Post­transcriptional transfer RNA modifications as modulators of heterologous protein production

Heterologous protein production strains the translation system, causing biotic stresses that impact negatively on protein quality and yield. Transla-tion is modulated by post-transcriptional transfer RNA (tRNA) modifications (PTMs) that alter tRNA isoacceptor recognition and codon-anticodon base pairing interactions, affecting the processivity and fidelity of translation. As PTMs are dynamic and readily respond to biotic stresses, the main hypothesizes is that careful editing of PTM levels will prime translation towards enhanced heterologous protein production. This reasoning is further supported by preliminary results that show significant host cell PTM adaptation following long-term expression of a heterologous protein. In this project, we will assess which PTM changes are drivers of translation and devise a PTM-based strategy for enhancing the yield and quality of heterologous protein production. To this end, differently tagged reporter constructs have been generated for monitoring translation in real-time using Saccharomyces cerevisiae as the model system. By means of quantitative mass spectrometry and ribosome profiling, PTM levels will be characterized and translational changes will be studied to obtain a consolidated dataset from which specific translational events are assigned to key PTMs. The effect of these translational drivers will be independently validated in a simplified cell-free translation system. Finally, the above results will be employed to generate codon-optimized reporter constructs that, in a long-term competition study, will compete against native and codon-impaired versions, to unravel the real-life advantage of PTM-based codon optimization. Consequently, this project will further our insight into translational regulation and provide a proof-of-concept for PTM-based stress mitigation and enhancement of heterologous protein production in cell factories