Molecular Basis for Anaerobic Growth of <i>Saccharomyces cerevisiae</i> on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

TLDR

Yeast xylose metabolism is generally limited to aerobic conditions because the two‑step oxidoreductase pathway creates a redox imbalance, and even in engineered strains redox balancing remains a bottleneck that can be partially alleviated by external NADH sinks such as acetoin. The authors combined transcriptome profiling of aerobic chemostat cultures on xylose‑glucose mixtures and xylose alone with global gene‑expression and metabolic‑flux analysis of anaerobic chemostat cultures on xylose‑glucose mixtures to uncover the molecular basis of anaerobic xylose growth. They found that the engineered strain exhibits markedly elevated transcript levels and fluxes in central carbon, pentose‑phosphate, and glycerol pathways, up‑regulation of redox‑related genes that boost cytosolic NADPH production and NADH consumption, and that growth on xylose is ultimately limited by ATP production rather than redox balance, as evidenced by acetoin‑mediated ATP increases without changes in xylose uptake or NADPH synthesis.

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.