Enantioselective bioconversion using Escherichia coli cells expressing Saccharomyces cerevisiae reductase and Bacillus subtilis glucose dehydrogenase.
Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni- NTA and desalting column chromatography. It evidenced an optimum temperature of 45 degrees C and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.
Park
Hyun Joo
HJ
Division of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Korea.
Jung
Jihye
J
Choi
Hyejeong
H
Uhm
Ki-Nam
KN
Kim
Hyung Kwoun
HK
D000090:Acetoacetates / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D001709:Biotechnology / Q000379:methods*; D002087:Butyrates / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D042843:Glucose 1-Dehydrogenase / Q000235:genetics*; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
D000090:Acetoacetates; D002087:Butyrates; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C121244:ethyl 4-chloro-3-hydroxybutanoate; D009249:NADP; C111982:ethyl 4-chloro-3-oxobutanoate; D010088:Oxidoreductases; C432953:GRE2 protein, S cerevisiae; D042843:Glucose 1-Dehydrogenase