Systems Biology of Recombinant Protein Production by Fungi

Doctoral thesis, 2014

Systems biology has emerged as a highly potent tool for studying biological processes over the last decades. However, its application to complex metabolic processes such as protein secretion is still at the infant stage. Saccharomyces cerevisiae and Aspergillus oryzae are two important fungal cell factories which occupy significant proportions of recombinant protein productions, whereas various bottlenecks and undiscovered mechanisms limit their full potential as robust hosts. In this thesis, systems biology approaches were applied to explore these two organisms in respect of protein production. By utilizing and engineering the yeast endogenous heme synthesis, we demonstrated the possibility for efficient production of complex proteins (e.g. multimer with a prosthetic group) by yeast. Applying inverse metabolic engineering, we identified many genomic variants that may contribute to improve protein secretion in yeast. Specifically, we examined the effect of a single point mutation on VTA1 encoding a regulatory protein in the MVB pathway in endocytosis. Our result suggests that the VTA1S196I mutation might help to accelerate nutrient uptake via endocytosis, which subsequently enhanced protein synthesis and secretion. Oxygen is an important element associated with normal cellular metabolism as well as protein production. We studied how Rox1p, a heme-dependent transcription repressor of many hypoxia-induced genes, affect protein production in yeast, under aerobic conditions. By knocking out ROX1, we observed a 100% increase in the α-amylase production. Through genome wide transcriptome analysis we identified several Rox1p targets and based on this suggested their roles in improving protein productions. Lastly, applying comparative genomics study, we enriched the list of core protein components involved in the secretory machinery of A. oryzae. To verify the list, several high α-amylase producing strains were constructed. The transcriptional responses of these strains to α-amylase production were studied using microarray, through which several strategies including overexpressing the up-regulated cell wall proteins EglD and Cwp1 and knocking out the genes encoding extracellular proteins competing for the secretory pathway, were proposed.