UC San Diego

E. coli has been highly favored as a model organism and platform production strain because of its high rate of growth under simple culture conditions and its genetic tractability allowing introduction of foreign genes to expand its native metabolic capabilities. However, we lack understanding of how sequence features affect effective protein expression in E. coli, as well as the burden on the host cell during heterologous protein expression. In this dissertation we make use of next-generation sequencing to peer into the cell at the genomic, transcription, and translation level. We integrate the data across multiple scales in order to better understand protein expression in E. coli under normal growth conditions, whilst expressing heterologous proteins, and after adaptation to oxidative stress. Firstly, we examine the translation dynamics of native proteins in the cell under normal growth conditions to reveal the causes and functions of programmed translational pauses along the transcript. Secondly, we investigate the transcriptome of E. coli whilst expressing a large library of heterologous proteins to identify 4 major host cell responses which vary widely across these proteins, Fear vs Greed, Metal Homeostasis and Respiration, Protein Folding, and Amino Acid and Nucleotide Biosynthesis. Lastly, we use Adaptive Laboratory Evolution to increase tolerance to oxidative stress, commonly found to be generated during high levels of protein expression. We make use of genomic resequencing, transcriptomics, and ribosome profiling to achieve a systems level understanding of the adaptations which occur in response to oxidative stress. As a whole, this work improves our understanding of E. coli as a platform protein production strain through A) identifying fundamental constraints on translation rates in native proteins, B) classifying host cell responses during expression of a variety of heterologous proteins to identify target areas for further research, and C) elucidating tolerance adaptations and mechanisms to oxidative stress, a common endogenous and exogenous stress during industrial biotechnology.