COFACTOR REGENERATION SYSTEM

Abstract

The present invention relates to cofactor regeneration systems, components and uses thereof and methods for generating and regenerating cofactors. The cofactor regeneration system comprises a first electron transfer component selected from a polypeptide comprising a NADH:acceptor oxido-reductase or NADPH:acceptor oxido-reductase, a second electron transfer component selected from a hydrogenase moiety and/or non-biological nanoparticles and an electronically conducting surface. The first and second electron transfer components are immobilised on the electrically conducting surface, and the first and second electron transfer components do not occur together in nature as an enzyme complex.

Claims

1-42. (canceled)

43. A method for manufacturing a cofactor regeneration system, the method comprising: a. providing: a first electron transfer component selected from one or more polypeptides comprising a NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase; and a second electron transfer component selected from a hydrogenase moiety, a non-biological nanoparticle or a combination thereof; and an electronically conducting surface; wherein the first and second electron transfer components do not occur together in nature as an enzyme complex; and b. immobilising the first and second electron transfer components on the electronically conducting surface so that in use electrons flow: from the first electron transfer component via the electronically conducting surface to the second electron transfer component; or from the second electron transfer component via the electronically conducting surface to the first electron transfer component.

44. The method according to claim 43, wherein the first electron transfer component comprises a NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase that has activity in an assay which comprises: a. adsorbing the NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase onto an electrode which is immersed in an electrochemical cell solution containing buffered electrolyte; b. holding the electrode at a constant potential of 412 mV while an increasing concentration of NAD.sup.+ or NADP.sup.+ is injected into said electrochemical cell solution; and c. recording a change in the electrocatalytic current magnitude at the electrode.

45. The method according to claim 43, wherein the second electron transfer component comprises a hydrogenase or non-biological nanoparticle that has an electrocatalytic H.sup. reduction current, electrocatalytic Hz oxidation current, or a combination thereof, within 100 mV below or above the thermodynamic potential of the 2W/H.sub.2 couple under the experimental conditions, when tested in an assay comprising: a. attaching the hydrogenase or non-biological nanoparticle to an electrode such that the catalyst can exchange electrons directly with the electrode; b. immersing the electrode in an electrochemical cell solution containing buffered electrolyte saturated with Hz; and c. cycling the electrode potential between lower and upper limiting potentials.

46. The method according to claim 43, wherein the first electron transfer component comprises an iron sulfur cluster, a heme cluster, a flavin moiety or a combination thereof; or wherein the hydrogenase comprises an iron sulfur cluster; optionally wherein the flavin moiety is a flavin mononucleotide or a flavin adenine dinucleotide.

47. The method according to claim 43, wherein the first electron transfer component is a diaphorase.

48. The method according to claim 43, wherein the first electron transfer component comprises an amino acid sequence having at least 20% sequence identity to one or more sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

49. The method according to claim 43, wherein the first electron transfer component comprises an amino acid sequence having at least 70% sequence identity to one or more sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68; or wherein the second electron transfer component comprises an amino acid sequence having at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72.

50. A method for regenerating a cofactor, the method comprising: a. adding a cofactor selected from NAD.sup.+, NADH, NADP.sup.+, NADPH or a combination thereof to a cofactor regeneration system, wherein the cofactor generation system comprises: i. a first electron transfer component selected from one or more polypeptides comprising a NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase; ii. a second electron transfer component selected from a hydrogenase moiety, a non-biological nanoparticle or a combination thereof; and iii. an electronically conducting surface; and wherein the first and second electron transfer components are immobilised on the electronically conducting surface; and wherein the first and second electron transfer components do not occur together in nature as an enzyme complex; and wherein the cofactor regeneration system is configured so that in use, electrons flow: from the first electron transfer component via the electronically conducting surface to the second electron transfer component; or from the second electron transfer component via the electronically conducting surface to the first electron transfer component; and b. obtaining a regenerated cofactor.

51. The method according to claim 50 further comprising harvesting the regenerated cofactor.

52. The method according to claim 50, wherein the first electron transfer component comprises a NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase that has activity in an assay which comprises: a. adsorbing the NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase onto an electrode which is immersed in an electrochemical cell solution containing buffered electrolyte; b. holding the electrode at a constant potential of 412 mV while an increasing concentration of NAD.sup.+ or NADP.sup.+ is injected into said electrochemical cell solution; and c. recording a change in the electrocatalytic current magnitude at the electrode.

53. The method according to claim 50, wherein the second electron transfer component comprises a hydrogenase or non-biological nanoparticle that has an electrocatalytic H.sup.+ reduction current, electrocatalytic H.sub.2 oxidation current, or a combination thereof, within 100 mV below or above the thermodynamic potential of the 2H.sup.+/H.sub.2 couple under the experimental conditions, when tested in an assay comprising: a. attaching the hydrogenase or non-biological nanoparticle to an electrode such that the catalyst can exchange electrons directly with the electrode; b. immersing the electrode in an electrochemical cell solution containing buffered electrolyte saturated with Hz; and c. cycling the electrode potential between lower and upper limiting potentials.

54. The method according to claim 51, wherein the regenerated cofactor is harvested by removal of the cofactor regeneration system by filtration, centrifugation, sedimentation, or a combination thereof

55. The method according to claim 50, wherein the method is carried out under anaerobic conditions.

56. The method according to claim 50, wherein the first electron transfer component comprises an iron sulfur cluster, a heme cluster, a flavin moiety or a combination thereof; or wherein the hydrogenase comprises an iron sulfur cluster.

57. The method according to claim 50, wherein the first electron transfer component is a diaphorase.

58. The method according to claim 50, wherein the first electron transfer component comprises an amino acid sequence having at least 20% sequence identity to one or more sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

59. The method according to claim 50, wherein the first electron transfer component comprises an amino acid sequence having at least 70% sequence identity to one or more sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68; or wherein the second electron transfer component comprises an amino acid sequence having at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72.

60. A cofactor regeneration system comprising: a. a first electron transfer component selected from one or more polypeptides comprising a NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase; and b. a second electron transfer component, wherein the second electron transfer component is a hydrogenase moiety; and c. an electronically conducting surface; and wherein the first and second electron transfer components are immobilised on the electronically conducting surface; and wherein the first and second electron transfer components do not occur together in nature as an enzyme complex; and wherein the cofactor regeneration system is configured so that in use, electrons flow: from the first electron transfer component via the electronically conducting surface to the second electron transfer component; or from the second electron transfer component via the electronically conducting surface to the first electron transfer component, optionally wherein the first electron transfer component comprises an iron sulfur cluster, a heme cluster, a flavin moiety or a combination thereof; or optionally wherein the hydrogenase comprises an iron sulfur cluster.

61. The cofactor regeneration system according to claim 60, wherein the first electron transfer component comprises a NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase that has activity in an assay which comprises: a. adsorbing the NADH:acceptor oxido-reductase or a NADPH:acceptor oxido-reductase onto an electrode which is immersed in an electrochemical cell solution containing buffered electrolyte; b. holding the electrode at a constant potential of -412 mV while an increasing concentration of NAD.sup.+ or NADP.sup.+ is injected into said electrochemical cell solution; and c. recording a change in the electrocatalytic current magnitude at the electrode.

62. The cofactor regeneration system according to claim 60, wherein the second electron transfer component comprises a hydrogenase or non-biological nanoparticle that has an electrocatalytic H.sup.+ reduction current, electrocatalytic H.sub.2 oxidation current, or a combination thereof, within 100 mV below or above the thermodynamic potential of the 2H.sup.+/H.sub.2 couple under the experimental conditions, when tested in an assay comprising: a. attaching the hydrogenase or non-biological nanoparticle to an electrode such that the catalyst can exchange electrons directly with the electrode; b. immersing the electrode in an electrochemical cell solution containing buffered electrolyte saturated with H.sub.2; and c. cycling the electrode potential between lower and upper limiting potentials.

Description

EXAMPLES

Example 1

NADH Cofactor Regeneration System

[0219] All steps were carried out in an anaerobic glove box (Glove Box Technology or MBraun) under an atmosphere of N.sub.2. Particles of pyrolytic graphite were prepared by abrasion of a piece of pyrolytic graphite with emery paper. These particles were immersed in 50 mM potassium phosphate buffer and suspended by sonication (5 minutes, ultrasonic bath). An aliquot of the particle suspension was removed. To this aliquot was added an aliquot of Escherichia coli hydrogenase 2 (component (ii), 10.sup.12 moles) and an aliquot of Ralstonia eutropha diaphorase (component (i), HoxFU, 10.sup.11 moles). The particles and enzymes were left at 4 C. for 10 minutes to allow the enzyme components to adsorb onto the particles. Centrifugation (5 minutes, benchtop centrifuge) was used to separate the particles and to remove excess unadsorbed enzyme. The enzyme-modified particles were then resuspended in 1 mM NAD.sup.+ in potassium phosphate buffer (50 mM, pH 7.0). The particle suspension was placed in a vial sealed with a septum and the headspace of the vial was exchanged for H.sub.2 gas via inlet and outlet needles. Aliquots were removed at specific time intervals for analysis for NAD.sup.+/NADH content. Each aliquot was centrifuged to remove particles, removed from the anaerobic glove box, and examined using ultra-violet/visible spectroscopy. NADH generation was observed.

Example 2

NAD.SUP.+ Cofactor Regeneration System

[0220] All steps were carried out in an anaerobic glove box (Glove Box Technology or MBraun) under an atmosphere of N2. Pyrolytic graphite particles modified with Escherichia coli hydrogenase 2 (component (ii)) and Ralstonia eutropha diaphorase (HoxFU, component (i)) were prepared as described in Example 1. After collection of the enzyme-modified particles by centrifugation, the particles were resuspended in 1 mM NADH in potassium phosphate buffer (50 mM, pH 7.0). The particle suspension was placed in a vial sealed with a septum containing N2 gas. Aliquots were removed at specific time intervals for analysis for NAD.sup.+/NADH content. Each aliquot was centrifuged to remove particles and examined using ultra-violet/visible spectroscopy. NAD.sup.+ generation was observed.

Example 3

Preparation of Wild-Type and Variant R. eutropha diaphorase (HoxFU)

[0221] For purification of wild type HoxFU and variants, R. eutropha cells containing plasmid pHoxFU harboring the genes hoxFUIhypA2B2F2CDEX (the hoxF gene was equipped at the 3 end with a sequence encoding the Strep-tag II peptide) were grown heterotrophically in a mineral salts medium containing a mixture of 0.2% (w/v) fructose and 0.2% (v/v) glycerol supplemented with 1 M NiCl.sub.2 and 1 M ZnCl.sub.2. (Lauterbach et al. PLoS ONE doi:10.1371/journal.pone.0025939). Cells were harvested at an optical density at 436 nm of 9 to 11 and washed with 50 mM potassium phosphate (K-PO.sub.4) buffer, pH 7.0 containing 50 mM succinate. The resulting cell pellet was resuspended in two volumes of resuspension buffer (50 mM Tris-HCl, 150 mM KCl, 5% glycerol, pH 8.0 containing Protease Inhibitor (EDTA-free, Roche). After two passages through a chilled French pressure cell at 6.2 MPa, the suspension was centrifuged at 100,000g for 45 min. The soluble extract was applied to a 2 mL Strep-Tactin Superflow column (IBA), washed with 6 mL of resuspension buffer and eluted with the same buffer containing 5 mM desthiobiotin (Lauterbach et al. PLoS ONE doi:10.1371/joumal.pone.0025939). Fractions containing HoxFU protein were pooled, concentrated and subsequently used for immobilization on graphite particles.

[0222] Diaphorase (HoxFU) variants were isolated as described above except that the hoxF sequence on plasmid pHoxFU was altered by genetic engineering which resulted in the production of HoxFU variants containing specific amino acid exchanges, to improve, inter alia, the NADP(H) binding affinity.

Example 4

Preparation of Soluble Extract HoxHYFUI2 (SH I64A)

[0223] The R. eutropha HF210 strains with the plasmid pGE747 for production of the SHI.sub.64 derivative were grown heterotrophically in a mineral salts medium containing a mixture of 0.2% (w/v) fructose and 0.2% (v/v) glycerol (FGN medium), which were harvested at an optical density at 436 nm of 9 to 11. For preparing soluble extract HoxHYFUI2 of the SHI.sub.64 derivative, the cells were resuspended in two volumes of 50 mm Tris-HCl, 150 mm KCl, pH 8.0 buffer containing a protease inhibitor cocktail (EDTA-free Protease Inhibitor, Roche). Cells were broken by two passages through a chilled French pressure cell at 6.2 MPa and the resulting suspension was centrifuged at 100,000 g for 45 min. The supernatant (soluble extract) was applied for preparing particles.

[0224] Example 5

Use of Cofactor Regeneration System of the Present Invention to Regenerate NADH for a Dehydrogenase

[0225] Pyrolytic graphite particles modified with Escherichia coli hydrogenase 2 (component (ii)) and Ralstonia eutropha diaphorase (HoxFU, component (i)) were prepared in an anaerobic glove box (Glove Box Technology or MBraun) as described in Example 1. After collection of the enzyme-modified particles by centrifugation, the particles were added to a solution containing S-Lactate dehydrogenase (Sigma, 0.5 mg/mL) and pyruvate (3 mM). To this suspension was added NAD.sup.+ (0.2 mM), and the suspension was equilibrated with H.sub.2 gas at atmospheric pressure. The formation of lactate was detected by Attentuated Total Reflectance Fourier Transform InfraRed spectroscopy using a diamond Attentuated Total Reflectance accessory (DurasampIIR II, SensIR Technologies).

Example 6

Use of Cofactor Regeneration System to Supply NADH to an NADH-Dependent Dehydrogenase Enzyme

[0226] The product of the dehydrogenase enzyme is a high value fine chemical or pharmaceutical product. A reactor is supplied with particles modified with E. coli hydrogenase 2 (component (ii)) and R. eutropha HoxFU (component (i)). The reactor is also supplied with the dehydrogenase enzyme, the substrate of the dehydrogenase enzyme (500 mM), NAD.sup.+ (1 mM), and H.sub.2. After a certain period of time, the product of the dehydrogenase reaction is collected from the reactor(eg by solvent extraction).

Example 7

Regeneration of NADH for Supply to a Dehydrogenase

[0227] The cofactor regeneration system of the present invention is placed in a solution of NAD.sup.+ (eg 0.2 mM) under an atmosphere comprising mainly H.sub.2 (eg H.sub.2 gas, or 90% H.sub.2/10% N.sub.2). An NADH-dependent dehydrogenase (eg lactate dehydrogenase) is added to the solution (or is attached to the electronically conducting surface). The substrate for the dehydrogenase (eg pyruvate for lactate dehydrogenase) is placed in the solution. The product of the dehydrogenase reaction (eg S-lactate for S-lactate dehydrogenase) can be collected continuously or batchwise.

Example 8

Regeneration of NADH for Supply to a P450 Monoxygenase

[0228] The cofactor regeneration system of the present invention is placed in a solution (free or immobilised on an electrically conducting surface) of NAD.sup.+ (eg 0.2 mM) under an atmosphere comprising mainly H.sub.2 with a small amount of O.sub.2 (eg 99% H.sub.2/1% O.sub.2). An NADH-dependent cytochrome P450 mono-oxygenase enzyme is added to the solution (or is attached to the electronically conducting surface). The substrate for the cytochrome P450 mono-oxygenase is placed in the solution. The product of the cytochrome P450 mono-oxygenase reaction can be collected continuously or batchwise.

Example 9

Regeneration of NAD.SUP.+ for Supply to a Dehydrogenase

[0229] The cofactor regeneration system of the present invention is placed in a solution of NADH (eg 0.2 mM) under an under an inert atmosphere (eg N.sub.2, argon) or an atmosphere containing low level H.sub.2 (eg 1%). An NAD.sup.+-dependent dehydrogenase (eg alcohol dehydrogenase) is added to the solution (or is attached to the electronically conducting surface). The substrate for the dehydrogenase (eg ethanol for alcohol dehydrogenase) is placed in the solution. The product of the dehydrogenase reaction (eg acetaldehyde for alcohol dehydrogenase) can be collected continuously or batchwise.

Example 10

NADPH Cofactor Regeneration System

[0230] All steps were carried out in an anaerobic glove box (Glove Box Technology or MBraun) under an atmosphere of N2. Particles of pyrolytic graphite were prepared by abrasion of a piece of pyrolytic graphite with emery paper. These particles were immersed in 100 mM bis-Tris buffer, pH 6.0 and suspended by sonication (5 minutes, ultrasonic bath). An aliquot of the particle suspension was removed. To this aliquot was added an aliquot of D. vulgaris Miyazaki F hydrogenase (component (ii)) and an aliquot of a variant of Ralstonia eutropha diaphorase (HoxHYFU, D467S E341A) (component (i)). The particles and enzymes were left at 4 C. for 10 minutes to allow the enzyme components to adsorb onto the particles. Centrifugation (5 minutes, benchtop centrifuge) was used to separate the particles and to remove excess unadsorbed enzyme. The enzyme- modified particles were then resuspended in 2 mM NADP.sup.+ in bis-Tris buffer (100 mM, pH 6.0). The particle suspension was placed in a vial sealed with a septum and the headspace of the vial was exchanged for H.sub.2 gas via inlet and outlet needles. Aliquots were removed at specific time intervals for analysis for NADP.sup.+/NADPH content. Each aliquot was centrifuged to remove particles, removed from the anaerobic glove box, and examined using ultra-violet/visible spectroscopy. NADPH generation was observed.

Example 11

Use of Cofactor Regeneration System of the Present Invention to Regenerate NADH for Yeast Alcohol Dehydrogenase

[0231] Pyrolytic graphite particles modified with Escherichia coli hydrogenase 2 (component (ii)) and a soluble extract of Ralstonia eutropha diaphorase (HoxHYFU, I64A variant, component (i)) were prepared in an anaerobic glove box (Glove Box Technology or MBraun) according to the methodology described in Example 1. After collection of the enzyme-modified particles by centrifugation, the particles were then added to a solution containing yeast alcohol dehydrogenase and heptanal (10 mM). To this suspension was added NAD.sup.+ (2 mM), and the suspension was equilibrated with H.sub.2 gas at atmospheric pressure. Ethyl acetate was added to the final solution to extract the product. After thorough mixing, the organic and aqueous phases were separated by centrifugation. The formation of 1-heptanol in the organic phase was confirmed by gas chromatography detection.

Example 12

NADH Cofactor Regeneration System with Co-Expressed Soluble Extract

[0232] Cells from a strain of Ralstonia eutropha H.sub.16 incorporating plasmids encoding the membrane bound hydrogenase and HoxFU are broken open. The membrane bound hydrogenase is solubilized from the membrane by the addition of detergent Triton X-100 to crude cell extracts. A subsequent high-speed centrifugation step leads to a soluble extract containing both membrane bound hydrogenase (component (ii)) and HoxFU (component (i)). A soluble cell extract of an Escherichia coli strain with overexpressed alcohol dehydrogenase is added to the Ralstonia eutropha soluble extract. Carbon-based particles are added to the soluble extract and left for 30 minutes at 4 C. The particle suspension is warmed to 30 C. and the substrate for the alcohol dehydrogenase is added to a concentration of 500 mM. NAD.sup.+ is also added to a concentration of 1 mM and H.sub.2 gas is gently bubbled into the solution. After 10 hours the product of the alcohol dehydrogenase reaction is collected by solvent extraction.