the degradation products of -glucan by NMR showed that it was a laminarinase (EC 3.2.1.6). A yeast-based cloning method for positively acting regulatory proteins was set up, and two regulatory genes of the T. reesei cellulases, ace1 and ace2, were isolated. The isolation was based on the ability of the encoded proteins to activate expression of a reporter gene, which was linked to the full-length promoter of the major cellulase gene cbh1 in yeast. No homologs of the new regulatory proteins were detected outside the Mycota. The DNA-binding properties of the regulatory proteins were studied both in vitro and in vivo in yeast. Deletion of the ace1 gene resulted in slower radial growth of the fungus on cellulose-containing plates. However, although isolated as an activator, ACEI was later shown to act as a repressor of hydrolase expression. ACEII, on the other hand, was shown to be an activator of cellulase expression. However, it is certainly not the only one, since its deletion did not result in a cellulase-negative phenotype. Additionally, a sugar permease-encoding gene was isolated from T. reesei by complementation. The yeast strain used as a host was deleted for the major hexose transporter genes (hxt1-7, gal2), and additionally engineered for xylose utilisation. The T. reesei permease complemented the growth defect of the mutant strain on xylose-maltose medium. However, adaptive mutation(s) were needed in the host to enable growth on xylose of the permease-expressing strain. The same, engineered yeast strain was used as a host for the native S. cerevisiae hexose transporter genes HXT1, HXT2, HXT4 and HXT7, and the kinetics of xylose transport were studied. The affinities of the permeases for xylose varied, Km values of 190-900 mM were detected. Interesting differences were obtained in the levels of inhibition by the presence of glucose. The single-Hxt strains exhibited a biphasic growth mode on xylose media, where an initially very slow growth was followed by exponential growth after a lag of several days.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Penttilä, Merja, Supervisor
Award date6 Aug 2004
Place of PublicationEspoo
Publisher
Print ISBNs951-38-6400-6
Electronic ISBNs951-38-6401-4
Publication statusPublished - 2004
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

molecular cloning
Saccharomyces cerevisiae
hydrolysis
xylose
yeasts
Trichoderma reesei
biomass
endo-1,4-beta-glucanase
regulatory proteins
cellulose
genes
beta-glucanase
hexoses
transporters
enzymes
fungi
cellulases
degradation
gene deletion
binding properties

Keywords

  • Saccharomyces cerevisiae
  • Trichoderma reesei
  • Hypocrea jecorina
  • expression-cloning
  • reporter gene
  • cellulase
  • activator
  • ACEI
  • ACEII
  • EGV
  • permease
  • Hxt
  • xylose
  • uptake
  • xylose utilisation
  • bio-ethanol

Cite this

@phdthesis{981264a34ac54d988ad583adf9e87861,
title = "Yeast Saccharomyces cerevisiae as a tool in cloning and analysis of fungal genes: Applications for biomass hydrolysis and utilisation: Dissertation",
abstract = "The baker s yeast, Saccharomyces cerevisiae has been employed by man for centuries in manufacturing of bread, beer, and wine. In science, it has become a useful tool as well. In this work, methods were developed in order to study the molecular biology of the cellulolytic filamentous fungus Trichoderma reesei with the aid of yeast. Cellulose is the most abundant carbon source in nature, and its enzymatic degradation is essential for carbon turnover. In addition, cellulose is used as a raw material in microbial processes. In this work, a previously unknown cellulase-encoding gene was cloned by expression in yeast and detection of hydrolysis halos on substrate plates. This EGV enzyme consists of an exceptionally small core domain, a cellulose-binding domain, and a linker region connecting the two. EGV belongs to family GH45 of glycosyl hydrolases. Additionally, a gene encoding a beta -1,3-1,4-glucanase enzyme was cloned and studied. The enzyme was produced in insect cells, and analysis of the degradation products of -glucan by NMR showed that it was a laminarinase (EC 3.2.1.6). A yeast-based cloning method for positively acting regulatory proteins was set up, and two regulatory genes of the T. reesei cellulases, ace1 and ace2, were isolated. The isolation was based on the ability of the encoded proteins to activate expression of a reporter gene, which was linked to the full-length promoter of the major cellulase gene cbh1 in yeast. No homologs of the new regulatory proteins were detected outside the Mycota. The DNA-binding properties of the regulatory proteins were studied both in vitro and in vivo in yeast. Deletion of the ace1 gene resulted in slower radial growth of the fungus on cellulose-containing plates. However, although isolated as an activator, ACEI was later shown to act as a repressor of hydrolase expression. ACEII, on the other hand, was shown to be an activator of cellulase expression. However, it is certainly not the only one, since its deletion did not result in a cellulase-negative phenotype. Additionally, a sugar permease-encoding gene was isolated from T. reesei by complementation. The yeast strain used as a host was deleted for the major hexose transporter genes (hxt1-7, gal2), and additionally engineered for xylose utilisation. The T. reesei permease complemented the growth defect of the mutant strain on xylose-maltose medium. However, adaptive mutation(s) were needed in the host to enable growth on xylose of the permease-expressing strain. The same, engineered yeast strain was used as a host for the native S. cerevisiae hexose transporter genes HXT1, HXT2, HXT4 and HXT7, and the kinetics of xylose transport were studied. The affinities of the permeases for xylose varied, Km values of 190-900 mM were detected. Interesting differences were obtained in the levels of inhibition by the presence of glucose. The single-Hxt strains exhibited a biphasic growth mode on xylose media, where an initially very slow growth was followed by exponential growth after a lag of several days.",
keywords = "Saccharomyces cerevisiae, Trichoderma reesei, Hypocrea jecorina, expression-cloning, reporter gene, cellulase, activator, ACEI, ACEII, EGV, permease, Hxt, xylose, uptake, xylose utilisation, bio-ethanol",
author = "Anu Saloheimo",
year = "2004",
language = "English",
isbn = "951-38-6400-6",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
number = "541",
address = "Finland",
school = "University of Helsinki",

}

Yeast Saccharomyces cerevisiae as a tool in cloning and analysis of fungal genes : Applications for biomass hydrolysis and utilisation: Dissertation. / Saloheimo, Anu.

Espoo : VTT Technical Research Centre of Finland, 2004. 89 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Yeast Saccharomyces cerevisiae as a tool in cloning and analysis of fungal genes

T2 - Applications for biomass hydrolysis and utilisation: Dissertation

AU - Saloheimo, Anu

PY - 2004

Y1 - 2004

N2 - The baker s yeast, Saccharomyces cerevisiae has been employed by man for centuries in manufacturing of bread, beer, and wine. In science, it has become a useful tool as well. In this work, methods were developed in order to study the molecular biology of the cellulolytic filamentous fungus Trichoderma reesei with the aid of yeast. Cellulose is the most abundant carbon source in nature, and its enzymatic degradation is essential for carbon turnover. In addition, cellulose is used as a raw material in microbial processes. In this work, a previously unknown cellulase-encoding gene was cloned by expression in yeast and detection of hydrolysis halos on substrate plates. This EGV enzyme consists of an exceptionally small core domain, a cellulose-binding domain, and a linker region connecting the two. EGV belongs to family GH45 of glycosyl hydrolases. Additionally, a gene encoding a beta -1,3-1,4-glucanase enzyme was cloned and studied. The enzyme was produced in insect cells, and analysis of the degradation products of -glucan by NMR showed that it was a laminarinase (EC 3.2.1.6). A yeast-based cloning method for positively acting regulatory proteins was set up, and two regulatory genes of the T. reesei cellulases, ace1 and ace2, were isolated. The isolation was based on the ability of the encoded proteins to activate expression of a reporter gene, which was linked to the full-length promoter of the major cellulase gene cbh1 in yeast. No homologs of the new regulatory proteins were detected outside the Mycota. The DNA-binding properties of the regulatory proteins were studied both in vitro and in vivo in yeast. Deletion of the ace1 gene resulted in slower radial growth of the fungus on cellulose-containing plates. However, although isolated as an activator, ACEI was later shown to act as a repressor of hydrolase expression. ACEII, on the other hand, was shown to be an activator of cellulase expression. However, it is certainly not the only one, since its deletion did not result in a cellulase-negative phenotype. Additionally, a sugar permease-encoding gene was isolated from T. reesei by complementation. The yeast strain used as a host was deleted for the major hexose transporter genes (hxt1-7, gal2), and additionally engineered for xylose utilisation. The T. reesei permease complemented the growth defect of the mutant strain on xylose-maltose medium. However, adaptive mutation(s) were needed in the host to enable growth on xylose of the permease-expressing strain. The same, engineered yeast strain was used as a host for the native S. cerevisiae hexose transporter genes HXT1, HXT2, HXT4 and HXT7, and the kinetics of xylose transport were studied. The affinities of the permeases for xylose varied, Km values of 190-900 mM were detected. Interesting differences were obtained in the levels of inhibition by the presence of glucose. The single-Hxt strains exhibited a biphasic growth mode on xylose media, where an initially very slow growth was followed by exponential growth after a lag of several days.

AB - The baker s yeast, Saccharomyces cerevisiae has been employed by man for centuries in manufacturing of bread, beer, and wine. In science, it has become a useful tool as well. In this work, methods were developed in order to study the molecular biology of the cellulolytic filamentous fungus Trichoderma reesei with the aid of yeast. Cellulose is the most abundant carbon source in nature, and its enzymatic degradation is essential for carbon turnover. In addition, cellulose is used as a raw material in microbial processes. In this work, a previously unknown cellulase-encoding gene was cloned by expression in yeast and detection of hydrolysis halos on substrate plates. This EGV enzyme consists of an exceptionally small core domain, a cellulose-binding domain, and a linker region connecting the two. EGV belongs to family GH45 of glycosyl hydrolases. Additionally, a gene encoding a beta -1,3-1,4-glucanase enzyme was cloned and studied. The enzyme was produced in insect cells, and analysis of the degradation products of -glucan by NMR showed that it was a laminarinase (EC 3.2.1.6). A yeast-based cloning method for positively acting regulatory proteins was set up, and two regulatory genes of the T. reesei cellulases, ace1 and ace2, were isolated. The isolation was based on the ability of the encoded proteins to activate expression of a reporter gene, which was linked to the full-length promoter of the major cellulase gene cbh1 in yeast. No homologs of the new regulatory proteins were detected outside the Mycota. The DNA-binding properties of the regulatory proteins were studied both in vitro and in vivo in yeast. Deletion of the ace1 gene resulted in slower radial growth of the fungus on cellulose-containing plates. However, although isolated as an activator, ACEI was later shown to act as a repressor of hydrolase expression. ACEII, on the other hand, was shown to be an activator of cellulase expression. However, it is certainly not the only one, since its deletion did not result in a cellulase-negative phenotype. Additionally, a sugar permease-encoding gene was isolated from T. reesei by complementation. The yeast strain used as a host was deleted for the major hexose transporter genes (hxt1-7, gal2), and additionally engineered for xylose utilisation. The T. reesei permease complemented the growth defect of the mutant strain on xylose-maltose medium. However, adaptive mutation(s) were needed in the host to enable growth on xylose of the permease-expressing strain. The same, engineered yeast strain was used as a host for the native S. cerevisiae hexose transporter genes HXT1, HXT2, HXT4 and HXT7, and the kinetics of xylose transport were studied. The affinities of the permeases for xylose varied, Km values of 190-900 mM were detected. Interesting differences were obtained in the levels of inhibition by the presence of glucose. The single-Hxt strains exhibited a biphasic growth mode on xylose media, where an initially very slow growth was followed by exponential growth after a lag of several days.

KW - Saccharomyces cerevisiae

KW - Trichoderma reesei

KW - Hypocrea jecorina

KW - expression-cloning

KW - reporter gene

KW - cellulase

KW - activator

KW - ACEI

KW - ACEII

KW - EGV

KW - permease

KW - Hxt

KW - xylose

KW - uptake

KW - xylose utilisation

KW - bio-ethanol

M3 - Dissertation

SN - 951-38-6400-6

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -