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

Abstract The noncanonical amino acid l-azidohomoalanine (Aha) can replace methionine in ribosomal translation under tightly controlled conditions. Aha has been commonly employed to functionalize proteins by the bioorthogonal conjugation with alkynes, the so-called “click chemistry”. However, its application for scalable recombinant protein production is currently hampered by the high price. To address this shortcoming, a biosynthesis cascade from inexpensive sodium azide (NaN3) as the precursor had been reported in the literature. Here, we advance the approach to a scalable bioprocess for the biosynthesis of Aha in Escherichia coli. We biosynthesized Aha via a two-enzyme cascade reaction using the cellular metabolite l-homoserine and NaN3 as the precursors. First, l-homoserine-O-acetyltransferase acetylates l-homoserine, then O-acetyl-l-homoserine sulfhydrylase catalyzes the conversion of the activated O-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 O-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.