Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

Abstract

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...

Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol

“…Due to the high cost of the cofactor, use of the cosubstrate glucose 6-phosphate and lower stability of PICs, strain A is favoured for industrial applications. Zhang et al (2006) increased TTN from 59 to 402 by decreasing cofactor concentration with BpG6PDH regeneration, which is in the same range as our results. A further increase of TTN to more than 4000 was demonstrated by further decreasing the cofactor concentration and simultaneously increasing the cell density, but this led to a loss of activity of 90 %.…”

“…BpG6PDH-coupled regeneration was more efficient than that of BmGDH. External NADP + is necessary for efficient synthesis, if PICs are used, which was also observed by Zhang et al (2006). The lowering of the cofactor concentration from 1 to 0.2 for strain B and to 0.1 for strain A decreased activity to 47 and 48 %, respectively, with simultaneous increase of TTN from 39 to 195 and 391 and almost full conversion of acetophenone, respectively.…”

“…In this work, a maximum TTN of 871 for strain A and 2066 for strain B was reached with activities of 10 and 5 % compared to higher cofactor concentrations. Zhang et al (2009) obtained TTNs of up to 4200 with an initial addition of 0.005 mM NADP + ; however, the influence of cofactor concentration on synthetic activity was not discussed. Both authors used up to 60 mM substrate; however, acetophenone is not soluble at this concentration and the addition of organic solvents or the use of two-phase systems would be necessary to increase substrate concentration.…”

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD + -Auxotrophic Mutant

“…It has been demonstrated that about 20% total cellular NAD(H) in wild-type cells is freely accessible and the (4). In several studies, the cellular cofactor concentration has been increased through manipulating the NAD ϩ biosynthetic pathway (12,16) or feeding permeabilized cells (18,36,37). The NAD(H) level was increased up to 6-fold previously (12).…”

Microbial transformation of quinic acid to shikimic acid by Bacillus megaterium

“…Cofactor recycling plays a key role in the NADPHdependent enzyme biocatalysis; therefore, it is necessary to optimize the reaction condition for proper cofactor recycling [21,22]. To study the effect of cofactor generation, different concentrations of glucose (0.05 to 0.4 M) were used in the reaction mixture.…”