2026-10-12T14:40:07Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34021109 2026-09-29T04:13:27Z category_4 category_146 category_13 category_14 category_19 category_21 category_873 category_734 category_69 category_106 category_133 portal_63 item_type_6 month_year_09_2026

Metabolic Engineering and Bioprocess Optimization Strategies Promote l‑Azidohomoalanine Biosynthesis in Fed-Batch Bioreactor Culture Claudia Lacombe (25135106) Gerald Striedner (443201) Birgit Wiltschi (172810) Biochemistry Space Science Genetics Molecular Biology Pharmacology Biotechnology Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Inorganic Chemistry Science Policy Plant Biology tightly controlled conditions interest become viable inexpensive sodium azide concentration routinely used cell dry mass batch bioreactor cultures batch bioreactor culture approximately three times homoserine sulfhydrylase catalyzes maximum aha production cellular metabolite l biosynthesized aha via toxic precursor nan 3 </ sub target proteins study advances scalable bioprocess scalable applications ribosomal translation quantitative analysis nucleophilic attack next level high price global replacement functionalize proteins established methods escherichia coli earlier studies currently hampered commonly employed coli independent bioorthogonal conjugation >- acetyl 4 mm 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-<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. 2026-09-29T00:00:00Z Text Journal contribution 10.1021/acsomega.6c05952.s001 https://figshare.com/articles/journal_contribution/Metabolic_Engineering_and_Bioprocess_Optimization_Strategies_Promote_l_Azidohomoalanine_Biosynthesis_in_Fed-Batch_Bioreactor_Culture/34021109 CC BY-NC 4.0