Simultaneous synthesis of enantiomerically pure (S)-amino acids and (R)-amines using coupled transaminase reactions.
For the simultaneous synthesis of enatiomerically pure (S)-amino acids and (R)-amines from corresponding alpha-keto acids and racemic amines, an alpha/omega-transaminase coupled reaction system was designed using favorable reaction equilibrium shift led by omega-transaminase reaction. Cloned tyrB, aspC and avtA, and omegataA were co-expressed in E. coli BL21(DE3) using pET23b(+) and pET24ma, respectively. The coupled reaction produced the (S)-amino acids with 73-90% (> 99% ee(S)) of conversion yield and resolved the racemic amines with 83-99% ee(R) for 5 to 10 hours. In designing the coupled reactions in the cell, alanine and pyruvate were efficiently used in the cell as an amine donor for the alanine transaminase and an amino acceptor for the omega-transaminase, respectively, resulting in an alanine-pyruvate shuttling system. The common problem of the low equilibrium constant of the alpha-transaminase can be efficiently overcome by the coupling with the omega-transaminase. However, overcoming the product inhibition of omega-transaminase by the ketone by-product and increasing the decarboxylation rate of the oxaloacetate produced during the transaminase reaction become barriers to further improving the overall reaction rate and the yield of the coupled reactions.
Cho
Byung-Kwan
BK
Interdisciplinary Program for Biochemical Engineering and Biotechnology, School of Chemical Engineering, and Institute of Molecular Biology and Genetics, Seoul National University, Seoul Korea.
Cho
Ho Jung
HJ
Park
Sung-Hee
SH
Yun
Hyungdon
H
Kim
Byung-Gee
BG
D000588:Amines / Q000737:chemistry / Q000378:metabolism*; D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005240:Feasibility Studies; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic; D013045:Species Specificity; D013237:Stereoisomerism; D000637:Transaminases / Q000737:chemistry / Q000378:metabolism*
D000588:Amines; D000596:Amino Acids; D009097:Multienzyme Complexes; D000637:Transaminases