Metabolic Engineering and Bioprocess Optimization Strategies Promote l‑Azidohomoalanine Biosynthesis in Fed-Batch Bioreactor Culture

First, l-homoserine-<i>O</i>-acetyltransferase acetylates l-homoserine, then <i>O</i>-acetyl-l-homoserine sulfhydrylase catalyzes the conversion of the activated <i>O</i>-acetyl-l-homoserine to Aha by the nucleophilic attack of NaN₃. In contrast to earlier studies, we focused on optimizing the biosynthesis of Aha in fed-batch bioreactor cultures of E. coli independent of its incorporation into target proteins.

We established methods for the quantitative analysis of NaN₃, the <i>O</i>-acetyl-l-homoserine intermediate and Aha to monitor the biosynthesis reaction. Combining gene-, strain- and process-engineering, we achieved a maximum Aha production of 1.4 mM or 0.05 mmol per gram of cell dry mass. Our study advances the biosynthesis of Aha to the next level as it demonstrates that the concentration routinely used for the global replacement of methionine can be exceeded by approximately three times.

Nevertheless, further optimization is required to improve the conversion of the toxic precursor NaN₃ such that the cobiosynthesis of Aha and its direct incorporation into a protein of interest become viable for scalable applications.