Scale-down study of adaptation in Saccharomyces cerevisiae chemostat cultures for heterologous protein production

Naia Risager Wright*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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Microbes are today used for the production of a wide range of commercial products including pharmaceuticals, food ingredients and chemicals. The development of stable microbial production processes takes place in the laboratory, whereas the actual industrial production is done in large-scale reactors that range from hundreds to thousands of cubic meters in volume. This disparity between the conditions where the microbial processes are developed and the conditions where the industrial production takes place can result in unnecessarily expensive process development and in worst-case lead to wrong decisions in terms of investments for new products and bioprocesses. Scale-up is therefore a critical step in process development. In order to facilitate better scale-up of production processes, a deeper understanding of the complex interplay between the environment in the production reactors and the physiology of the microbial production organisms is necessary.

This thesis addresses the production of heterologous insulin in the yeast Saccharomyces cerevisiae cultured in chemostat mode. The overall aim of the study is to investigate the interplay between the physiology of the organism and the environment in industrial bioreactors. In particular, I focus on feast-famine conditions and how these conditions influence the physiology of the organism in comparison to a constant glucose-limited environment. I apply a unified approach, integrating experimental scale-down simulators, in depth omics analysis and sensing of population heterogeneity in order to address this question.

My results reveal that adaptation and phenotypic differentiation of the recombinant strain occur in the glucose-limited environment. Fluctuations in glucose availability and feast-famine conditions can on the other hand completely prevent this phenotypic transition. I therefore propose that the phenotypic adaptation and occurrence of subpopulations are a result of the selective pressure of the glucose-limited environment and the burden of the heterologous insulin production.

I present different hypotheses and tests carried out in order to investigate possible mechanisms behind the physiological adaptation including a test for genetic mutations, investigation of chronological aging hallmarks and application of genome-scale metabolic modelling. However, further studies are needed in order to clarify the exact mechanism.

With this study, I have improved our knowledge and understanding of the interplay between the environment in production reactors and the physiology of heterologous production hosts. This knowledge can be used for better design and scale-up of industrial production processes in the future.
Original languageEnglish
Place of PublicationKgs. Lyngby, Denmark
PublisherDTU Bioengineering
Number of pages205
Publication statusPublished - 2021


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