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KMID : 1094720230280020300
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Biotechnology and Bioprocess Engineering
2023 Volume.28 No. 2 p.300 ~ p.309
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Biocatalytic Cascade for Synthesis of Sitagliptin Intermediate Employing Coupled Transaminase
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Taresh P. Khobragade
Amol D. Pagar
Pritam Giri
Sharad Sarak
Jeon Hyun-Woo
Joo Sang-Woo
Mostafa M. Mostafa
Park Bu-Soo
Yun Hyung-Don
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Abstract
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Transaminases (TAs) are employed in synthesizing various enantiopure β-amino acids, key precursors for various pharmaceuticals. Sitagliptin, an oral hyperglycemic drug, is a well-known example. Herein, we developed the coupled enzyme cascade to synthesize the sitagliptin intermediate by fusing two different TAs to regenerate the amino donor. In a cascade system, ethyl 3-oxo-4-(2,4,5-trifluorophenyl) butanoate (1) was converted by esterase from Pseudomonas stutzeri (EstPS) to respective β-keto acid (2) which was subsequently converted by first TA to sitagliptin intermediate (3) using (S)-α-MBA as an amino donor and the acetophenone formed in the reaction was recycled by the second TA to (S)-α-MBA. A single whole-cell system was established by the co-expression of esterase and TA fusion protein. The whole-cell biotransformation reaction was performed with varying substrate concentrations from 50?200 mM. The excellent conversion of the product was achieved, ranging from 62-to 100% at the expense of only 25 mM (S)-α-MBA. Notably, our designed system with fusion protein can produce ∼5-fold higher product at the expense of 0.5 equivalent (S)-α-MBA. Finally, a preparative scale reaction was performed with 98% conversion.
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KEYWORD
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transaminase, esterase, sitagliptin, fusion protein, two cell system, single cell system, promoter engineering
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