Production of xylitol and other five-carbon sugars and sugar alcohols from D-glucose with Saccharomyces cerevisiae

Merja Toivari, A.N. Miasnikov, Peter Richard, Laura Ruohonen, Merja Penttilä

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

Abstract

We constructed recombinant Saccharomyces cerevisiae strains deficient in either transketolase or phosphoglucose isomerase activities for production of xylitol, ribitol or ribose from D-glucose via the pentose phosphate pathway (PPP) intermediates D-xylulose 5-phosphate, D-ribulose 5-phosphate or D-ribose 5-phosphate. The transketolase-deficient strains accumulated PPP sugar-phosphates and excreted D-ribulose/D-ribose to the culture medium. Introduction of xylitol dehydrogenase encoding gene XYL2 from Pichia stipitis increased ribitol and xylitol production and concomitantly decreased the amount of D-ribulose/D-ribose. A sugar phosphate phosphatase Dog1p increased ribitol production 1.6-fold, but did not enhance xylitol excretion. Deletion of the xylulokinase encoding gene XKS1 increased the amount of xylitol produced to 50% of the total sugar alcohols (ribitol plus xylitol) produced. The highest yield of xylitol and ribitol obtained with the various transketolase deficient strains studied was 3.6% (w/w) of consumed D-glucose. S. cerevisiae strains deficient in phosphoglucose isomerase activity are unable to grow on D-glucose unless a NADPH-consuming reaction is introduced into the cell (1, 2). We introduced two NADPH-consuming reactions, a NADPH-utilizing glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis (GapB) and a NAD-dependent glutamate dehydrogenase (GDH2) of S. cerevisiae into the pgi1-mutant strain in order to produce 5-carbon sugars and sugar alcohols from D-glucose via the PPP. Both of these enzymes enabled growth on D-glucose of the pgi1-mutant strains, as shown for the GDH2 overexpression previously (1). The Dog1p phosphatase was, however, needed for production of 5-carbon sugars and sugar alcohols. On higher D-glucose concentrations the expression of DOG1 together with either GapB or GDH2 reduced growth. The Dog1p may create a futile cycle of phosphorylation and dephosphorylation of glucose 6-phosphate resulting in ATP depletion.
Original languageEnglish
Title of host publication3rd European Federation of Biotechnology Conference
Subtitle of host publicationPhysiology of Yeasts and Filamentous Fungi PYFF3
Place of PublicationEspoo
PublisherVTT Technical Research Centre of Finland
Pages156
ISBN (Electronic)978-951-38-6314-2
ISBN (Print)978-951-38-6313-5
Publication statusPublished - 2007
Event3rd European Federation of Biotechnology Conference : Physiology of Yeasts and Filamentous Fungi - Helsinki, Finland
Duration: 13 Jun 200716 Jun 2007

Publication series

NameVTT Symposium
PublisherVTT
Number245
ISSN (Print)0357-9387
ISSN (Electronic)1455-0873

Conference

Conference3rd European Federation of Biotechnology Conference
Abbreviated titlePYFF3
CountryFinland
CityHelsinki
Period13/06/0716/06/07

Fingerprint

sugar alcohols
xylitol
Saccharomyces cerevisiae
sugars
ribose
glucose
phosphates
transketolase
carbon
ribulose
pentoses
NADP (coenzyme)
glucose-6-phosphate isomerase
sugar phosphates
Scheffersomyces stipitis
xylulose
mutants
glucose 6-phosphate
glyceraldehyde-3-phosphate dehydrogenase
dephosphorylation

Cite this

Toivari, M., Miasnikov, A. N., Richard, P., Ruohonen, L., & Penttilä, M. (2007). Production of xylitol and other five-carbon sugars and sugar alcohols from D-glucose with Saccharomyces cerevisiae. In 3rd European Federation of Biotechnology Conference : Physiology of Yeasts and Filamentous Fungi PYFF3 (pp. 156). [P98] Espoo: VTT Technical Research Centre of Finland. VTT Symposium, No. 245
Toivari, Merja ; Miasnikov, A.N. ; Richard, Peter ; Ruohonen, Laura ; Penttilä, Merja. / Production of xylitol and other five-carbon sugars and sugar alcohols from D-glucose with Saccharomyces cerevisiae. 3rd European Federation of Biotechnology Conference : Physiology of Yeasts and Filamentous Fungi PYFF3 . Espoo : VTT Technical Research Centre of Finland, 2007. pp. 156 (VTT Symposium; No. 245).
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abstract = "We constructed recombinant Saccharomyces cerevisiae strains deficient in either transketolase or phosphoglucose isomerase activities for production of xylitol, ribitol or ribose from D-glucose via the pentose phosphate pathway (PPP) intermediates D-xylulose 5-phosphate, D-ribulose 5-phosphate or D-ribose 5-phosphate. The transketolase-deficient strains accumulated PPP sugar-phosphates and excreted D-ribulose/D-ribose to the culture medium. Introduction of xylitol dehydrogenase encoding gene XYL2 from Pichia stipitis increased ribitol and xylitol production and concomitantly decreased the amount of D-ribulose/D-ribose. A sugar phosphate phosphatase Dog1p increased ribitol production 1.6-fold, but did not enhance xylitol excretion. Deletion of the xylulokinase encoding gene XKS1 increased the amount of xylitol produced to 50{\%} of the total sugar alcohols (ribitol plus xylitol) produced. The highest yield of xylitol and ribitol obtained with the various transketolase deficient strains studied was 3.6{\%} (w/w) of consumed D-glucose. S. cerevisiae strains deficient in phosphoglucose isomerase activity are unable to grow on D-glucose unless a NADPH-consuming reaction is introduced into the cell (1, 2). We introduced two NADPH-consuming reactions, a NADPH-utilizing glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis (GapB) and a NAD-dependent glutamate dehydrogenase (GDH2) of S. cerevisiae into the pgi1-mutant strain in order to produce 5-carbon sugars and sugar alcohols from D-glucose via the PPP. Both of these enzymes enabled growth on D-glucose of the pgi1-mutant strains, as shown for the GDH2 overexpression previously (1). The Dog1p phosphatase was, however, needed for production of 5-carbon sugars and sugar alcohols. On higher D-glucose concentrations the expression of DOG1 together with either GapB or GDH2 reduced growth. The Dog1p may create a futile cycle of phosphorylation and dephosphorylation of glucose 6-phosphate resulting in ATP depletion.",
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Toivari, M, Miasnikov, AN, Richard, P, Ruohonen, L & Penttilä, M 2007, Production of xylitol and other five-carbon sugars and sugar alcohols from D-glucose with Saccharomyces cerevisiae. in 3rd European Federation of Biotechnology Conference : Physiology of Yeasts and Filamentous Fungi PYFF3 ., P98, VTT Technical Research Centre of Finland, Espoo, VTT Symposium, no. 245, pp. 156, 3rd European Federation of Biotechnology Conference , Helsinki, Finland, 13/06/07.

Production of xylitol and other five-carbon sugars and sugar alcohols from D-glucose with Saccharomyces cerevisiae. / Toivari, Merja; Miasnikov, A.N.; Richard, Peter; Ruohonen, Laura; Penttilä, Merja.

3rd European Federation of Biotechnology Conference : Physiology of Yeasts and Filamentous Fungi PYFF3 . Espoo : VTT Technical Research Centre of Finland, 2007. p. 156 P98 (VTT Symposium; No. 245).

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

TY - CHAP

T1 - Production of xylitol and other five-carbon sugars and sugar alcohols from D-glucose with Saccharomyces cerevisiae

AU - Toivari, Merja

AU - Miasnikov, A.N.

AU - Richard, Peter

AU - Ruohonen, Laura

AU - Penttilä, Merja

PY - 2007

Y1 - 2007

N2 - We constructed recombinant Saccharomyces cerevisiae strains deficient in either transketolase or phosphoglucose isomerase activities for production of xylitol, ribitol or ribose from D-glucose via the pentose phosphate pathway (PPP) intermediates D-xylulose 5-phosphate, D-ribulose 5-phosphate or D-ribose 5-phosphate. The transketolase-deficient strains accumulated PPP sugar-phosphates and excreted D-ribulose/D-ribose to the culture medium. Introduction of xylitol dehydrogenase encoding gene XYL2 from Pichia stipitis increased ribitol and xylitol production and concomitantly decreased the amount of D-ribulose/D-ribose. A sugar phosphate phosphatase Dog1p increased ribitol production 1.6-fold, but did not enhance xylitol excretion. Deletion of the xylulokinase encoding gene XKS1 increased the amount of xylitol produced to 50% of the total sugar alcohols (ribitol plus xylitol) produced. The highest yield of xylitol and ribitol obtained with the various transketolase deficient strains studied was 3.6% (w/w) of consumed D-glucose. S. cerevisiae strains deficient in phosphoglucose isomerase activity are unable to grow on D-glucose unless a NADPH-consuming reaction is introduced into the cell (1, 2). We introduced two NADPH-consuming reactions, a NADPH-utilizing glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis (GapB) and a NAD-dependent glutamate dehydrogenase (GDH2) of S. cerevisiae into the pgi1-mutant strain in order to produce 5-carbon sugars and sugar alcohols from D-glucose via the PPP. Both of these enzymes enabled growth on D-glucose of the pgi1-mutant strains, as shown for the GDH2 overexpression previously (1). The Dog1p phosphatase was, however, needed for production of 5-carbon sugars and sugar alcohols. On higher D-glucose concentrations the expression of DOG1 together with either GapB or GDH2 reduced growth. The Dog1p may create a futile cycle of phosphorylation and dephosphorylation of glucose 6-phosphate resulting in ATP depletion.

AB - We constructed recombinant Saccharomyces cerevisiae strains deficient in either transketolase or phosphoglucose isomerase activities for production of xylitol, ribitol or ribose from D-glucose via the pentose phosphate pathway (PPP) intermediates D-xylulose 5-phosphate, D-ribulose 5-phosphate or D-ribose 5-phosphate. The transketolase-deficient strains accumulated PPP sugar-phosphates and excreted D-ribulose/D-ribose to the culture medium. Introduction of xylitol dehydrogenase encoding gene XYL2 from Pichia stipitis increased ribitol and xylitol production and concomitantly decreased the amount of D-ribulose/D-ribose. A sugar phosphate phosphatase Dog1p increased ribitol production 1.6-fold, but did not enhance xylitol excretion. Deletion of the xylulokinase encoding gene XKS1 increased the amount of xylitol produced to 50% of the total sugar alcohols (ribitol plus xylitol) produced. The highest yield of xylitol and ribitol obtained with the various transketolase deficient strains studied was 3.6% (w/w) of consumed D-glucose. S. cerevisiae strains deficient in phosphoglucose isomerase activity are unable to grow on D-glucose unless a NADPH-consuming reaction is introduced into the cell (1, 2). We introduced two NADPH-consuming reactions, a NADPH-utilizing glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis (GapB) and a NAD-dependent glutamate dehydrogenase (GDH2) of S. cerevisiae into the pgi1-mutant strain in order to produce 5-carbon sugars and sugar alcohols from D-glucose via the PPP. Both of these enzymes enabled growth on D-glucose of the pgi1-mutant strains, as shown for the GDH2 overexpression previously (1). The Dog1p phosphatase was, however, needed for production of 5-carbon sugars and sugar alcohols. On higher D-glucose concentrations the expression of DOG1 together with either GapB or GDH2 reduced growth. The Dog1p may create a futile cycle of phosphorylation and dephosphorylation of glucose 6-phosphate resulting in ATP depletion.

M3 - Conference abstract in proceedings

SN - 978-951-38-6313-5

T3 - VTT Symposium

SP - 156

BT - 3rd European Federation of Biotechnology Conference

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Toivari M, Miasnikov AN, Richard P, Ruohonen L, Penttilä M. Production of xylitol and other five-carbon sugars and sugar alcohols from D-glucose with Saccharomyces cerevisiae. In 3rd European Federation of Biotechnology Conference : Physiology of Yeasts and Filamentous Fungi PYFF3 . Espoo: VTT Technical Research Centre of Finland. 2007. p. 156. P98. (VTT Symposium; No. 245).