The nitroaromatic non-standard amino acid (nsAA) - para-nitro L-phenylalanine (pNPhe) - is known to be immunogenic, with a demonstrated ability to elicit strong and targeted immune responses towards proteins that contain it. This feature could be useful for the design of next-generation vaccine candidates. Biosynthesis of pNPhe by live bacterial cells offers a versatile platform for manufacturing multiple distinct end products: pNPhe as a chemical building block, antigens incorporating pNPhe as candidate subunit vaccines, or engineered live bacteria capable of producing nitrated antigens as candidate live vaccines. However, to realize these options, we must improve the performance of the de novo biosynthetic pathway that we previously engineered, particularly the selectivity and end-point titer of pNPhe.
We modularized the pathway into three components: 1) central carbon metabolism feeding a heterologous operon to produce the amine precursor, 2) an amine-oxidizing N-oxygenase, and 3) an orthogonal amino-acyl synthetase and tRNA pair for site-specific incorporation. Prioritizing the bottleneck amine-oxidation step, we identified strategies that improved the activity of diiron-containing N-oxygenases and increased pNPhe titers by over 5-fold in a chorismate overproducer strain. To investigate incorporation efficiency, we dosed pNPhe into diverse E. coli strains and found varying incorporation efficiency and reporter translation fluorescent intensity, suggesting the need to explore for strain suitable for both biosynthesis and incorporation. Next, we were encouraged to observe a doubling in incorporated protein production for our improved biosynthesis and incorporation strain when compared to supplemented pNPhe. Our next step is to demonstrate autonomous synthesis and incorporation of pNPhe into bacterial antigens. Overall, our work advances an autonomous platform for biosynthesizing an immunogenic non-standard amino acid and incorporating it into antigens, with potential applications in bacterial vaccine development.
