In situ insertion of one or two types of hydroxy-rich noncanonical amino acids into one protein

Noncanonical amino acids (ncAAs) possess unique properties owing to their distinct functional groups. The predominant method for inserting ncAAs into proteins is genetic code expansion, which mimics the natural translation process and requires exogenous ncAAs supplementation. However, microbial cells often lack specific transporters for ncAAs uptake across the cell membrane and do not recognise them as essential nutrients, leading to limited insertion efficiency. To address these challenges, we developed an in situ insertion method to enhance ncAAs insertion efficiency and explored the feasibility of co-inserting two types of ncAAs into a single protein. Initially, orthogonal translation systems specific to the hydroxy-rich ncAAs 5-hydroxytryptophan (5-HTP) and 4-hydroxyisoleucine (4-HiL) were constructed and introduced into microbial cells. Subsequently, the biosynthetic pathways for 5-HTP and 4-HiL were engineered in Escherichia coli, which harboured the corresponding orthogonal translation systems. Furthermore, a co-insertion strategy was developed based on the dual use of UGA and UAG codons. In situ co-insertion of 5-HTP and 4-HiL into one protein was achieved by introducing biosynthetic pathways and orthogonal translation systems for 5-HTP and 4-HiL into the same microbial cell. These findings establish a robust framework for the precise and scalable insertion of ncAAs in advanced protein design.