Ribosome profiling reveals ribosome pausing in Bacillus subtilis

The optimization of protein production focusses on increasing the mRNA levels of the protein of interest, removal of proteases and the induction of protein folding chaperones. However, almost no research has been done on improving translation of the protein of interest, other than simple codon optimization. As far as we know, no ribosome profiling study has been performed focused at the production of industrial relevant enzymes. In Chapter 2, we describe the setting up and testing of the Ribo-seq procedure for B. subtilis, and the investigation of ribosome pausing during the secretion of α-amylase under repeated batch fermentation conditions. The stringent response, which reduces protein translation, is activated in the stationary phase of growth when B. subtilis is most active in the production of secreted enzymes. In Chapter 3, we have investigated how blocking the stringent response affects amylase production and various ribosome profiles. In Chapter 4, we tried to determine whether the ribosome pausing sites found in the amyM mRNA are caused by amino acid sequence motifs or by the nucleotide sequence of the mRNA. In the final experimental chapter, Chapter 5, we have benchmarked several different translation fixation conditions to further optimize Ribo-seq.