1.Abstract Over the last decades, the biopharmaceutical market has become a significant segment of the global pharmaceutical market, with antibodies and antibody fragments being the leading building blocks. However, also proteins and enzymes are conquering the biopharmaceutical market now. Mainly due to the possibility of resembling human-like glycosylation, mammalian cells are still the most widely used expression system for the production of biopharmaceuticals. However, implementation of less complex microorganisms, such as yeasts or bacteria, would be highly desirable to simplify the upstream processes and improve productivity. The methylotrophic yeast P. pastoris is commonly used for the production of industrial and biopharmaceutical enzymes. This yeast offers many advantages such as biomass growth up to very high cell density, easiness of genetic manipulation, availability of strong and tightly regulated promoters and the possibility of producing up to gram per litre of recombinant protein, both intracellularly and extracellularly. Nevertheless, its implementation for biopharmaceuticals production is not well established yet, mainly due to the need of circumventing methanol implementation as induction substrate and due to the distance from the native human-like post-translational modifications, above all glycosylation. Methanol is a high-degree reductant with high heat of combustion and high oxygen consumption. Therefore its replacement with a different substrate is highly desirable above all for large scale fermentations. Yeast hyperglycosylation leads to the production of non-human like glycosylated proteins thus limiting their implementation as biopharmaceuticals. Additionally, the presence of non-uniform glycoforms hampers downstream processing and conjugation approaches. Therefore the goal of this Thesis consists in solving the above mentioned issues with the aim of rendering P. pastoris a more suitable expression host for production of biopharmaceuticals. In compliance with the goal of this Thesis, the following methodology based on the application of three different engineering methods namely, process, strain and product engineering was applied. In the first chapter of this Thesis, -The biopharmaceutical market-, an overview is provided about this branch of the pharmaceutical market and the recent advances with the three main microbials, namely Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli for full length antibodies and antibody fragments production. In the second chapter of this Thesis, -Avoiding methanol requirement- I describe a process engineering approach based on the design of a mixed-feed strategy for optimizing the recombinant expression of phospholipase C enzyme (PLC) in a P. pastoris strain harbouring an AOX1 de-repression promoter variant. Using this strain methanol as inducer can be omitted. In the third chapter -Reducing hyperglycosylation- I describe two different engineering approaches, namely strain and product glycoengineering, which can be applied to reduce yeast hyperglycosylation. These methods were tested for the expression of a more uniformly and less glycosylated horseradish peroxidase (HRP) isovariants. Since HRP is a heme containing enzyme, I describe and compare two different approaches based on metabolic engineering and on cofactor media supplementation for enhancing heme availability, as described in the third chapter of this Thesis -Increasing product specific cofactor availability-. Over-expression of heme pathway genes versus hemin supplementation in growth media was tested for improving final yields of active HRP. In conclusion, in compliance with the described methodology, I was able to identify the most suitable engineering approach to be applied for solving each of the above mentioned pitfalls. In particular I found out that: - process engineering in combination with implementation of a de-repressed strain allows to circumvent methanol implementation; - product glycoengineering proves to be a valid strategy for reducing enzyme hyperglycosylation; - cofactor supplementation in media proves to be a valid approach to increase product specific cofactor availability. Therefore the presented Thesis represents a basis for the combination of these engineering approaches and may work as fundamental knowledge for successful expression of recombinant biopharmaceuticals in P. pastoris.