Metabolic Engineering of Recombinant Protein Production by Saccharomyces cerevisiae

Doctoral thesis, 2012

The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and it also provides a platform for the production of many heterologous proteins of medical or industrial interest. In this thesis, random and rational approaches, such as vector design, host engineering, fermentation analysis, UV Mutation, coupled with high-throughput systems biology techniques (including whole genomic sequencing, microarray analysis and flux analysis) and integrated analysis (Reporter feature technique), were employed to engineer cellular properties more effectively and purposefully to construct cell factories for protein production. We reported that insulin production mainly depends on the expression level of the gene, whereas amylase tends to achieve higher secretion at lower growth conditions in order to reduce ER stress. Moreover, based on large data generated and systems biology tools, we proposed several models to address unknown questions regarding recombinant protein production: i) the futile cycle of protein folding in the ER and the thermodynamic model of non-stoichiometric production of reactive oxygen species explains the oxidative stress that occurred during recombinant protein production, and ii) the final electron acceptor for protein folding and the electron transferring model at anaerobic condition proposed potential electron consuming pathway for protein folding in the ER. Our research provided a deep understanding of the processing of protein secretory pathway, potential targets for future engineering, as well as shed lights for basic cellular metabolisms.