Metabolic Engineering of Central Carbon Metabolism in Saccharomyces cerevisiae The contribution of systems biology to physiological studies

Doctoral thesis, 2012

Saccharomyces cerevisiae is one of the most well characterized yeast of large industrial interest due to several attractive features such as its capability to efficiently convert glucose into ethanol and carbon dioxide at high flux, its amenability to genetic modification and the presence of extensive knowledge databases. For these reasons, it is often considered a suitable cell factory for the production of different classes of compounds. The central carbon metabolism of S. cerevisiae has been object of numerous studies aiming at elucidating the complex mechanisms underlying the tight cellular balance arising as a consequence of a wide variety of regulatory pathways and phenomena, such as the Crabtree effect. To engineer efficient cell factories, a deep knowledge of cellular metabolism and its regulatory mechanisms is of fundamental importance to further de-regulate regulatory circuits hampering the reaching of desired characteristics. In this perspective, metabolic engineering and systems biology can supply valid and more efficient approaches for a global understanding of the yeast cell. Although the central carbon metabolism of S. cerevisiae has been object of numerous investigations, the design of intuitive metabolic engineering strategies has often encountered several hurdles due to the tight regulation exerted by the cell. In this doctoral thesis, the contribution of systems biology and metabolic engineering to gaining new insight into the central carbon metabolism of S. cerevisiae is addressed. Different metabolic engineering approaches to re-wire the glycolytic flux are presented. While the first and most direct approach is based on a deletion in the lower part of glycolysis through the construction of a phosphoglycerate mutase (Δgpm1) mutant, a more elaborated approach is described in the expression of the Aspergillus nidulans phosphoketolase pathway in S. cerevisiae. Fermentation technology as well as tools within systems biology, such as DNA microarrays and 13C flux analysis, were used as tools for the characterization of the recombinant phenotypes, highlighting the challenges faced by the re-wiring of essential pathways, thus indicating the robustness and the primary role in metabolism of the glycolytic pathway. To undertake a different approach to investigate the central carbon metabolism of S. cerevisiae, a high-throughput based comparison with the Crabtree negative yeast Scheffersomyces stipitis (Pichia stipitis) was performed. Integrative, system-level analysis of the two yeasts growing aerobically under glucose excess and glucose limitation conditions contributed to gain insight into a different regulation of the central carbon metabolism of the two yeasts. What emerges from the different works performed is that physiological studies based on metabolic engineering benefit from systems biology methodologies such as transcriptomics, fluxomics and metabolomics, supporting the characterization of recombinant and wild-type strains and helping to bridge the gap between genotype and phenotype. As both microarrays and RNAseq have been used to characterize transcriptomes of different yeast strains, an attempt to address and compare the performances of the two transcriptomic platforms is presented in the last chapter of this thesis where a technical comparison between the two methodologies is described, addressing the contribution of the different steps involved in RNA-seq analysis to obtain biologically meaningful data.