Proteins are often produced using microbial cell factories for academic or industrial purposes. Protein production is however not an open-and-shut procedure. Production yields often vary in an unpredictable and context dependent manner, limiting the rational design of a straightforward production experiment.

This thesis gives an overview of how proteins are biosynthesised in bacterial cells and how this knowledge is used to produce proteins recombinantly in a host organism such as Escherichia coli. In the present investigation, we reason that unpredictable and poor protein production yields could result from incompatibility between the vector derived 5’ UTR and the 5’ end of the cloned CDS which leads to an unevolved translation initiation region (TIR). Data presented in this thesis show that an unevolved TIR could work more efficiently and yield more produced protein if subjected to synthetic evolution. Clones with an engineered synthetically evolved TIR showed enhanced protein production in both small- and large-scale production setups. This engineering method could lower production expenses, which in turn would result in increased functional determination of proteins and expanded availability of protein-based medicine to people globally.