Engineering an E. coli Biosynthetic Platform for Synthesis of Light-Sensitive Non-Standard Amino Acids

• Alice Rousseau, Chemical Engineering, University of Delaware

• Aditya Kunjapur, Chemical & Biomolecular Engineering, University of Delaware

Bacterial metabolic processes can be harnessed to produce valuable chemical compounds, including non-standard amino acids (nsAAs) that extend beyond the standard twenty amino acids found in nature. Many photo-responsive or photo-caged nsAAs possess an ortho-nitro functional group that enables light-induced protein cleavage following site-specific incorporation, facilitating targeted manipulation of protein structure and function, enabling the development of light-responsive engineered living materials with applications in programmable biomaterials and protein engineering. However, current access to these chemistries is limited to multistep, low-yield chemical synthesis. The development of biosynthetic platforms in E. coli could provide a scalable and sustainable alternative.

This project seeks to screen candidate N-oxygenases (NOs) capable of oxidizing the amino group of various amino-benzoic acids. We performed assays of the stability of the target products after exogenous supplementation across multiple engineered candidate strains to identify an optimal chassis. High-performance liquid chromatography (HPLC) analysis revealed background degradation, identifying a host strain with improved pathway compound stability. Next, we employed bioprospecting to mine genomic databases for diverse N-oxygenase homologs, resulting in the construction of a library of NOs. The resulting NO library was characterized by sequence diversity and enzyme expression. Candidate NOs were then functionally evaluated through in vivo conversion assays following co-expression with a helper plasmid to support oxidation in our optimized host background. Assays were conducted under optimized growth and induction conditions, with product formation monitored by HPLC and enzyme activity characterized by product accumulation. Together, these studies established a foundational framework integrating chassis optimization and N-oxygenase screening for future biosynthetic production of light-sensitive non-standard amino acids.