A family of customizable tethering molecules for tethering cofactors such as, but not necessarily limited to, nicotinamine adenine dinucleotide (NAD+/NADH, NAD(P)+/NAD(P)H) to substrates or structures formed from or including graphene-like materials is described. The tethered cofactor can then be used, for example, as biosensors employed for clinical diagnostic, food industry, medical drug development and environmental and military applications, as well as in reagentless biofuel cells for power generation.

The invention discloses a genetically engineered bacterium in which the gene encoding adenine deaminase on the genome of the bacterium is knocked out or/and the gene encoding the enzyme in the NAD.sup.+ anabolic pathway is integrated on the genome of the bacterium. The invention also discloses a construction method of the above-mentioned genetically engineered bacteria. The gene encoding adenine deaminase on the genome of the host strain is knocked out to obtain a strain with high NAD.sup.+ yield. Or the expression cassettes of the gene encoding the enzyme in the NAD.sup.+ synthesis pathway are constructed separately, and then the enzyme encoding The gene expression cassette is integrated into the genome of the host strain whose gene encoding adenine deaminase is knocked out to construct a strain with high NAD.sup.+ production. The application of the above genetically engineered bacteria is disclosed. A method of producing NAD.sup.+ is disclosed.

The invention discloses a genetically engineered bacterium in which the gene encoding adenine deaminase on the genome of the bacterium is knocked out or/and the gene encoding the enzyme in the NAD.sup.+ anabolic pathway is integrated on the genome of the bacterium. The invention also discloses a construction method of the above-mentioned genetically engineered bacteria. The gene encoding adenine deaminase on the genome of the host strain is knocked out to obtain a strain with high NAD.sup.+ yield. Or the expression cassettes of the gene encoding the enzyme in the NAD.sup.+ synthesis pathway are constructed separately, and then the enzyme encoding The gene expression cassette is integrated into the genome of the host strain whose gene encoding adenine deaminase is knocked out to construct a strain with high NAD.sup.+ production. The application of the above genetically engineered bacteria is disclosed. A method of producing NAD.sup.+ is disclosed.

A process for the enzymatic regeneration of the redox cofactors NAD.sup.+/NADH and NADP.sup.+/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula R.sub.1C(O)COOH is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a compound of general formula R.sub.2CH(OH)R.sub.3 is oxidized and wherein R.sub.1, R.sub.2 and R.sub.3 in the compounds have different meanings.

A process for the enzymatic regeneration of the redox cofactors NAD.sup.+/NADH and NADP.sup.+/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula R.sub.1C(O)COOH is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a compound of general formula R.sub.2CH(OH)R.sub.3 is oxidized and wherein R.sub.1, R.sub.2 and R.sub.3 in the compounds have different meanings.

A method of using an electrochemical cell, specifically a membrane bioreactor, to provide electrons to an electron transport chain capable of generating a proton gradient for performing ATP regeneration from ADP. Such an electron transport chain may be part of, or contained within, a synthetic membrane, or may be prepared by the suitable disruption of living cells. Electrons provided by the electrochemical cell are passed to the electron transport system via a suitable electron carrier, such as NADH2, FMNH2, FADH2, reduced ubiquinone(s), thiols, or other electron carriers or biological reducing equivalents that are compatible with the components of the electron transport chain performing ATP regeneration.

In an enzymatic synthesis of 3′,3′-cGAMP, other types of cyclic dinucleotides, c-di-GMP and c-di-AMP, are produced as by-products. One problem to be solved in order to establish a practical method for enzymatic synthesis of 3′,3′-cGAMP is suppression of production of these other types of cyclic dinucleotides during the synthesis. As a result of intensive studies, the inventors of the present invention found a variation of 3′,3′-cGAMP synthase by which the production of c-di-GMP and c-di-AMP is suppressed, and established a 3′,3′-cGAMP enzymatic synthesis system using this variation of the enzyme to complete the present invention. This enzyme brings about significantly reduced production of c-di-GMP and c-di-AMP, compared to the wild-type 3′,3′-cGAMP synthase. Accordingly, a production method using this enzyme makes it possible to reduce the production of other types of cyclic dinucleotides in comparison to conventional enzymatic synthesis methods, and efficiently synthesize 3′,3′-cGAMP.

In an enzymatic synthesis of 3′,3′-cGAMP, other types of cyclic dinucleotides, c-di-GMP and c-di-AMP, are produced as by-products. One problem to be solved in order to establish a practical method for enzymatic synthesis of 3′,3′-cGAMP is suppression of production of these other types of cyclic dinucleotides during the synthesis. As a result of intensive studies, the inventors of the present invention found a variation of 3′,3′-cGAMP synthase by which the production of c-di-GMP and c-di-AMP is suppressed, and established a 3′,3′-cGAMP enzymatic synthesis system using this variation of the enzyme to complete the present invention. This enzyme brings about significantly reduced production of c-di-GMP and c-di-AMP, compared to the wild-type 3′,3′-cGAMP synthase. Accordingly, a production method using this enzyme makes it possible to reduce the production of other types of cyclic dinucleotides in comparison to conventional enzymatic synthesis methods, and efficiently synthesize 3′,3′-cGAMP.

Polynucleotide synthesis performed with a substrate independent polymerase such as terminal deoxynucleotidyl transferase (TdT) is regulated by controlling the oxidation state of a metal cofactor. The oxidation state of the metal cofactor is changed to +2, thus activating the polymerase, by applying a voltage with electrodes or by introducing a chemical redox reagent. Addressable polynucleotide synthesis creates polynucleotides with different arbitrary sequences through use of spatial control of cofactor oxidation states to add nucleotides only at selected locations on an array. Control of metal oxidation states is regulated by selective activation of a microelectrode array, controlled addition of redox reagents to specific locations on the array, or controlled activation of photocatalysts at specific locations on the array. Scavengers in solution prevent cofactors distant from the selected locations from catalyzing polymerase activity and thereby maintain the localized effect of polymerase activation.

Polynucleotide synthesis performed with a substrate independent polymerase such as terminal deoxynucleotidyl transferase (TdT) is regulated by controlling the oxidation state of a metal cofactor. The oxidation state of the metal cofactor is changed to +2, thus activating the polymerase, by applying a voltage with electrodes or by introducing a chemical redox reagent. Addressable polynucleotide synthesis creates polynucleotides with different arbitrary sequences through use of spatial control of cofactor oxidation states to add nucleotides only at selected locations on an array. Control of metal oxidation states is regulated by selective activation of a microelectrode array, controlled addition of redox reagents to specific locations on the array, or controlled activation of photocatalysts at specific locations on the array. Scavengers in solution prevent cofactors distant from the selected locations from catalyzing polymerase activity and thereby maintain the localized effect of polymerase activation.