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Redox balancing in recombinant strains of Saccharomyces cerevisiae

Anderlund, Mikael LU (1998)
Abstract
In metabolically engineered Saccharomyces cerevisiae expressing Pichia stipitis XYL1 and XYL2 genes, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, xylitol is excreted as the major product during anaerobic xylose fermentation and only low yields of ethanol are produced. This has been interpreted as a result of the dual cofactor dependence of XR and the exclusive use of NAD+ by XDH. The excretion of xylitol was completely stopped and the formation of glycerol and acetic acid were reduced in xylose utilising S. cerevisiae strains cultivated in oxygen-limited conditions by expressing lower levels of XR than of XDH. The expression level of XYL1 and XYL2 were controlled by changing the promoters and transcription... (More)
In metabolically engineered Saccharomyces cerevisiae expressing Pichia stipitis XYL1 and XYL2 genes, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, xylitol is excreted as the major product during anaerobic xylose fermentation and only low yields of ethanol are produced. This has been interpreted as a result of the dual cofactor dependence of XR and the exclusive use of NAD+ by XDH. The excretion of xylitol was completely stopped and the formation of glycerol and acetic acid were reduced in xylose utilising S. cerevisiae strains cultivated in oxygen-limited conditions by expressing lower levels of XR than of XDH. The expression level of XYL1 and XYL2 were controlled by changing the promoters and transcription directions of the genes. A new functional metabolic pathway was established when Thermus thermophilus xylA gene was expressed in S. cerevisiae. The recombinant strain was able to ferment xylose to ethanol when cultivated on a minimal medium containing xylose as only carbon source. In order to create a channelled metabolite transfer in the two first steps of the xylose metabolism, XYL1 and XYL2 were fused in-frame and expressed in S. cerevisiae. When the fusion protein, containing a linker of three amino acids, was coexpressed together with native XR and XDH monomers, enzyme complexes consisting of chimeric and native subunits were formed. The total activity of these complexes exhibited 10 and 9 times higher XR and XDH activity, respectively, than the original conjugates, consisting of only chimeric subunits. This strain produced less xylitol and the xylitol yield was lower than with strains only expressing native XR and XDH monomers. In addition, more ethanol and less acetic acid were formed. A new gene encoding the cytoplasmic transhydrogernase from Azotobacter vinelandii was cloned. The enzyme showed high similarity to the family of pyridine nucleotide-disulphide oxidoreductase. To analyse the physiological effect of transhydrogenation between the two coenzyme systems NADP(H) and NAD(H) during anaerobic growth, S. cerevisiae was transformed with a plasma membrane bound AB-transhydrogenase from E. coli and with a cytoplasmic BB-transhydrogenase from A. vinelandii. Expression of both types changed the intracellular nucleotide levels. The NADPH/NADP+ ratio was reduced while the NADH/NAD+ ratio was almost constant. An increased formation of 2-oxoglutarate, glycerol and acetate was observed during anaerobic glucose fermentation. (Less)
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author
opponent
  • Gustafsson, Lena, Göteborg University, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
xylose isomerase, xylitol dehydrogenase, xylose reductase, glycerol, xylitol, xylose, E. coli, A. vinelandii, T. thermophilus, S. cerevisiae, P. stipitis, transhydrogenase., Microbiology, bacteriology, virology, mycology, Mikrobiologi, bakteriologi, virologi, mykologi
pages
160 pages
publisher
Department of Applied Microbiology, Lund University
defense location
Lund University, Kemicentrum, Sölvegatan 39, Lund, Sweden
defense date
1998-09-28 10:15
external identifiers
  • Other:ISRN: LUTKDH/TKMB--98/1029--SE
language
English
LU publication?
yes
id
4e23d45c-b940-42e4-a493-05ef49baec69 (old id 38961)
date added to LUP
2007-06-20 11:14:07
date last changed
2016-09-19 08:45:01
@phdthesis{4e23d45c-b940-42e4-a493-05ef49baec69,
  abstract     = {In metabolically engineered Saccharomyces cerevisiae expressing Pichia stipitis XYL1 and XYL2 genes, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, xylitol is excreted as the major product during anaerobic xylose fermentation and only low yields of ethanol are produced. This has been interpreted as a result of the dual cofactor dependence of XR and the exclusive use of NAD+ by XDH. The excretion of xylitol was completely stopped and the formation of glycerol and acetic acid were reduced in xylose utilising S. cerevisiae strains cultivated in oxygen-limited conditions by expressing lower levels of XR than of XDH. The expression level of XYL1 and XYL2 were controlled by changing the promoters and transcription directions of the genes. A new functional metabolic pathway was established when Thermus thermophilus xylA gene was expressed in S. cerevisiae. The recombinant strain was able to ferment xylose to ethanol when cultivated on a minimal medium containing xylose as only carbon source. In order to create a channelled metabolite transfer in the two first steps of the xylose metabolism, XYL1 and XYL2 were fused in-frame and expressed in S. cerevisiae. When the fusion protein, containing a linker of three amino acids, was coexpressed together with native XR and XDH monomers, enzyme complexes consisting of chimeric and native subunits were formed. The total activity of these complexes exhibited 10 and 9 times higher XR and XDH activity, respectively, than the original conjugates, consisting of only chimeric subunits. This strain produced less xylitol and the xylitol yield was lower than with strains only expressing native XR and XDH monomers. In addition, more ethanol and less acetic acid were formed. A new gene encoding the cytoplasmic transhydrogernase from Azotobacter vinelandii was cloned. The enzyme showed high similarity to the family of pyridine nucleotide-disulphide oxidoreductase. To analyse the physiological effect of transhydrogenation between the two coenzyme systems NADP(H) and NAD(H) during anaerobic growth, S. cerevisiae was transformed with a plasma membrane bound AB-transhydrogenase from E. coli and with a cytoplasmic BB-transhydrogenase from A. vinelandii. Expression of both types changed the intracellular nucleotide levels. The NADPH/NADP+ ratio was reduced while the NADH/NAD+ ratio was almost constant. An increased formation of 2-oxoglutarate, glycerol and acetate was observed during anaerobic glucose fermentation.},
  author       = {Anderlund, Mikael},
  keyword      = {xylose isomerase,xylitol dehydrogenase,xylose reductase,glycerol,xylitol,xylose,E. coli,A. vinelandii,T. thermophilus,S. cerevisiae,P. stipitis,transhydrogenase.,Microbiology,bacteriology,virology,mycology,Mikrobiologi,bakteriologi,virologi,mykologi},
  language     = {eng},
  pages        = {160},
  publisher    = {Department of Applied Microbiology, Lund University},
  school       = {Lund University},
  title        = {Redox balancing in recombinant strains of Saccharomyces cerevisiae},
  year         = {1998},
}