Metabolic engineering and random mutagenesis for improved xylose utilisation of Saccharomyces cerevisiae

Abstract: In this thesis I have summarised my work on the analysis and improvement of xylose utilisation by recombinant S. cerevisiae. A metabolic flux model was developed and used to analyse the intracellular fluxes in the recombinant xylose utilising S. cerevisiae TMB 3001 cultivated in chemostat at various dilution rates and xylose/glucose concentrations. Xylose uptake increased with xylose concentration in the feed and with decreasing dilution rate. The fraction of NADH-mediated xylose reduction increased with increasing dilution rate and with increasing xylose concentration in the feed. The flux between ribulose 5-phosphate and xylulose 5-phosphate was very low during all cultivation experiments with xylose/glucose in the feed. The aerobic conversion of xylose to pyruvate does not introduce new thermodynamic bottlenecks compared with glucose conversion to pyruvate. The thermodynamics of the reactions catalysed by xylose reductase and xylitol dehydrogenase are favourable over a larger metabolite concentration range if NADPH is used for xylose reduction and NAD+ is used for xylitol oxidation, respectively. Xylitol excretion in recombinant xylose utilising S. cerevisiae TMB 3001 is halted by addition of electron acceptors like acetoin and acetaldehyde which are reduced in NADH-coupled reactions. Also furfural, which is present in lignocellulosic hydrolysates, decreased xylitol formation. Xylitol and also glycerol by-product formation would be prevented if enzymatic activities for phosphoketolase, phosphate acetyl transferase and acetaldehyde dehydrogenase (acylating) were introduced in S. cerevisiae. In such a hypothetical pathway, excessive NADH formed either from xylitol oxidation or amino acid synthesis would be regenerated to NAD+ by converting acetyl-phosphate, formed in the phosphoketolase reaction, to ethanol using phosphate acetyl transferase, acetaldehyde dehydrogenase (acylating) and alcohol dehydrogenase. Random mutagenesis was successfully used to develop the recombinant S. cerevisiae TMB 3400 that has a maximum specific growth rate of 0.14 h-1 on xylose, compared to 0.025 h-1 for its parental strain, the non-mutated, recombinant S. cerevisiae TMB 3399. S. cerevisiae TMB 3400 showed elevated mRNA expression levels of genes involved in transport, initial xylose metabolism and in the pentose phosphate pathway. The biomass yield of S. cerevisiae TMB 3400 on xylose, 0.43 g biomass g xylose-1, was lower than that on glucose, 0.47 g biomass g glucose-1.