High Cell Density Cultivation to Produce Heterologous Proteins by S. cerevisiae Strains: a Holistic Approach to Investigate and Optimize the System

Abstract

The research of this PhD thesis has been focused on the study and realization of high cell density cultivation (HCDC) systems with auxotrophic strains of the yeast Saccharomyces cerevisiae, these latter to be used as hosts for heterologous proteins (HEPs) production. Considering the complexity of the HCDC systems and that their performance depends on the strong interactions between biological and environmental determinants, the study of such systems was carried out by means an holistic approach or by considering the cultivation system as a whole and its behavior explicable on the basis of the intimate interconnection of its parts. In this light, the work has been aimed not only at improving the HCDC performance but also elucidating the physiology expressed by yeast when it proliferates in this type of system. The HCDC study has been carried out in an aerated fed-batch reactor which allows the proliferating biomass to be accumulated so as to achieve high cell density (HCDC). This is possible due to the extension of working time and the control of by-products, or catabolite repression effects through controlled conditions for the substrate supply. Initially, the experimental activity has been directed to find out the host for heterologous proteins expression. For this purpose a systematic investigation has been made considering six strains belonging to the CEN.PK family of the yeast S. cerevisiae, characterized by different types and number of auxotrophies. This preliminary investigation gave a clear result on the best candidate to be used as host for recombinant protein production. Indeed, the selected strain, CEN.PK113-5D, produced an amount of biomass which was, approximately, three-fold higher than the other strains tested, and similar to that obtained with S. cerevisiae industrial strains which did not carry any auxotrophy. Based on this result, the operative conditions of the bioprocess were optimized. Particularly the selected host has been tested in different operative conditions to find those values of specific growth rate ensured high volumetric productivity and biomass yield. Therefore, CEN.PK113-5D was transformed for the production of interleukin-1β, used as a model protein and subsequently, for two proteins of great interest for the agri-food field: Lipase A from Bacillus subtilis and an endoglucanase from Paenibacillus barcinonensis. Fed-batch tests were combined with physiological studies to asses Reactive Oxygen Species, catalase, cell viability and presence of by-product into the culture medium during fed-batch runs. The results of this work were implemented by expressing in CEN.PK 113-5D three human proteins: hemoglobin, myoglobin and neuroglobin. In this way, the effects of the production of a wide range of recombinant proteins of different origin, on the yeast growth, has been investigated. It is worth noticing here the special interest, from an applied point of view, of the positive effect obtained with the expression of human myoglobin and neuroglobin on the growth characteristics of the recombinant yeast strains and its possible relationship with a better oxygen transfer. This part of the work has been performed at the Chalmers University in Goteborg (Sweden). During the work, particular interest was reserved to the study concerning the limits encountered in the cultivation of microorganisms at high cell densities. Indeed, fermentation runs of the S. cerevisiae strains investigated in fed-batch, were characterized by a peculiar decay in the specific growth rate and metabolic fluctuation with a limit of cell density hardly to be overcome, that for CEN.PK 113-5D was about 100 g l-1. Further, it has been highlighted that growth decay phenomena were related to the accumulation in the medium of not yet identified, inhibitory compounds. The experimental approach of this thesis work has been implemented with the development of an innovative mathematical model built up on the System Dynamic principles. This model was capable to represent the growth dynamics of S. cerevisiae strains in batch and fed-batch reactors. The model has been developed through the explicit representation of the two main pathways of the glucose catabolism in yeast, respiration and fermentation. Pyruvate was identified as the key intermediate of sugar metabolism in yeast. Moreover, other components exerting negative feedback effect on cell proliferation were modeled. Model results highlighted that in order to obtain a good fitting between the simulation curves and the experimental data, it was essential to consider a negative feedback effector represented by the secretion of inhibitory compounds along the fermentation runs and as such capable to describe entirely the yeast growth in the fed-batch reactor. [edited by author]