Enzyme‐Loaded Nanoreactors Enable the Continuous Regeneration of Nicotinamide Adenine Dinucleotide in Artificial Metabolisms

Abstract Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme for numerous biocatalytic pathways. While in nature, NAD+ is continuously regenerated from NADH by enzymes, all synthetic NAD+ regeneration strategies require a continuous supply of expensive reagents and generate byproducts, making these strategies unattractive. In contrast, we present an artificial enzyme combination that produces NAD+ from oxygen and water continuously; no additional organic substrates are required once a minimal amount pyruvate is supplied. Three enzymes, i.e., LDH, LOX, and CAT, are covalently encapsulated into a substrate‐permeable silica nanoreactor by a mild fluoride‐catalyzed sol–gel process. The enzymes retain their activity inside of the nanoreactors and are protected against proteolysis and heat. We successfully used NAD+ from the nanoreactors for the continuous production of NAD+ i) to sense glucose in artificial glucose metabolism, and ii) to reduce the non‐oxygen binding methemoglobin to oxygen‐binding hemoglobin. This latter conversion might be used for the treatment of Methemoglobinemia. We believe that this versatile tool will allow the design of artificial NAD+‐dependent metabolisms or NAD+‐mediated redox‐reactions.


Introduction
Nicotinamide adenine dinucleotide (NAD + ;t he oxidized form of NAD) is an attractive bio-based oxidizing agent in synthesis,h owever,i ts regeneration from NADH (the reduced form of NAD) requires stoichiometric amounts of reagents or the use of organometallic catalysts.T od ate,n o regeneration strategy that works without additional reagents in stoichiometric amounts has been reported. It is desirable to use NAD + as an oxidizing agent only in small amounts and that the NAD + can be regenerated throughout the synthesis.
We demonstrate the continuous production of NAD + by an artificial enzyme-combination inside as ubstrate-permeable and robust silica nanoreactor by water and oxygen as the necessary stoichiometric reagents with am inimal amount of pyruvate,w ithout any additional substrates and without generating byproducts.
Living cells need ac ontinuous supply of NAD + but also have the capability to regenerate the NAD + . [1] If the NAD + / NADH couple is used in synthetic chemistry for either oxidation or reduction, stoichiometric use of NAD + /NADH is necessary. [2] As ustainable regeneration strategy of the coenzymes would also reduce the cost [3] of natural or artificial metabolisms. [4] Chemical, electrochemical, photocatalytic, and enzymatic strategies for both NAD + and NADH regeneration have been proposed. [3,5] Chemical regeneration of NADH by reducing agents such as NaBH 4 is effective, [6] but its high reactivity is problematic.E lectrochemical regeneration is usually mediator-dependent, side reactions to 1,6-NADH or the NAD 2 dimer have been described, and fouling of electrodes can occur. [7] Photocatalytic regeneration became arecent focus that uses light as agreen energy source but UVlight might be harmful to some processes. [8] Most methods for the NADH regeneration are non-selective,a nd other redoxsensitive compounds interfere. [3,5] In contrast, the enzymatic regeneration of NADH is the only selective method reported to date. [3,5] However,the low stability of enzymes and their difficult isolation from ahomogeneous reaction mixture are significant challenges.E ncapsulation or immobilization of the enzymes is essential to increase stability and ease purification. However,i tis accompanied by denaturation and reduced enzyme activity, for example,a cidic/basic pH, non-selective chemistry,o r organic solvents. [9] We present aN AD + -regeneration strategy that relies on three enzymes co-encapsulated into semipermeable silica nanoreactors (SiNRs) prepared via amild fluoride-catalyzed sol-gel chemistry in the microemulsion. High encapsulation efficiencies with conserved enzyme activity were achieved. Thea rtificial enzyme combination of lactate dehydrogenase (LDH), lactate oxidase (LOX), and catalase (CAT) only need am inimal amount of pyruvate to continuously produce NAD + from NADH as the additional substrates oxygen and water are available in the mixture (Figure 1). TheSiNRs were obtained as an aqueous dispersion and were combined exemplarily with the NAD + -dependent i) fluorometric glucose detection and ii)the recovery of hemoglobin from methemoglobin. In combination with the possibility of reusing the SiNRs,t he herein presented strategy for continuous NAD + regeneration is an attractive strategy for the use in various NAD + -dependent reactions.

Results and Discussion
We realized the continuous production of NAD + by the combination of lactate dehydrogenase (LDH), lactate oxidase (LOX), and catalase (CAT)e ncapsulated in as ubstratepermeable silica nanoreactor (Figure 1a). From these enzymes,L DH produces NAD + and lactate from NADH and pyruvate.The lactate is recycled by LOXtopyruvate without the need for ac oenzyme or cofactor, producing hydrogen peroxide as the only byproduct (Figure 1b), which is removed by disproportionation with CATt oo xygen and water. The enzyme cascade was initiated by the addition of NADH and am inimal amount of pyruvate to continuously produce NAD + (if consumed by areaction) (Figure 1b). Reproduction of pyruvate by LDH/LOX, "self-fueling", enables the continuous NAD + regeneration without the need to further supply pyruvate.T he remaining substrates water and oxygen are present in the open aqueous system.
To investigate the compatibility of the artificial enzyme set, the NAD + production was conducted in as olution containing LDH or amixture of LDH, LOX, and CAT. When the ratio of NADH:pyruvate was set to 2:1, 50 %conversion of NADH to NAD + by LDH alone was achieved, as pyruvate was converted to lactate.I nc ontrast, the mixture of LDH, LOX, and CATa chieved the full conversion of NADH, which demonstrates the recycling of pyruvate by LOX (Figure 1c). [10] Subsequently,t he three enzymes were covalently encapsulated into the substrate-permeable silica nanoreactor by am ild sol-gel process (Figure 2a). Unlike the conventional sol-gel process that relies on alkaline or acidic catalysts,w e used fluoride (F À )a sacatalyst to retain the enzyme activity ( Figure 1b). [11] This artificial enzyme combination, confined in the same nanoreactor,e nabled an efficient cascade reaction, modular handling,a nd easy separation from reactants with the possibility for reuse. [12] Encapsulation of LDH, LOX,a nd CATi nto the semipermeable silica nanoreactor was achieved by af luoridecatalyzed sol-gel process ( Figure 2). As ac ontrol, as ilica nanoreactor,l oaded with LDH alone,w as prepared. We cocondensated tetraethyl orthosilicate (TEOS) as the matrix Figure 1. Continuous NAD + -production and regeneration by an artificial enzyme set. a) The combination of LDH (lactate dehydrogenase), LOX (lactate oxidase), and CAT( catalase) encapsulated in semipermeable silica nanoreactors catalyze an NAD + -dependent reaction A!B. b) Enzyme reactions inside the silica nanoreactor:LDH converts NADH to NAD + and pyruvate to lactate. LOX recycles the pyruvate from lactate using water and oxygen and produces H 2 O 2 .CAT partially recycles H 2 O 2 to water and oxygen. c) The reaction of lactate to pyruvate:i fthe pyruvate is added in nonstoichiometric amounts (NADH:pyruvate = 2:1) the single enzyme (LDH) can oxidize 50 %of the NADH to NAD + (blue curve), while the enzyme combination (LDH, LOX, CAT) achieves the full conversion (red curve). material with 3-aminopropyl-trimethoxysilane (APTMS) as ac ovalent anchor for the enzymes in aw ater-in-oil microemulsion with potassium fluoride (KF) as the catalyst, preserving the pH-value at 7.4. During the process,t he enzymes were covalently attached to the silica network by N-(3-dimethyl aminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry. Circular dichroism (CD) spectra of enzymes in the fluoride solution show no significant change in their secondary structure (LDH and LOXf or Figure S1), (CAT for the previous report [13] ). Conventional sol-gel chemistry relies on acidic or alkaline catalysis,which may result in the denaturation of proteins and loss of enzymatic activity in some cases. [11] Fore xample,w hen ammonia was used as ac atalyst for the condensation, no LDH enzyme activity was detected in the resulting nanoreactor ( Figure S2). Besides,with the fluoridecatalyzed sol-gel chemistry,ahigher enzyme loading efficiency than conventional surface-immobilization of enzymes was achieved (see below and ref. [14]).
As the LOXreaction produces hydrogen peroxide,ahigh amount of CATi se ssential to disproportionate hydrogen peroxide into water and oxygen in the same nanoreactor and there is an eed to protect LDH from the risk of oxidative denaturation. In the following,w ea djusted the molar equivalents of the enzymes to LDH:LOX:CAT = 3:1:15. Theh ydrodynamic diameters of the silica nanoreactors were determined by dynamic light scattering (DLS) to be between 200 and 270 nm (LDH@SiNRs:a verage diameter:2 70 nm, PDI = 0.404;L DH/LOX/CAT@SiNRs average diameter: 200 nm, PDI = 0.420, Figures S3 and S4). From thermogravimetric analysis (TGA), ahigh enzyme loading was estimated by aweight loss of ca. 20 %organic contents in the self-fueled nanoreactor during the thermal degradation compared to the empty nanoreactor ( Figure S5). 29 Si solid-state NMR spectra showed the successful formation of the silica network by condensation of TEOS (Q 2 ,Q 3, and Q 4 )and APTMS (T 2 and T 3 )w ith am olar ratio of 2:1b yt he sol-gel process (Figure 2c). Besides,F T-IR confirms the formation of silicon oxide by detecting two strong bands for Si-OH (780 cm À1 )and Si-O-Si (1040 cm À1 )( Figure S6). Additionally,d istinct amide vibrations at 1550 cm À1 were detected for the loading of enzymes.M easurements of the surface area of the nanoreactors by Brunauer-Emmett-Teller (BET) gas adsorption showed aBET surface area of 31.5 m 2 g À1 and pore volume of 0.08 cm 3 g À1 ,w hich was sufficient for the diffusion of small molecular substrates and products to the enzymes through the silica matrix. [15] All enzyme-loaded silica nanoreactors exhibited ah igh enzyme activity,w hich is evident in the substrate-permeability of the silica matrix. Theenzymatic activity (k cat /K m )(with k cat = turnover number, K m = Michaelis-Menten constant) of LDH in the self-fueled nanoreactors (LDH/LOX/CAT@-SiNRs) was 2.3-fold higher than that of the LDH@SiNRs for forward reactions (pyruvate + NADH ! lactate + NAD + ) (Figure 3a,b). In particular, at wo-fold higher k cat value was observed in LDH in the LDH/LOX/CAT@SiNRs.T oexplore the reason for the increased k cat value,w ei nvestigated the product inhibition of LDH. According to the literature,t he forward reaction of LDH can be inhibited by lactate as the product. [16] Our results show aslightly decreased velocity for the forward reaction of native LDH (10 %f or 1:20 of pyruvate:lactate), which is probably caused by the increasing amount of lactate in the mixture.Interestingly,much stronger effect of the product inhibition was observed for the LDH@SiNRs (25 %f or a2 :1 of pyruvate:lactate ratio, Figure S7), presumably due to the local accumulation of the produced lactate inside the nanoreactors.I nc ontrast, lactate was quickly eliminated by the reaction of LOXi nt he LDH/ LOX/CAT@SiNRs,w hich we believe the main contribution to the increased k cat value.
Thea ctivity of LOXd id not differ remarkably for both single LOX-loaded nanoreactors and LDH/LOX/CAT@-SiNRs in terms of K m and k cat ( Figures S8 and S9), because no substrate recycling (by LDH) occurred during the reaction.
We determined the changes of the enzymatic activity after exposure to 60 and 70 8 8C(below melting temperature (T m )of the native enzyme) and 80 8 8C( above T m )f or 15 min (Figure 3c). Theenzymatic assay was performed at room temperature.T he native LDH significantly lost its activity after heating to 80 8 8C(above T m )for 30 min to less than 10 %ofthe initial activity.I nc ontrast, the encapsulated LDH showed much higher stability when heated to 80 8 8Cf or 2h with ca. 40 %r esidual activity ( Figure S10). Regarding such higher stability of the encapsulated LDH, previous studies have claimed that immobilized proteins by multipoint attachment could increase the resistance against heat, organic solvents,or denaturing agents,p resumably due to the prevention of structural denaturation. [17] It is consistent with our previous results that the encapsulated glucose oxidase and betaglucosidase in silica nanoreactors show higher stability than their native states. [9,14] Thee ncapsulated LOXa lso showed higher preserved activity than their native form at high temperatures.U nlike encapsulated LDH, the native LDH lost its activity at 70 8 8C( Figure S11). On the other hand, both native CATand encapsulated CATd id not significantly lose their enzymatic activity after exposure to 80 8 8Cf or 15 min ( Figure S12).
To further explore the structural stability of encapsulated enzymes,n ano differential scanning fluorimetry (NanoDSF) measurements were performed. [12,18] NanoDSF is based on changes in the intrinsic fluorescence of aromatic amino acids (i.e., Tr p) in the protein structure during thermal denaturation. Thef olded and unfolded proteins exhibit ad ifferent emission ratio at 330 nm and 350 nm. We compared the structural stability of the enzymes in solution, surfaceimmobilized, or encapsulated in silica nanoreactors.I nt he case of LDH, the initial emission ratios of 350 nm/330 nm were not significantly different between LDH in solution and the encapsulated LDH (0.82 and 0.83, respectively) (Figure S13), which indicated as imilar folding state of native LDH in solution and the LDH after encapsulation. However, as ignificant difference between the pure LDH and the encapsulated LDH was detected after cooling to room temperature:w hile the native enzyme did not show any residual activity,the encapsulated enzyme in the silica matrix was still active,which might be attributed to partial refolding during the cooling procedure ( Figure S14). According to the results,t he encapsulated LDH recovered almost its initial fluorescence ratio of 350 nm/330 nm after the cooling,w hile native LDH did not.
ForL OX,b oth the initial emission ratios at 350 nm/ 330 nm and melting temperature were similar between dissolved and encapsulated LOX( 68 8 8C) ( Figure S15). The stability of the encapsulated CATwas found to be more stable than native CAT( 4 8 8Ci ncreased melting temperature in encapsulated CAT; Figures S16 and S17). This further underlines the mild conditions of the encapsulation procedure for av ariety of enzymes.
Proteolytic resistance of encapsulated enzymes (LDH and LOX) in nanoreactors was investigated by exposure to Proteinase K( EC 3.4.21.64;2 8.9 kDa): unlike the dissolved and surface-immobilized enzymes,t he nanoreactors with loaded enzymes did not lose their activity in the presence of Proteinase K ( Figure 3d for LDH and Figure S18 for LOX). This data further underlines the encapsulation of the enzymes and indicates that Proteinase Kc annot penetrate the silica matrix due to its high molar mass.Additionally,noleakage of the enzymes from the nanoreactors was detected:t he enzymatic activity remained unchanged after extensive washing of the dispersion, proving the covalent attachment of the enzyme inside of the silica matrix ( Figure S19 and S20). In previous studies,only very little enzyme loading was achieved when the crosslinker (EDC,N HS) was omitted during the preparation of the silica nanoreactors. [15] Thec ontinuous production of NAD + was studied at different ratios of pyruvate:NADH at an initial molar ratio of pyruvate:N ADH = 2:1. Thed ecreasing absorbance monitored the conversion of NADH into NAD + at 340 nm. Additional NADH was added after the first equivalent was consumed. In the case of LDH@SiNRs,o nly two cycles produced NAD + ;a fterward, no oxidation of NADH was observed due to the lack of pyruvate and possible enzyme deactivation ( Figure S21). In contrast, the self-fueled nanoreactors (LDH/LOX/CAT@SiNRs) enabled the continuous regeneration of NAD + for at least eight cycles (Figure 4a). To initiate each cycle,o nly additional NADH was added to the dispersion, no further substrates or cofactors were necessary to produce NAD + .A fter eight cycles,t he dispersion was centrifuged to separate the nanoreactors from the reactant. After re-dispersion the nanoreactors in an ew reaction cocktail, four additional NADH additions obtained the successful conversion to NAD + ,w hich seems not to be the limit. Notably,t he reaction velocity decreased from addition to addition (Figures 4a and S22), which can be attributed to the changed NAD + concentration in the reaction mixture that alters the equilibrium constant (K)a fter each addition of NADH and reduces the reaction rates ( Figure S23). This was supported by conducting the NAD + regeneration at different ratios of NADH:NAD + ,proving adecreased conversion rate of NADH, when the amount of NAD + was increased ( Figure S24). After recycling the nanoreactors,t he rate of the NAD + production was increased to ca. 80 %ofthe initial velocity of the 1st cycle;p robably only 80 %r ecovery was obtained due to loss of the material during the washing procedures ( Figure S25). We confirmed the protection-effect of CATinside of the nanoreactors.The NADH oxidation was studied with ananoreactor,loaded only with LDH and LOX (but without CAT). Ther eaction rates decreased gradually, and the NAD + production ceased after the 6th cycle due to side-reactions with hydrogen peroxide ( Figure S26).
Thee fficiency of enzyme-loaded nanoreactors was demonstrated by conducting the NADH oxidation at an extremely low pyruvate concentration, i.e., pyruvate:NADH = 1:100, which would lead to only 1% NAD + conversion by the LDH, which resembles ac hange of 0.002 in the absorbance at 340 nm. Using the self-fueled nanoreactors,a lmost quantitative conversion from NADH to NAD + was achieved (Figure 4b), proving that only aminimal amount of pyruvate was sufficient to produce NAD + ,o nly relying on feeding with water and oxygen as substrates for the enzyme catalysis cascade (water and oxygen are present throughout the reaction cycles as the reaction proceeds under ambient air).
Thec onfinement effect of several enzymes in the same nanoreactor was compared to separately encapsulated enzymes in different nanoreactors.Wecompared the kinetics for the NAD + production for the LDH@SiNRs alone with two single enzyme nanoreactors (LDH and LOX) and the nanoreactors encapsulated LDH and LOX. Single LDH nanoreactors exhibited the lowest reaction rate to NAD + as the concentration of pyruvate decreases during the process (Figure 4c,g reen). Thea ddition of LOX( in as eparate nanoreactor) increased the reaction kinetics by afactor of ca. 1.1, as pyruvate was constantly regenerated by LOX ( Figure 4c,b lack). However,t he confinement of both LDH and LOXi nt he same nanoreactors further increased the NAD + regeneration by af actor of ca. 2.3 as the local pyruvate concentration remains high throughout the whole enzyme cascade (Figure 4c,red).
TheN AD + -producing nanoreactors can be applied in artificial enzyme cascades,f or example,f or glucose detection. [19] Glucose dehydrogenase is an NAD + -dependent enzyme and catalyzes the conversion of glucose to gluconod-lactone.I naconsecutive step,w eu sed peroxidase,w hich converted Amplex red into resorufin, generating af luorescence signal at 555 nm excitation and 595 nm emission, which is as imple glucose sensor (Figure 5a). Them olar ratio of NADH:pyruvate:Amplex red was 1:1:10, that is,amaximum of 10 %c onversion would be achieved without NAD +regeneration. Using the NAD + -producing nanoreactor,h igh conversion to resorufin was achieved (Figure 5b). In contrast, when the LDH@SiNRs were used, only al ow conversion (< 10 %) was achieved, which was equivalent to the amount of NAD + in the reaction mixture,a sn oc ontinuous regeneration was possible.T he results underline that the NAD +regeneration module can be combined with natural or artificial NAD + -dependent enzymatic pathways,which opens the possibility for catalysis in artificial cells or reactors. [20] With the redox potential of "NAD + + H + + 2e À ! NADH" (À320 mV) and hemoglobin A( À52 to À71 mV), [21] the NAD + -regeneration module was also capable of reducing methemoglobin to hemoglobin. Hemoglobin is an essential protein in most vertebrates for oxygen-transport, and it is amajor component of red blood cells.The heme group is responsible for the binding and release of oxygen. Methemoglobinemia is as erious disease in which the hemoglobin is autoxidized to the methemoglobin, and thus it cannot bind to oxygen. [22] In natural erythrocytes,t he reduction of methemoglobin to hemoglobin is achieved by the NADH-dependent methemoglobin reductase. [23] The reduction of methemoglobin would be of high interest not only for natural red blood cells but also for the design of synthetic hemoglobin-based oxygen carriers (Figure 6a). [24] As the oxidation of NADH to NAD + by LDH is at woelectron process,t wo heme groups might be involved to reduce Fe 3+ into Fe 2+ .LDH/LOX/CAT@SiNRs were used to produce hemoglobin from methemoglobin (0.125 mM) by the help of NADH (5 and 50 mM). TheU V/visible spectrum shows the formation of oxyhemoglobin (oxygen-bound form): Figure 5. Utilization of LDH/LOX/CAT@SiNRs in NAD + -dependent enzyme pathways. a) Glucose-sensing:G lucose is astarting substrate for glucose dehydrogenase, producingglucono-d-lactone. The peroxidase converts Amplex red into resorufin. Regenerationo fNAD + from NADH is necessary for the continuousprogress of the reaction. b) Monitoring of the fluorescence of resorufin during artificial glucose metabolismusing LDH@SiNRsorLDH/LOX/CAT@SiNRs. The molar ratio of NADH, pyruvate, and Amplex red was 1:1:10 (5 mM:5 mM:50 mM). The normalized value of 100 %att he y-axis indicates the raw value for the fluorescence of 33 000. Figure 6. Rapid reduction of methemoglobin to hemoglobin with NAD + -regenerationn anoreactors. a) Areaction scheme for the reduction of methemoglobin to hemoglobin. b) UV/Vis spectra showing the reduction of methemoglobin (8 mg mL À1 ;1 25 mM) to oxyhemoglobin at different concentrations of NADH with LDH/LOX/CAT@SiNRs. c) Reduction of methemoglobin to oxyhemoglobin with 10 mM NADH by LDH/LOX/CAT@SiNRs, LDH@SiNRs, and only NADH. Absorbance at 580 nm indicates production of oxyhemoglobin. d) UV/Vis spectra showing the reduction of metmyoglobin to oxymyoglobinw ith 10 mM NADH by LDH/LOX/CAT@SiNRs. two strong absorbance bands at 540 nm and 580 nm, together with adecreased absorbance at 630 nm, which are typical for oxyhemoglobin, were detected (Figure 6b). [25] This finding suggested that our NAD + regeneration system can be used for hemoglobin reconstitution, relying on NADH-consumption and NAD + production. Additionally,w ed emonstrated that the NAD + -regeneration module (LDH/LOX/CAT) was able to produce hemoglobin from methemoglobin more rapidly at the early stage of the reaction, even though NADH alone would be able to produce hemoglobin gradually (Figure 6c and Figure S27). Theprevious reports described the oxidation of NADH to NAD + without catalysis triggers the reduction of Fe 3+ to Fe 2+ , [26] whereas much higher and faster methemoglobin reduction was obtained when the NAD + -regeneration module was used in this study.W einvestigated the consumption of NADH for the reduction of methemoglobin:arapid consumption of NADH was observed by the reaction of LDH/LOX/CAT@SiNRs ( Figure S28). In contrast, when NADH was used alone or only LDH@SiNR was added, no significant change in the NADH concentration was detected. This implies that the reduction mainly relies on enzymatic reactions.T hese results add an ew function to LDH/LOX/ CAT@SiNRs and make it ap romising replacement for methemoglobin reductase in artificial hemoglobin regeneration. It will open potential applications for treatment of Methemoglobinemia,combined with other oxygen carriers as hemoglobin reconstitution modules,and other enzyme-mediated reactions.
In addition, the rapid conversion of metmyoglobin to oxymyoglobin was also possible using the LDH/LOX/CAT@-SiNR in the presence of 10 mM NADH (Figure 6d). Afaster reduction of metmyoglobin by the use of the enzyme modules (1.76-fold) was achieved when compared to hemoglobin. Myoglobin is akey oxygen carrier in the skeletal muscle tissue of many vertebrates and in almost all mammals. [27] Unlike hemoglobin, myoglobin has as ingle heme group.D ue to higher oxygen affinity than hemoglobin, myoglobin is an attractive oxygen carrier for blood substitutes. [27b] Moreover, the prevention of myoglobin autooxidation is an important challenge in solving the problem of meat discoloration. [28] Thus,t hese results could also possibly open an ew avenue in the meat industry.

Conclusion
An enzymatic NAD + -regeneration nanoreactor was developed. Thee nzymes LDH, LOX, and CATwere encapsulated into the silica nanoreactors by fluoride-catalyzed sol-gel chemistry retaining their enzymatic activities and stability. This "all-in-one" module allows their use in any NAD +dependent pathway,t ogether with easy separation from the reaction mixture and the possibility of recycling. Then anoreactors,l oaded with the three enzymes,o perate without external fuelling or additional reagents through self-recycling of all organic substrates.Only oxygen and water are needed. TheN AD + -regeneration was implemented into NAD + -dependent pathways,e .g., artificial glucose metabolism. The NAD + -regeneration module might be used in biocatalytic reactions or for the development of artificial mitochondria as artificial organelles in living or synthetic cells.Moreover,the self-fuelled module can quickly recover the irreversibly oxidized methemoglobin (metmyoglobin) to the reduced hemoglobin (myoglobin), implying the potential for an ew treatment to Methemoglobinemia and catalysing other redox reactions.