Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy.

(31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts.     

Enhancing the flux of D-glucose to the pentose phosphate pathway in Saccharomyces cerevisiae for the production of D-ribose and ribitol

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.