Khobragade_2024_Methods.Mol.Biol_2844_197

The promoter is an essential component of an expression system since it regulates the transcriptional beginning of related genes. The optimal expression level can be achieved by employing a promoter engineering approach. Typically, creating a library of T7 promoters allows for titratable protein expression. In the process of making beta-amino acid (sitagliptin intermediate) from beta-keto ester, esterase from Pseudomonas stutzeri (Est PS) is used to convert the beta-keto ester to beta-keto acid. Subsequently, transaminase from Ilumatobacter coccineus (TAIC) transforms the beta-keto acid to its corresponding beta-amino acid. Here, we describe the optimization of the expression levels of Est PS for the maximum production of sitagliptin intermediate. The different promoter strengths for Est PS were built into the T7 promoters of the pET15b vector. With the help of these new co-expressing entire cells, the expressed enzyme ratio for each enzyme was determined. As the strength of the promoter of Est PS decreases, the expression level also decreases (from 100% to 10%). Conversely, the TAIC expression level is increased. This developed system produced a higher sitagliptin intermediate than enzymes' unoptimized expression level.