Enhancing the flux of D-glucose to the pentose phosphate pathway in Saccharomyces cerevisiae for the production of D-ribose and ribitol
- 423 Downloads
Phosphoglucose isomerase-deficient (pgi1) strains of Saccharomyces cerevisiae were studied for the production of D-ribose and ribitol from D-glucose via the intermediates of the pentose phosphate pathway. Overexpression of the genes coding for NAD+-specific glutamate dehydrogenase (GDH2) of S. cerevisiae or NADPH-utilising glyceraldehyde-3-phosphate dehydrogenase (gapB) of Bacillus subtilis enabled growth of the pgi1 mutant strains on D-glucose. Overexpression of the gene encoding sugar phosphate phosphatase (DOG1) of S. cerevisiae was needed for the production of D-ribose and ribitol; however, it reduced the growth of the pgi1 strains expressing GDH2 or gapB in the presence of higher D-glucose concentrations. The CEN.PK2-1D laboratory strain expressing both gapB and DOG1 produced approximately 0.4 g l−1 of D-ribose and ribitol when grown on 20 g l−1 (w/v) D-fructose with 4 g l−1 (w/v) D-glucose. Nuclear magnetic resonance measurements of the cells grown with 13C-labelled D-glucose showed that about 60% of the D-ribose produced was derived from D-glucose. Strains deficient in both phosphoglucose isomerase and transketolase activities, and expressing DOG1 and GDH2 tolerated only low D-glucose concentrations (≤2 g l−1 (w/v)), but produced 1 g l−1 (w/v) D-ribose and ribitol when grown on 20 g l−1 (w/v) D-fructose with 2 g l−1 (w/v) D-glucose.
KeywordsSugar alcohols Pentose sugars Saccharomyces cerevisiae Pentose phosphate pathway D-ribose Ribitol NMR
Dr. Peter Richard is acknowledged for providing the gapB plasmid. Dr. Kari Koivuranta is acknowledged for providing the transketolase-deficient strain H2926. M.Sc. Ritva Verho is thanked for the strain H2493. Prof. Eckhard Boles is thanked for providing the GDH2 plasmid YEpMSP3-T. Dr. Marilyn Wiebe is acknowledged for helpful discussions and critical reading of the manuscript. Pirjo Tähtinen, Outi Könönen, and Merja Helanterä are thanked for excellent technical assistance. Helena Simolin and Marita Ikonen are thanked for the Dionex HPLC analysis.
The financial support from Danisco sweeteners and Tekes, the Finnish Funding Agency for Technology and Innovation (project 4007/96), and Finnish Academy (Centre of Excellence in White Biotechnology—Green Chemistry, project 118573) is gratefully acknowledged.
- Ciriacy M, Breitenbach I (1979) Physiological effects of seven different blocks in glycolysis in Saccharomyces cerevisiae. J Bacteriol 139:152–160Google Scholar
- Maitra PK (1971) Glucose and fructose metabolism in a phosphoglucoseisomeraseless mutant of Saccharomyces cerevisiae. J Bacteriol 107:759–769Google Scholar
- Sasajima K, Yoneda M (1989) Production of D-ribose by microorganisms. In: Vandamme EJ (ed) Biotechnology of vitamins, pigments and growth factors. Elsevier, New York, pp 167–197Google Scholar
- Sherman F, Fink G, Hicks JB (1983) Methods in yeast genetics. Cold Springs Harbor Laboratory, Cold Springs Harbor, NYGoogle Scholar
- Vinopal RT, Hillman JD, Schulman H, Reznikoff WS, Fraenkel DG (1975) New phosphoglucose isomerase mutants of Escherichia coli. J Bacteriol 122:1172–1174Google Scholar