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Genetic Traits Beneficial for Xylose Utilization by Recombinant Saccharomyces cerevisiae

Bengtsson, Oskar LU (2008)
Abstract
Saccharomyces cerevisiae ferments hexoses in lignocellulosic hydrolysates under anaerobic conditions with high rates and ethanol yields. However, S. cerevisiae is naturally unable to utilize the pentose fraction of the hydrolysates. Xylose is the most abundant pentose sugar, and although recombinant S. cerevisiae strains capable of ethanolic xylose fermentation have been obtained through metabolic engineering, there still remains the challenge of transferring the acquired knowledge into an industrial context. The present thesis describes an enhancement of the xylose utilization capability of recombinant S. cerevisiae through several approaches. A series of integrating plasmids was constructed in order to facilitate improvements of the... (More)
Saccharomyces cerevisiae ferments hexoses in lignocellulosic hydrolysates under anaerobic conditions with high rates and ethanol yields. However, S. cerevisiae is naturally unable to utilize the pentose fraction of the hydrolysates. Xylose is the most abundant pentose sugar, and although recombinant S. cerevisiae strains capable of ethanolic xylose fermentation have been obtained through metabolic engineering, there still remains the challenge of transferring the acquired knowledge into an industrial context. The present thesis describes an enhancement of the xylose utilization capability of recombinant S. cerevisiae through several approaches. A series of integrating plasmids was constructed in order to facilitate improvements of the XR-XDH xylose utilization pathway. The aerobic xylose growth rate of recombinant S. cerevisiae was distinctly improved when utilizing the TDH3 promoter for XR expression instead of the ADH1 promoter. Traditionally, xylose-utilizing S. cerevisiae with the xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis produce low ethanol yields due to the formation of xylitol by-product, which has been attributed to the different cofactor preferences of the NAD(P)H-dependent XR and the NAD+-dependent XDH. With the purpose of engineering the enzyme towards NADH-preference, the cofactor binding site of P. stipitis XR was altered by site-directed mutagenesis. Native P. stipitis XR and several mutants were kinetically characterized, and the effects of the mutations were investigated in vivo. The K270R mutation enabled increased ethanol yields, decreased xylitol yields and higher xylose consumption rates in anaerobic xylose fermentations. Additionally, traits important for ethanolic xylose fermentation were investigated at the transcriptional level. Four recombinant S. cerevisiae strains with enhanced xylose utilization capabilities were compared with two control strains in a genome-wide transcription analysis. Thirteen genes, with altered expression levels in all improved strains, were singled out for further analysis. It was found that the overexpression of SOL3 and TAL1, and the deletion of YLR042C, MNI1 and RPA49, enhanced the aerobic xylose growth by recombinant S. cerevisiae. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Thevelein, Johan, Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, and Department of Molecular Microbiology, VIB, Belgium
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Saccharomyces cerevisiae, metabolic engineering, fermentation, xylose, ethanol, promoter, xylose reductase, cofactor, validation, transcriptome
defense location
Room B, Kemicentrum, Getingevägen 60, Lund university, Faculty of Engineering
defense date
2008-12-19 10:15
ISBN
978-91-7422-211-1
language
English
LU publication?
yes
id
2a90b009-dedd-4f33-9b3a-ad1770aad1dd (old id 1270414)
date added to LUP
2008-11-25 11:13:39
date last changed
2016-09-19 08:45:16
@misc{2a90b009-dedd-4f33-9b3a-ad1770aad1dd,
  abstract     = {Saccharomyces cerevisiae ferments hexoses in lignocellulosic hydrolysates under anaerobic conditions with high rates and ethanol yields. However, S. cerevisiae is naturally unable to utilize the pentose fraction of the hydrolysates. Xylose is the most abundant pentose sugar, and although recombinant S. cerevisiae strains capable of ethanolic xylose fermentation have been obtained through metabolic engineering, there still remains the challenge of transferring the acquired knowledge into an industrial context. The present thesis describes an enhancement of the xylose utilization capability of recombinant S. cerevisiae through several approaches. A series of integrating plasmids was constructed in order to facilitate improvements of the XR-XDH xylose utilization pathway. The aerobic xylose growth rate of recombinant S. cerevisiae was distinctly improved when utilizing the TDH3 promoter for XR expression instead of the ADH1 promoter. Traditionally, xylose-utilizing S. cerevisiae with the xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis produce low ethanol yields due to the formation of xylitol by-product, which has been attributed to the different cofactor preferences of the NAD(P)H-dependent XR and the NAD+-dependent XDH. With the purpose of engineering the enzyme towards NADH-preference, the cofactor binding site of P. stipitis XR was altered by site-directed mutagenesis. Native P. stipitis XR and several mutants were kinetically characterized, and the effects of the mutations were investigated in vivo. The K270R mutation enabled increased ethanol yields, decreased xylitol yields and higher xylose consumption rates in anaerobic xylose fermentations. Additionally, traits important for ethanolic xylose fermentation were investigated at the transcriptional level. Four recombinant S. cerevisiae strains with enhanced xylose utilization capabilities were compared with two control strains in a genome-wide transcription analysis. Thirteen genes, with altered expression levels in all improved strains, were singled out for further analysis. It was found that the overexpression of SOL3 and TAL1, and the deletion of YLR042C, MNI1 and RPA49, enhanced the aerobic xylose growth by recombinant S. cerevisiae.},
  author       = {Bengtsson, Oskar},
  isbn         = {978-91-7422-211-1},
  keyword      = {Saccharomyces cerevisiae,metabolic engineering,fermentation,xylose,ethanol,promoter,xylose reductase,cofactor,validation,transcriptome},
  language     = {eng},
  title        = {Genetic Traits Beneficial for Xylose Utilization by Recombinant Saccharomyces cerevisiae},
  year         = {2008},
}