Promotion of the Asymmetric Reduction of Prochiral Ketone with Recombinant E. coli Through Strengthening Intracellular NADPH Supply by Modifying EMP and Introducing NAD Kinase

2021 ◽  
Author(s):  
Hui-Jun Du ◽  
Wei Luo ◽  
Bright Appiah ◽  
Zhi-Cheng Zou ◽  
Zhong-Hua Yang ◽  
...  
Keyword(s):  
Asymmetric Reduction ◽  
Nad Kinase ◽  
E Coli ◽  
Prochiral Ketone

Promotion of the microalgal photo-biocatalytic asymmetric reduction of prochiral ketone by NADPH metabolic regulation

2016 ◽  
Vol 11 (4) ◽  
pp. 533-538 ◽  
Author(s):  
Wei Luo ◽  
Xin-Xing Deng ◽  
Zhi-Wei Gong ◽  
Zhong-Hua Yang
Keyword(s):  
Metabolic Regulation ◽  
Asymmetric Reduction ◽  
Prochiral Ketone

scholarly journals Characterization of a Carbonyl Reductase from Rhodococcus erythropolis WZ010 and Its Variant Y54F for Asymmetric Synthesis of (S)-N-Boc-3-Hydroxypiperidine

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3117 ◽  
Author(s):  
Xiangxian Ying ◽  
Jie Zhang ◽  
Can Wang ◽  
Meijuan Huang ◽  
Yuting Ji ◽  
...  
Keyword(s):  
Asymmetric Reduction ◽  
Rhodococcus Erythropolis ◽  
Catalytic Efficiency ◽  
Carbonyl Reductase ◽  
Specific Enzyme ◽  
Practical Applications ◽  
Whole Cells ◽  
E Coli ◽  
Irreversible Reduction

The recombinant carbonyl reductase from Rhodococcus erythropolis WZ010 (ReCR) demonstrated strict (S)-stereoselectivity and catalyzed the irreversible reduction of N-Boc-3-piperidone (NBPO) to (S)-N-Boc-3-hydroxypiperidine [(S)-NBHP], a key chiral intermediate in the synthesis of ibrutinib. The NAD(H)-specific enzyme was active within broad ranges of pH and temperature and had remarkable activity in the presence of higher concentration of organic solvents. The amino acid residue at position 54 was critical for the activity and the substitution of Tyr54 to Phe significantly enhanced the catalytic efficiency of ReCR. The kcat/Km values of ReCR Y54F for NBPO, (R/S)-2-octanol, and 2-propanol were 49.17 s−1 mM−1, 56.56 s−1 mM−1, and 20.69 s−1 mM−1, respectively. In addition, the (S)-NBHP yield was as high as 95.92% when whole cells of E. coli overexpressing ReCR variant Y54F catalyzed the asymmetric reduction of 1.5 M NBPO for 12 h in the aqueous/(R/S)-2-octanol biphasic system, demonstrating the great potential of ReCR variant Y54F for practical applications.

Asymmetric Synthesis of Ethyl (R)-4-Chloro-3-Hydroxybutanoate with a Two Recombinant Escherichia coli System

2013 ◽  
Vol 690-693 ◽  
pp. 1188-1192
Author(s):  
Ke Ju Jing ◽  
Wen Ting Ban
Keyword(s):  
Escherichia Coli ◽  
Butyl Acetate ◽  
Asymmetric Reduction ◽  
Glucose Dehydrogenase ◽  
Reaction System ◽  
Phase Reaction ◽  
Aldehyde Reductase ◽  
Two Phase ◽  
E Coli ◽  
Final Yield

The asymmetric reduction of ethyl 4-chloro-acetoacetate (CAAE) to ethyl (R)-4-chloro-3- hydroxybutanoate (R-CHBE) biocatalysed by the aldehyde reductase of Sporobolomyces salmonicolor expressed in E. coli M15 (pQE30-alr) in combination with regeneration of NADPH by the glucose dehydrogenase of Bacillus megaterium expressed in E. coli M15 (pQE30-gdh) was reported. The bioreduction was carried out in a two-phase reaction system with n-butyl acetate as an organic solvent. Bioconversion of 300 mmol CAAE with a final yield of 97.5 % and an enantiometric excess of 99 % was achieved without the addition of cofactor NADPH.

scholarly journals Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

2003 ◽  
Vol 69 (2) ◽  
pp. 933-937 ◽  
Author(s):  
Masaru Wada ◽  
Ayumi Yoshizumi ◽  
Yumiko Noda ◽  
Michihiko Kataoka ◽  
Sakayu Shimizu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Double Bond ◽  
Enzymatic Synthesis ◽  
Asymmetric Reduction ◽  
Glucose Dehydrogenase ◽  
Enantiomeric Excess ◽  
Old Yellow Enzyme ◽  
Chiral Compound ◽  
E Coli

ABSTRACT A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD+, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.

scholarly journals First Archaeal Inorganic Polyphosphate/ATP-Dependent NAD Kinase, from Hyperthermophilic Archaeon Pyrococcus horikoshii: Cloning, Expression, and Characterization

2005 ◽  
Vol 71 (8) ◽  
pp. 4352-4358 ◽  
Author(s):  
Haruhiko Sakuraba ◽  
Ryushi Kawakami ◽  
Toshihisa Ohshima
Keyword(s):  
Average Length ◽  
Gel Filtration ◽  
Nad Kinase ◽  
Inorganic Polyphosphate ◽  
Genome Database ◽  
Filtration Method ◽  
Pyrococcus Horikoshii ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Chromatography Step

ABSTRACT The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90°C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95°C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.

scholarly journals Asymmetric reduction of ketones with recombinant E. coli whole cells in neat substrates

2011 ◽  
Vol 47 (44) ◽  
pp. 12230 ◽  
Author(s):  
Andre Jakoblinnert ◽  
Radoslav Mladenov ◽  
Albert Paul ◽  
Fabrizio Sibilla ◽  
Ulrich Schwaneberg ◽  
...  
Keyword(s):  
Asymmetric Reduction ◽  
Whole Cells ◽  
E Coli ◽  
Reduction Of Ketones

scholarly journals NADP(H) Phosphatase Activities of Archaeal Inositol Monophosphatase and Eubacterial 3′-Phosphoadenosine 5′-Phosphate Phosphatase

2007 ◽  
Vol 73 (17) ◽  
pp. 5447-5452 ◽  
Author(s):  
Chikako Fukuda ◽  
Shigeyuki Kawai ◽  
Kousaku Murata
Keyword(s):  
Phosphatase Activity ◽  
Physiological Role ◽  
Inositol Polyphosphate ◽  
Nad Kinase ◽  
Inositol Monophosphatase ◽  
Tertiary Structures ◽  
E Coli ◽  
Fructose 1,6 Bisphosphatase ◽  
Fructose 1,6 Bisphosphate

ABSTRACT NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP+ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3′-phosphoadenosine 5′-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3′-phosphoadenosine 5′-phosphate phosphatase. Based on this fact, we found that 3′-phosphoadenosine 5′-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3′-phosphoadenosine 5′-phosphate phosphatase rather than as NADP(H) phosphatase.

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