Illuminating subcellular consequences of membrane protein production in Lactococcus lactis

Bacterial cells are often used for the production of a wide variety of proteins. After purification, the proteins can either be used for commercial (medical) purposes or to further study their function and structure. Proteins that are situated in the membrane are important for many biomolecular mechanisms, which is why there is a great interest from the scientific community to obtain these so-called membrane proteins in high quantity and purity. Next to the well-studied model bacterium Escherichia coli, Lactococcus lactis has been acknowledged as a suitable host for the production of membrane proteins. However, in both cases, the production of especially eukaryotic membrane proteins often suffers from multiple obstructions. It is therefore of importance to identify the bottlenecks that preclude the successful production of these proteins. Such knowledge would enable designing and examining compatible strategies to optimize the protein production process. The search for bottlenecks has been approached from an entirely new angle in this thesis. Rather than analyzing the final yield of the overproduced proteins, the subcellular location of the transcripts and their cognate membrane proteins was examined in single L. lactis cells using various fluorescence microscopy techniques. By zooming in to this level, it was observed that crucial aberrations occur with respect to the transcription as well as the localization of transcripts encoding selected eukaryotic membrane proteins. These findings have laid the foundation for the additional work presented in this thesis, in which the causes and the effects of transcript mislocalization were further examined and used as a guide for the development of protein production optimization strategies.