NADPH Regeneration by Glucose Dehydrogenase from<i>Gluconobacter scleroides</i>for<i>l</i>-Leucovorin Synthesis

Eguchi, Tamotsu; Kuge, Yukihiro; Inoue, Koshiro; Yoshikawa, Naohiro; Mochida, Kenichi; Uwajima, Takayuki

doi:10.1271/bbb.56.701

Cited by 38 publications

(20 citation statements)

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“…Efficient coenzyme recycling by recombinant enzymes converting the oxidized coenzyme back to its reduced form is essential for the productivity of reductive whole-cell biotransformations. In Escherichia coli , diverse strategies for cofactor regeneration have been applied: one-enzyme-coupled systems (Eguchi et al 1992; Ernst et al 2005; Seelbach et al 1996; Wichmann and Vasic-Racki 2005) and approaches taking advantage of the metabolism of the cell (Walton and Stewart 2004; Chin et al 2009; Fasan et al 2011; Blank et al 2008, 2010). …”

Section: Introductionmentioning

confidence: 99%

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Siedler

Bringer

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et al. 2011

Appl Microbiol Biotechnol

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Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by 13C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively.

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Section: Introductionmentioning

confidence: 99%

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Siedler

Bringer

Blank

et al. 2011

Appl Microbiol Biotechnol

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“…Accumulation of glucono-1,5-lactone in GDH regenerated processes over a time period of 2 h. Solid lines, NADH regeneration; dashed lines, NADPH regeneration. Data for modeling (glucose conversion rates, coenzyme concentration, pH) are taken from the indicated references: a, synthesis of l-6-hydroxynorleucine; [13] b, synthesis of actinol; [9] c, synthesis of l-leucovorin; [16] d, synthesis of methyl (R)-o-chloromandelate; [4] e, synthesis of l-carnitine; [12] f, synthesis of l-glyceraldehyde; [11] g, synthesis of (R)-ethyl 2-methyl 4-oxo-2-pentanoate; [10] h, synthesis of (R)-1-phenyl-A C H T U N G T R E N N U N G ethanol. [14] The pH-dependent glucono-1,5-lactone hydrolysis rates were taken from.…”

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confidence: 99%

Guidelines for the Application of NAD(P)H Regenerating Glucose Dehydrogenase in Synthetic Processes

et al. 2013

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Glucose dehydrogenase (GDH) is frequently used for the reduction of NAD + and NADP + in bench-and industrial-scale syntheses because the coenzyme regenerating system GDH is easy to apply, robust and relatively inexpensive. To optimize the application of this long known coenzyme regeneration system we investigated the commonly applied Bacillus GDH and characterized this enzyme by its kinetic features in the presence of substrates and products at pH 6.4 and 8.0. Three substrates/products were found to inhibit GDH considerably: (i) the reaction product glucono-1,5-lactone, (ii) the reduced coenzyme NAD(P)H and (iii) the oxidized coenzyme NAD(P)+ . The inhibition of GDH under several process conditions was modeled using the determined kinetic constants. It was found that the GDH regeneration system is strongly inhibited by the usually applied conditions. This study provides the rate equation of the GDH reaction and simulations of this coenzyme regenerating system leading to an improved prediction and, thus, to a faster scale-up and increased efficiency of NAD(P)H-dependent synthetic processes.

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“…Another aldehyde, geranial was obtained from geraniol (extractd from essential oils) in a twophase system using HLADH and regeneration of NAD + with acetaldehyde [204]. The substrate in the organic phase was converted while passing through a column containing a porous protein-based foam matrix (the aqueous phase) on which enzyme and coenzyme were coimmobilized.…”

Section: Enzymatic Syntheses Involving Alcoholsmentioning

confidence: 99%

Redox Enzymes Used in Chiral Syntheses Coupled to Coenzyme Regeneration

Leonida¹

2001

CMC

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Due to their ability to perform reactions in a stereo- and regiospecific manner, while functioning under mild conditions of temperature and pH, enzymes are increasingly used for chiral synthesis in the pharmaceutical industry, in medicinal chemistry, agriculture, cosmetic and food industry. The oxidoreductases as catalysts require a coenzyme (cofactor) which functions as an electron carrier. If used in stoichiometric amounts the cost of coenzymes makes synthetic applications of redox enzymes prohibitively expensive for industrial applications. The present review updates previous discussions about the objectives of cofactor regeneration as a requirement for the applicability of enzymatic redox reactions, and about the strategies customarily used to this end. Different types of chiral syntheses coupled successfully to coenzyme regeneration (amino acid synthesis, lactone synthesis, synthetic reactions involving alcohols, hydroxy acid and steroid synthesis, deracemization) are then discussed. New catalysts, cross-linked enzyme crystals, prepared from redox enzyme are presented in a small paragraph together with some of their applications. Special attention is given to whole cells used in this type of synthesis and their advantages and disadvantages are presented in one paragraph. Finally, different reactors, within which were conducted chiral syntheses catalyzed by oxireductases and coupled to cofactor regeneration, are briefly discussed.

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NADPH Regeneration by Glucose Dehydrogenase fromGluconobacter scleroidesforl-Leucovorin Synthesis

Cited by 38 publications

References 1 publication

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Guidelines for the Application of NAD(P)H Regenerating Glucose Dehydrogenase in Synthetic Processes

Redox Enzymes Used in Chiral Syntheses Coupled to Coenzyme Regeneration

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