PUB - Publikationen an der Universität Bielefeld

In the era of sustainability, synthetic biology has emerged as a game changing field, holding immense potential to create innovative solutions to restore the ecological balance. The ability to produce peptides and proteins that can aid in achieving sustainability goals greatly depends on the development of effective protein design methods. However, conventional protein design approaches are limited by the 20 standard amino acids. This limitation calls for the exploration of alternative strategies for protein design that can expand the range of amino acids and allow the creation of novel proteins with enhanced functionalities. This challenge can be addressed by incorporating xenobiology in protein design strategies. Xenobiology seeks to expand the genetic code beyond the four naturally occurring nucleotides (A, C, T and G) and the 20 standard amino acids to create new synthetic DNA and protein blocks. To incorporate non-standard amino acids (nsAA) during translation, an orthogonal aminoacyl/tRNA synthetase (aaRS) needs to be introduced to endow the cell with the ability to charge a tRNA that is able to suppress a repurposed codon with the nsAA of choice. To make this system work a perfect aaRS and a codon that can be repurposed without influencing endogenous translation, apart from the incorporation of the nsAA, are needed. Current evolution methods for aaRS are limited in library design and alternative continuous evolution systems are not straightforward and can be hard to evaluate. To address this first bottleneck, we established and validated a Nanopore sequencing guided phage-assisted evolution strategy. The evolution of a L-2-nitrophenylalanyl aaRS was used to demonstrate the power of this new evolution method. By designing a new software solution to filter sequencing errors, it was possible to deep sequence the complete CDS throughout the whole evolution. This resulted in the evaluation of the entire evolution by constructing evolutionary trees with data indicating the strength of every mutation enriched throughout the evolution. By performing the evolution in triplicates, it was shown that independent experiments produced proteins that show the same amino acid exchanges under the same selection pressure. This uncovered which mutation results in local and global maxima of the aaRS activity and showed that mutations outside the ligand binding pocket have a strong influence on enzyme activity and specificity. Next to the aaRS, the choice of the repurposed codon has a strong influence on protein production with the nsAA. While in the evolution the most common used codon, the amber stop codon, was used to incorporate the non-standard amino acid, genetically recoded organisms are an alternative to create codons that do not interfere with the translational machinery. Genetically recoded organism are strains with one or several codons exchanged to synonymous codons throughout all protein CDSs. In Syn61 Δ3, the amber stop codons and two of the serine codons are exchanged. In addition to production of non-standard amino acid containing proteins, strains also offer the ability to resist viral replication. When a phage enters Syn61 Δ3 cells, the 3 recoded codons cannot be read by the cell anymore, and the phage proteins are not produced, impairing viral replication. However, several phages bring their own tRNAs being able to replicate in the recoded strains. To block this replication, leucine-serine swapped tRNAs were developed in this work. Through library and selection, we identified viral leucine tRNAs that tolerate an anticodon swap to the forbidden serine codons. These tRNAs can out-compete the viral tRNAs and lead to leucine incorporation at the serine codons, which leads to misfolding of the phage proteins and blocking phage replication. This swap can also be used to contain genetic information, making this system combined with genetically recoded organism a powerful tool for xenobiology. Both publications demonstrate the high pace at which this research discipline develops. However, to make this field grow in a responsible way, biosafety and biosecurity regulations need to grow at the same pace. Since biosecurity governance is mainly limited to pathogen-related research, the assessment and mitigation on the xenobiology field needs to be done by the researchers. This work shows that there is a dramatic lack of knowledge regarding dual-use risks and little effective efforts to change this situation. To make xenobiology research live up to its potential, responsibility, safety and security need to become a crucial aspect in project planning and execution, starting by educating young scientists about these topics over establishing suitable frameworks in governance.