Efficient regeneration of NADPH using an engineered phosphite dehydrogenase

Johannes, Tyler W.; Woodyer, Ryan D.; Zhao, Huimin

doi:10.1002/bit.21168

Cited by 155 publications

(124 citation statements)

References 35 publications

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“…In general, cofactor recycling can be achieved by coupling a desired enzymatic reaction with an additional chemical, electrochemical, photocatalytic, or enzymatic reaction, and the enzymatic method is favored (1,5,9,13,16,17,22,32,39). Enzymatic cofactor recycling can be obtained by using "coupled-substrate" (8, 12, 30, 31) and "coupled-enzyme" (14,15,18,19,20,21,29,33,34) approaches. The latter is a more general approach and utilizes the first enzyme for the desired biotransformation and the second enzyme for cofactor regeneration.…”

mentioning

confidence: 99%

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

Zhang

O’Connor

Wang

et al. 2009

Appl Environ Microbiol

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The glucose dehydrogenase (GDH) from Bacillus subtilis BGSC 1A1 was cloned and functionally expressed in Escherichia coli BL21(pGDH1) and XL-1 Blue(pGDH1). Controlled permeabilization of recombinant E. coli BL21 and XL-1 Blue with EDTA-toluene under optimized conditions resulted in permeabilized cells with specific activities of 61 and 14 U/g (dry weight) of cells, respectively, for the conversion of NADP ؉ to NADPH upon oxidation of glucose. The permeabilized recombinant strains were more active than permeabilized B. subtilis BGSC 1A1, did not exhibit NADPH/NADH oxidase activity, and were useful for regeneration of both NADH and NADPH. Coupling of permeabilized cells of Bacillus pumilus Phe-C3 containing an NADPHdependent ketoreductase and an E. coli recombinant expressing GDH as a novel biocatalytic system allowed enantioselective reduction of ethyl 3-keto-4,4,4-trifluorobutyrate with efficient recycling of NADPH; a total turnover number (TTN) of 4,200 mol/mol was obtained by using E. coli BL21(pGDH1) as the cofactorregenerating microorganism with initial addition of 0.005 mM NADP ؉ . The high TTN obtained is in the practical range for producing fine chemicals. Long-term stability of the permeabilized cell couple and a higher product concentration were demonstrated by 68 h of bioreduction of ethyl 3-keto-4,4,4-trifluorobutyrate with addition of 0.005 mM NADP ؉ three times; 50.5 mM (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate was obtained with 95% enantiomeric excess, 84% conversion, and an overall TTN of 3,400 mol/mol. Our method results in practical synthesis of (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate, and the principle described here is generally applicable to other microbial reductions with cofactor recycling.Biocatalytic oxidoreductions are important reactions in asymmetric synthesis, and they have great potential for industrial production of enantiopure chemicals and pharmaceuticals (4,11,23,24,26). These reactions often require a stoichiometric amount of the expensive cofactor NAD(P)H or NAD(P) ϩ , and thus practical applications require efficient recycling of the necessary cofactor (1,5,9,13,16,17,22,32). In general, cofactor recycling can be achieved by coupling a desired enzymatic reaction with an additional chemical, electrochemical, photocatalytic, or enzymatic reaction, and the enzymatic method is favored (1,5,9,13,16,17,22,32,39). Enzymatic cofactor recycling can be obtained by using "coupled-substrate" (8, 12, 30, 31) and "coupled-enzyme" (14,15,18,19,20,21,29,33,34) approaches. The latter is a more general approach and utilizes the first enzyme for the desired biotransformation and the second enzyme for cofactor regeneration. Formate dehydrogenase (15, 18) and glucose dehydrogenase (GDH) (33, 34) are well-known enzymes used for regeneration of NADH and NADPH, respectively. The "coupled-enzyme" approach has been successfully used with two isolated enzymes (15,18,29,33,34) or whole cells (14,20,21) of a microorganism coexpressing the two necessary enzymes. While the use of isolated enzymes is c...

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mentioning

confidence: 99%

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

Zhang

O’Connor

Wang

et al. 2009

Appl Environ Microbiol

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“…Two typical NAD(P)H-recycling techniques are entrapped cofactors by membrane technologies or immobilized cofactors (Betancor et al, 2006;Johannes et al, 2007;Liu and Wang, 2007). For example, NAD linked with polyethelene glycol has been regenerated more than 80,000 and 600,000 times for production of L-leucine and L-phenylalanine, respectively (Hummel et al, 1987;Kragl et al, 1996;Wichmann and Vasic-Racki, 2005).…”

Section: Cost Analysis and Perspectivesmentioning

confidence: 99%

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Zhang

2010

Biotech & Bioengineering

165

137

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Cell-free synthetic (enzymatic) pathway biotransformation (SyPaB) is the assembly of a number of purified enzymes (usually more than 10) and coenzymes for the production of desired products through complicated biochemical reaction networks that a single enzyme cannot do. Cell-free SyPaB, as compared to microbial fermentation, has several distinctive advantages, such as high product yield, great engineering flexibility, high product titer, and fast reaction rate. Biocommodities (e.g., ethanol, hydrogen, and butanol) are low-value products where costs of feedstock carbohydrates often account for $30-70% of the prices of the products. Therefore, yield of biocommodities is the most important cost factor, and the lowest yields of profitable biofuels are estimated to be ca. 70% of the theoretical yields of sugar-to-biofuels based on sugar prices of ca. US$ 0.18 per kg. The opinion that SyPaB is too costly for producing low-value biocommodities are mainly attributed to the lack of stable standardized building blocks (e.g., enzymes or their complexes), costly labile coenzymes, and replenishment of enzymes and coenzymes. In this perspective, I propose design principles for SyPaB, present several SyPaB examples for generating hydrogen, alcohols, and electricity, and analyze the advantages and limitations of SyPaB. The economical analyses clearly suggest that developments in stable enzymes or their complexes as standardized parts, efficient coenzyme recycling, and use of low-cost and more stable biomimetic coenzyme analogs, would result in much lower production costs than do microbial fermentations because the stabilized enzymes have more than 3 orders of magnitude higher weight-based total turn-over numbers than microbial biocatalysts, although extra costs for enzyme purification and stabilization are spent.

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“…Formate dehydrogenase (FDH) (Bolivar et al, 2007;Gül-Karagüler et al, 2001;Seelbach et al, 1996;), phosphite dehydrogenase (PTDH) (Johannes et al, 2007;Relyea et al, 2005;Woodyer et al, 2006), alcohol dehydrogenase (ADH), glucose-6 phosphate dehydrogenase, glucose dehydrogenase(GDH) (Endo &Koizumi, 2001;Xu et al, 2007) are currently available systems.…”

Section: Coenzyme Regenerationmentioning

confidence: 99%

Protein Engineering Applications on Industrially Important Enzymes: Candida methylica FDH as a Case Study

Ordu¹,

Karagüler²

2012

Protein Engineering

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Efficient regeneration of NADPH using an engineered phosphite dehydrogenase

Cited by 155 publications

References 35 publications

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Protein Engineering Applications on Industrially Important Enzymes: Candida methylica FDH as a Case Study

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