Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae

Abstract

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.

FIG. 1.

Cytosolic bioreaction network of S. cerevisiae. The bold arrows indicate the recombinant phosphoketolase pathway. Direct hydrolysis of acetyl-P may be catalyzed, for example, by [H⁺]ATPase (36). Abbreviations: PK, phosphoketolase; PTA, phosphotransacetylase; ACDH, acetaldehyde dehydrogenase (acylating); ACH1, acetyl-CoA hydrolase; ALDx, aldehyde dehydrogenase isoenzymes.

FIG. 2.

Physiological parameters of S. cerevisiae TMB3001c expressing various combinations of the phosphoketolase pathway genes during the xylose consumption phase (30 h up to about 120 h) in anaerobic batch cultures with 50 g of glucose liter⁻¹ and 50 g of xylose liter⁻¹. Average and deviation for two independent experiments are shown.

FIG. 3.

Specific intracellular carbon fluxes (μmol g DW⁻¹ h⁻¹) during xylose consumption in anaerobic batch experiments. Fluxes for TMB3001c (upper values) and TMB3001c-p4PTA/p5EHADH2 (middle values) were estimated from the experimental data shown in Fig. 2. Average values of duplicate experiments with deviations within 10% are given. The lower values (italics) represent the flux distribution that is required for maximum theoretical ethanol production from the xylose uptake rate of TMB3001c.

FIG. 4.

Physiological parameters of TMBALD6c and TMBALD6c-p4PTA/p5EHADH2 during xylose consumption in anaerobic batch culture with 50 g of glucose liter⁻¹ and 50 g of xylose liter⁻¹. TMB3001c and TMB3001c-p4PTA/p5EHADH2 (white bars) data from Fig. 2 are shown for comparison. The average and deviation for two independent experiments are displayed.