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Promoter engineering‐mediated Tuning of esterase and transaminase expression for the chemoenzymatic synthesis of sitagliptin phosphate at the kilogram‐scale
Author(s) -
Khobragade Taresh P.,
Yu Seongseon,
Jung Hyunsang,
Patil Mahesh D.,
Sarak Sharad,
Pagar Amol D.,
Jeon Hyunwoo,
Lee Somin,
Giri Pritam,
Kim GeonHee,
Cho SungSu,
Park SeongHwa,
Park HyeJung,
Kang Heung M.,
Lee Sung R.,
Lee Myeong S.,
Kim Jeong H.,
Choi In S.,
Yun Hyungdon
Publication year - 2021
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27819
Subject(s) - sitagliptin , transaminase , substrate (aquarium) , chemistry , yield (engineering) , amino acid , enzyme , biotransformation , esterase , escherichia coli , biocatalysis , biochemistry , phosphate , nuclear chemistry , catalysis , biology , materials science , microbiology and biotechnology , ecology , metformin , gene , insulin , metallurgy , ionic liquid
Here, we report a bienzymatic cascade to produce β‐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 β‐amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from β‐amino acids with 82% yield and > 99% purity.