Molecular basis for anaerobic growth of Saccharomyces cerevisiae on xylose, investigated by global gene expression and metabolic flux analysis

Abstract

Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.

FIG. 1.

Bioreaction network of S. cerevisiae central carbon metabolism. Extracellular metabolites are indicated by uppercase letters. ETC, electron transport chain; acetyl-CoA, acetyl coenzyme A; FADH₂, reduced flavin adenine dinucleotide.

FIG. 2.

Comparison of molar carbon fluxes (ovals; values are expressed in millimoles per gram of biomass per hour) and transcript levels of the related enzymes (boxes; values are expressed in arbitrary units) in S. cerevisiae TMB3001 (upper values) and C1 (lower values) during anaerobic chemostat cultivation on 10 g of glucose per liter and 10 g of xylose per liter. The averages ± standard deviations from a DNA microarray analysis of two independent cultures are shown. Independent of the fold change, changes in gene expression were considered significant (indicated by boldface type) upon SAM analysis with an expected median false-positive rate of 1%. The standard deviations for all carbon fluxes were less than 10% of the averages from two independent cultures. Extracellular metabolites are indicated by uppercase letters. Fluxes from the following intracellular metabolites are required for biomass formation but are not shown explicitly: glucose 6-phosphate, ribose 5-phosphate, erythrose 4-phosphate, 3-phosphoglycerate, phosphoenolpyruvate, pyruvate, and acetate (24, 47).

FIG. 3.

Molar carbon fluxes (expressed in millimoles per gram of biomass per hour) during anaerobic exponential batch growth of TMB3001 on 10 g of glucose per liter (A) and during anaerobic exponential batch growth of C1 on 10 g of xylose per liter (B, upper values) or 10 g of xylose per liter plus 0.5 g of acetoin per liter (B, lower values). The averages for duplicate experiments are shown, and the standard deviations were less than 10% in all cases.