Non-Protein Amino Acid Production Using Recombinant E. colis

摘要

In the microbial and plant cells, L-Cys is synthesized from L-Ser by the two-step enzymatic pathway, in which serine acetyltransferase (SAT) catalyzes the formation of O-aeetyl-L-Ser (OAS) and CoA from L-Ser and aeetyl-CoA, while O-acetylserine sulfhydrylase-A (OASS) converts OAS with sulfide (SH~-) to L-Cys and acetic acid (1). Instead of sulfide, various nucleophiies such as derivatives of heterocyclic compounds such as pyrazole could be utilized as the substrate to produce non-protein amino acids (2-4). However, to synthesize effectively non-protein amino acids such as β-(pylazol-1-yl)- L-alanine (β-PA), regeneration of CoA to acetyl-CoA is necessary. Previously, we synthesized enzymatically β-PA using SATΔC20 (5), OASS-A, and phosphotransacetylase (PTA) with acetyl phosphate (AcP) as the substrate to regenerate acetyl-CoA. SATΔC20 is a mutant SAT deleting 20 amino acids from the C-terminus, which looses ability to form a complex with OASS and thus the OASS activity is not repressed in the presence of SAT (6-9). We optimized the reaction conditions on the basis of kinetic studies to synthesize β-PA from 200 mM L-Ser, 200 mM pyrazole, and 200 mM AcP with a yield higher than 70%. In this study, we intended to optimize β-PA production from L-Ser and pyrazole using recombinant Escherichia colis transformed with different plasmids.