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

• Saccharomyces cerevisiae is one of the most well characterized yeast of large industrial interest due to several attractivefeatures such as its capability to efficiently convert glucose into ethanol and carbon dioxide at high flux, its amenabilityto genetic modification and the presence of extensive knowledge databases. For these reasons, it is often considered asuitable cell factory for the production of different classes of compounds. The central carbon metabolism of S. cerevisiaehas been object of numerous studies aiming at elucidating the complex mechanisms underlying the tight cellular balancearising as a consequence of a wide variety of regulatory pathways and phenomena, such as the Crabtree effect. Toengineer efficient cell factories, a deep knowledge of cellular metabolism and its regulatory mechanisms is offundamental importance to further de-regulate regulatory circuits hampering the reaching of desired characteristics. Inthis perspective, metabolic engineering and systems biology can supply valid and more efficient approaches for a globalunderstanding of the yeast cell. Although the central carbon metabolism of S. cerevisiae has been object of numerousinvestigations, the design of intuitive metabolic engineering strategies has often encountered several hurdles due to thetight regulation exerted by the cell. In this doctoral thesis, the contribution of systems biology and metabolic engineeringto gaining new insight into the central carbon metabolism of S. cerevisiae is addressed. Different metabolic engineeringapproaches to re-wire the glycolytic flux are presented. While the first and most direct approach is based on a deletionin the lower part of glycolysis through the construction of a phosphoglycerate mutase (Δgpm1) mutant, a more elaboratedapproach is described in the expression of the Aspergillus nidulans phosphoketolase pathway in S. cerevisiae. Fermentationtechnology as well as tools within systems biology, such as DNA microarrays and 13C flux analysis, were used as tools forthe characterization of the recombinant phenotypes, highlighting the challenges faced by the re-wiring of essentialpathways, 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 basedcomparison with the Crabtree negative yeast Scheffersomyces stipitis (Pichia stipitis) was performed. Integrative, system-levelanalysis of the two yeasts growing aerobically under glucose excess and glucose limitation conditions contributed to gaininsight into a different regulation of the central carbon metabolism of the two yeasts. What emerges from the differentworks performed is that physiological studies based on metabolic engineering benefit from systems biologymethodologies such as transcriptomics, fluxomics and metabolomics, supporting the characterization of recombinantand wild-type strains and helping to bridge the gap between genotype and phenotype. As both microarrays and RNAseqhave been used to characterize transcriptomes of different yeast strains, an attempt to address and compare theperformances of the two transcriptomic platforms is presented in the last chapter of this thesis where a technicalcomparison between the two methodologies is described, addressing the contribution of the different steps involved inRNA-seq analysis to obtain biologically meaningful data.

Ämnesord

NATURVETENSKAP  -- Biologi -- Annan biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Other Biological Topics (hsv//eng)

Nyckelord

fermentation technology

Metabolic engineering

RNA-sequencing

Saccharomyces cerevisiae

DNA microarrays

13Cbased flux analysis

system biology

Scheffersomyces stipitis

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