An Improved Strain for Aromatic Aldehyde Stability and Incorporation: Recoded RARE

Aromatic aldehydes find widespread use as end products in fragrances and flavorings as well as intermediates in the synthesis of other valuable compounds. When incorporated into proteins via non-standard amino acids (nsAAs), aromatic aldehydes can also act as clickable handles for conjugation to hydrazine or hydrazide molecules. However, chemical synthesis of these compounds is complex and utilizes nonrenewable petroleum feedstocks. Although biosynthesis of aromatic aldehydes could help reduce reliance on petroleum, these pathways are limited by the instability of aromatic aldehydes within microbes due to endogenous oxidoreductase enzymes. In addition, nsAA incorporation in wild-type E. coli is hindered by competition with native translation termination systems. The reduced aromatic aldehyde reduction (RARE) strain ameliorates stability issues through translational knockouts of selected aldo-keto reductases and alcohol dehydrogenases in E. coli K-12 MG1655. Another engineered chassis, the C321 strain, has all amber UAG stop codons replaced with ochre stop codons UAA. This leaves the UAG codon free for translation by orthogonal aminoacyl-tRNA synthetases (AARSs)/tRNAs. The recoded RARE strain (RR3) combines the features of the RARE and C321 strains for improved incorporation of non-standard amino acids with aldehyde functionalities, along with targeted mutations to improve growth compared to C321. In this work, we sought to evaluate the recoded RARE strain’s stabilization of aromatic aldehydes and incorporation of selected nsAAs. RR3 and C321 cultures were supplemented with 1 mM para- and meta-formylphenylalanine (formylPhe) and monitored over 24 hours to compare the stability of nsAAs in each strain. Subsequent analysis via high performance liquid chromatography revealed up to 5-fold improvements in the retention of formylPhe after 24 hours in RR3 compared to C321. Incorporation of a well-studied nsAA, pAzF, into a fluorescent reporter protein, GFP, was monitored via fluorescence to compare the incorporation efficiency of three strains: RR3, C321, and BL21, a widely used microbial host for protein overexpression. When compared to BL21, the RR3 strain showed an up to 20-fold increase in fluorescence and up to 8-fold increase compared to C321, indicating an improved incorporation efficiency. We further tested a library of AARSs within RR3 and C321 for activity on para- and meta-formylPhe. After identifying a variant with activity on para-formylPhe, we were able to demonstrate improved incorporation of the nsAA into recoded GFP within the RR3 chassis compared to C321. The recoded-RARE strain presents an improved bacterial strain for incorporation of aromatic aldehyde non-standard amino acids and derivatives, ideal for genetic code expansion applications.