Advanced

Yeasts as cellular factories: Saccharomyces cerevisiae strains with improved inhibitor-tolerance to furaldehydes via a mutant alcohol dehydrogenase- and- Human ameloblastin production in Pichia pastoris

Laadan, Boaz LU (2008)
Abstract
Several yeast species, usually engineered by humans in order to produce compounds of biotechnological interests, are currently used as cellular factories. This thesis presents two main themes. In the first, Saccharomyces cerevisiae is used for bio-ethanol production from lignocellulose hydrolysates and in the second Pichia pastoris is used for the heterologous expression of human ameloblastin.



The production of bio-ethanol from lignocellulose requires solution of several technical issues, such as fermentation to ethanol in the presence of yeast inhibitors generated during the hydrolyzation step. In this thesis, two major furan inhibitors, HMF and furfural, were shown to be reduced and therefore detoxified by a mutated... (More)
Several yeast species, usually engineered by humans in order to produce compounds of biotechnological interests, are currently used as cellular factories. This thesis presents two main themes. In the first, Saccharomyces cerevisiae is used for bio-ethanol production from lignocellulose hydrolysates and in the second Pichia pastoris is used for the heterologous expression of human ameloblastin.



The production of bio-ethanol from lignocellulose requires solution of several technical issues, such as fermentation to ethanol in the presence of yeast inhibitors generated during the hydrolyzation step. In this thesis, two major furan inhibitors, HMF and furfural, were shown to be reduced and therefore detoxified by a mutated alcohol dehydrogenase 1 that was identified from an industrial isolate of S. cerevisiae. The mutant enzyme, which in its native state cannot reduce HMF but only furfural, was shown to acquire a larger substrate-binding pocket, suitable for accommodation of larger substrate molecules. Site-directed mutagenesis was used in order to investigate the roles of the different mutations, three of them being novel and three others having been reported previously. The physiological implications of over-expression of the different mutants are demonstrated by fermentation trials, and supported by enzyme activity measurements in-vitro.



Production of the human protein ameloblastin, which is required for the proper formation of enamel in mammals, was achieved by expression of the human ameloblastin gene in P. pastoris. Successful expression in this eukaryotic system was confirmed by mass spectrometry, but proteolysis and the lack of extracellular secretion are remaining problems that must be solved in order to achieve secretion of a native functional protein. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Prof. Albertyn, Jacobus, Dept. of Microbial, Biochemical & Food Biotechnology, University of the Free State, Bloemfontein, South Africa
organization
publishing date
type
Thesis
publication status
published
subject
pages
59 pages
defense location
Hall B, Kemicentrum
defense date
2008-04-11 10:00
ISBN
978-91-628-7466-7
language
English
LU publication?
yes
id
5d0ff2ad-23e5-4625-b80e-65b8f10b5fa4 (old id 1048575)
date added to LUP
2008-03-17 11:41:18
date last changed
2016-09-19 08:45:17
@phdthesis{5d0ff2ad-23e5-4625-b80e-65b8f10b5fa4,
  abstract     = {Several yeast species, usually engineered by humans in order to produce compounds of biotechnological interests, are currently used as cellular factories. This thesis presents two main themes. In the first, Saccharomyces cerevisiae is used for bio-ethanol production from lignocellulose hydrolysates and in the second Pichia pastoris is used for the heterologous expression of human ameloblastin.<br/><br>
<br/><br>
The production of bio-ethanol from lignocellulose requires solution of several technical issues, such as fermentation to ethanol in the presence of yeast inhibitors generated during the hydrolyzation step. In this thesis, two major furan inhibitors, HMF and furfural, were shown to be reduced and therefore detoxified by a mutated alcohol dehydrogenase 1 that was identified from an industrial isolate of S. cerevisiae. The mutant enzyme, which in its native state cannot reduce HMF but only furfural, was shown to acquire a larger substrate-binding pocket, suitable for accommodation of larger substrate molecules. Site-directed mutagenesis was used in order to investigate the roles of the different mutations, three of them being novel and three others having been reported previously. The physiological implications of over-expression of the different mutants are demonstrated by fermentation trials, and supported by enzyme activity measurements in-vitro. <br/><br>
<br/><br>
Production of the human protein ameloblastin, which is required for the proper formation of enamel in mammals, was achieved by expression of the human ameloblastin gene in P. pastoris. Successful expression in this eukaryotic system was confirmed by mass spectrometry, but proteolysis and the lack of extracellular secretion are remaining problems that must be solved in order to achieve secretion of a native functional protein.},
  author       = {Laadan, Boaz},
  isbn         = {978-91-628-7466-7},
  language     = {eng},
  pages        = {59},
  school       = {Lund University},
  title        = {Yeasts as cellular factories: Saccharomyces cerevisiae strains with improved inhibitor-tolerance to furaldehydes via a mutant alcohol dehydrogenase- and- Human ameloblastin production in Pichia pastoris},
  year         = {2008},
}