Abstract
Unnatural amino acids (unAAs) possess unique properties owing to their distinct functional groups, and their insertion into proteins can significantly alter protein function and properties. Currently, the predominant method for inserting unAAs into proteins is through genetic code expansion (GCE), which mimics the natural translation process within cells and necessitates the exogenous supplementation of unAAs. However, in many instances, microbial cells do not recognize unAAs as essential nutrients and lack specific transporters for their uptake across the cell membrane, thereby greatly reducing their insertion efficiency. To address this issue, our study developed an in situinsertion method for enhancing the efficiency of unAAs insertion into proteins and further explored the feasibility of simultaneously inserting two different unAAs into one protein. Firstly, the orthogonal translation system for hydroxy-rich unAAs 5-hydroxytryptophan (5-HTP) or 4-hydroxyisoleucine (4-HiL) were constructed and then transformed into microbial cells to achieve the insertion of 5-HTP or 4-HiL into sfGFP by feeding 5-HTP or 4-HiL. Subsequently, the biosynthetic pathways of 5-HTP or 4-HiL were constructed in E. coli which contained the corresponding orthogonal translation system, resulting in the in situ insertion of 5-HTP or 4-HiL into sfGFP.Further, we developed a co-insertion method based on codons UGA and UAG. Introduction of the biosynthetic pathways and the orthogonal translation systems of 5-HTP and 4-HiL in the same cells achieved the in situ co-insertion of 5-HTP and 4-HiL in one sfGFP. This work provided a representative example for in situinsertion of unAAs into protein to increase the insertion efficiency, and explored the possibility of co-inserting two types of unAAs into one protein.