Yeast engineering for pharmaceutical and nutraceutical purposes
Saccharomyces is a genus of fungi consisting of many species of yeast strains including S. cerevisiae and S. boulardii, and has been engineered for implementing economic processes for producing biofuels and chemicals. However, this microorganism has restricted capabilities for producing other biomolecules than ethanol due to a rigid metabolic flux through glycolysis and ethanol pathway during utilization of fermentable sugars. Since the ethanol pathway plays a key role in cytosolic acetyl-CoA synthesis and redox balance of Saccharomyces, new metabolic engineering approaches not to knock out its ethanol pathway but to minimize the overflow metabolism are needed for increasing productivities of acetyl-CoA derivatives, growth-dependent metabolites, and intracellular biomolecules. The overall goal of my thesis study is to manipulate sugar metabolisms in Saccharomyces yeast strains for developing optimal yeast cells capable of efficiently synthesizing value-added pharmaceutical and nutraceutical molecules by bypassing negative characteristics of the overflow metabolism. Two strategies for bypassing the native overflow metabolism in Saccharomyces are demonstrated in this study: i) utilization of non-fermentable sugar, and ii) elimination of the interaction between phosphofructokinase and fructose 2,6-bisphosphate. Xylose utilization allowed engineered S. cerevisiae to dysregulate glucose-dependent repressions on cytosolic acetyl-CoA synthesis, ethanol re-oxidation, and oxidative phosphorylation. Restoring transcription levels of these metabolisms in engineered yeasts led to efficient cytosolic acetyl-CoA synthesis by simultaneous co-utilization of xylose and ethanol. The positive xylose effect was expanded to both accumulative and diffusible isoprenoids, which are synthesized from cytosolic acetyl-CoA. The elimination of allosteric up-regulation on phosphofructokinase and consequently upper glycolysis down-regulation could be achieved by mutagenesis on PFK1 and PFK2 or deletion of PFK26 and PFK27. Down-regulated upper glycolysis increased the availabilities of sugar phosphate intermediates, such as glucose 6-phosphate and fructose 6-phopsphate. Higher glucose 6-phosphate availabilities led to efficient regeneration of redox cofactor NADPH, which is required to biosynthesize isoprenoids, fatty acids, and sterols, through oxidative pentose phosphate pathway and accordingly increased isoprenoid production during glucose utilization. Higher fructose 6-phosphate availabilities allowed engineered yeasts to synthesize more GDP-mannose and consequently contain more cell wall mannan, a potent prebiotic oligosaccharide. Metabolic engineering approaches on the mannan synthetic machinery further increased cell wall mannan content. I demonstrated that adhesive capacities of engineered yeasts against pathogenic bacteria could be modulated by engineering mannan content of yeast cell wall fractions.
Saccharomyces cerevisiae, Saccharomyces boulardii, Acetyl-Coenzyme A (Acetyl-CoA), Isoprenoid, Cofactor, Guanosine diphosphate mannose (GDP-mannose), Mannan oligosaccharide
University of Illinois - Urbana-Champaign
Networked Digital Library of Theses and Dissertations
University of Illinois at Urbana-Champaign