The transcriptome and flux profiling of Crabtree-negative hydroxy acid producing strains of Saccharomyces cerevisiae reveals changes in the central carbon metabolism

Saccharomyces cerevisiae is a yeast cell factory of choice for the production of many bio-based chemicals. However, it is also a Crabtree-positive yeast and so it shuttles a large portion of carbon into ethanol, even under aerobic conditions. To minimise the carbon loss, ethanol formation can be eliminated by deleting pyruvate decarboxylase (PDC) activity. Deletion of PDC genes has a profound impact on S. cerevisiae physiology, and it is not yet well understood how PDC-negative yeasts are affected when engineered to produce other products than ethanol. In this study, we introduced pathways for the production of three hydroxy acids (lactic, malic, or 3-hydroxypropionic acid) into an evolved PDC-negative strain. We characterised these strains via transcriptome and flux profiling to elucidate the effects that the production of these hydroxy acids has on the host strain. The expression of lactic acid and malic acid biosynthesis pathways improved the maximum specific growth rate (μ_max ) of the strain by 64 and 20% respectively, presumably due to NAD+ regeneration. On the contrary, the 3HP pathways expression decreased the μmax . All strains showed a very high flux (>90% of glucose uptake) into the oxidative pentose phosphate pathway under batch fermentation conditions. The transcriptional profile was least affected by the production of lactic acid and more by malic or 3-hydroxypropionic acids. The study, for the first time, directly compares the flux and transcriptome profiles of several different hydroxy acid producing strains of an evolved PDC-negative S. cerevisiae and suggests directions for future metabolic engineering. This article is protected by copyright. All rights reserved.