Exploring Yeast as a Cell Factory for the Production of Carboxylic Acids and Derivatives

University dissertation from Department of Chemistry, Lund University

Abstract: Baker’s yeast, Saccharomyces cerevisiae, is a promising cell factory for the sustainable utilization of renewable resources for the formation of products with commercial value. Among these, poly-3-D-hydroxybutyrate (PHB) is an extensively studied biopolymer naturally accumulated in some bacteria and archaea species through the formation of carbon granules. Its bio-based origin, biodegradability and applications in several industries makes it one of the most interesting biopolymers. In the present study, aerobic production of PHB from xylose was achieved in S. cerevisiae through the engineering of an optimized xylose oxido-reducing pathway and the expression of the genes involved in the PHB-producing pathway from the bacterium Cupriavidus necator. As anaerobicity is generally preferred in industrial applications, leading to an excess of NADH in the yeast metabolism, S. cerevisiae was further engineered by the introduction of a NADH-dependent acetoacetyl-CoA reductase from the bacterium Allochromatium vinosum. PHB formation clearly benefited from this modification and its formation from pure carbon sources under both anaerobic and oxygen-limited conditions was observed. The influence of nitrogen availability on PHB accumulation was also investigated. In contrast to the natural producers, PHB formation in S. cerevisiae was favored by high levels of nitrogen. These engineering strategies together resulted in one of the highest PHB contents reported in S. cerevisiae to date.The production of carboxylic acids, i.e. organic compounds that can be used as building blocks for a wide range of products, was also investigated in yeast due to its robustness and ability to grow at low pH. Cytosolic production of alpha-ketoglutarate (AKG) from xylose was attempted by rewiring the carbon flux towards the glyoxylate cycle in S. cerevisiae. Although AKG production was low, the study contributed to a deeper understanding of the mitochondrial and cytosolic formation of carboxylic acids in S. cerevisiae, revealing novel routes for their bio-production and for further optimization studies. In the last part of this work, AKG production was attempted by using a heterologous oxidative pathway that bypasses glycolysis and links xylose directly to the tricarboxylic acid cycle – the so-called Weimberg pathway. The Weimberg pathway was found to be partially active and highlighted the fact that the assembly and activity of the proteins converting xylonate into AKG require further development.