Promoter engineering-mediated Tuning of esterase and transaminase expression for the chemoenzymatic synthesis of sitagliptin phosphate at the kilogram-scale

Here, we report a bienzymatic cascade to produce beta-amino acids as an intermediate for the synthesis of the leading oral antidiabetic drug, sitagliptin. A whole-cell biotransformation using recombinant Escherichia coli coexpressing a esterase and transaminase were developed, wherein the desired expression level of each enzyme was achieved by promotor engineering. The small-scale reactions (30 ml) performed under optimized conditions at varying amounts of substrate (100-300 mM) resulted in excellent conversions of 82%-95% for the desired product. Finally, a kilogram-scale enzymatic reaction (250 mM substrate, 220 L) was carried out to produce beta-amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from beta-amino acids with 82% yield and > 99% purity.

Automated summary

A bienzymatic cascade was developed for the synthesis of beta-amino acids as an intermediate for sitagliptin production. Recombinant Escherichia coli were used for whole-cell biotransformation to achieve high conversion rates. The desired product was obtained with excellent conversions in small-scale reactions, and sitagliptin phosphate was synthesized with high yield and purity.