Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide

Abstract

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.

Fig. 1. Structures and enzymes for cofactor biosynthesis.

a Native NadD catalyzed synthesis of NaAD from ATP and NaMN. b The proposed NcdS to catalyze the condensation of CTP and NMN into NCD.

Fig. 2. Directed evolution of NadD for NCD biosynthesis.

a The principle of coupled colorimetric assay for NCD formation. The expected NCD synthesis activity converts CTP and NMN into NCD, and an NCD-dependent malic enzyme mutant Mae* is used to convert NCD into NCDH, which reduces NBT into formazan in the presence of PMS. b The ATP-binding domain of E. coli NadD (PDB ID: 1K4M). NAD and targeted amino acids in NadD are shown in stick mode. Blue, nitrogen; red, oxygen; orange, phosphorus; yellow, sulfur; gray, carbon in NadD; green, carbon in NAD. c The evolutionary tree of NadD for CTP preference in two rounds of mutagenesis and screening.

Fig. 3. Docking analysis for substrate in wild-type NadD and NcdS-2.

The docking poses for ATP (a) and CTP (b) with NadD. The docking poses for ATP (c) and CTP (d) with NcdS-2. The docking poses for NaMN (e) and NMN (f) with NadD. The docking poses for NaMN (g) and NMN (h) with NcdS-2. The protein was shown as cartoon mode and in light gray. The substrate and the key residues were shown as stick mode. Green, carbon atoms. Blue, nitrogen. Gray, hydrogen. Red, oxygen. The dashed line indicates the hydrogen bond between ligand and receptor. The number marked near the dashed line represents the distance between two atoms (unit, angstrom).

Fig. 4. Construction of NCD self-sufficient E. coli strains.

a The designed NCD biosynthesis pathway. FtNadE, CtCTPS*, and NcdS-2 are used for biosynthesis of NMN, CTP, and NCD, respectively. b HPLC profiles of cofactors in DH5α and engineered strains. XYC2002, DH5α with constitutive expression of NcdS-2; XYC2013, DH5α with constitutive expression of FtNadE, CtCTPS* and NcdS-2. c The MS spectra of intracellular NAD and NCD in XYC2013. Negative ion mode. Experimental monoisotopic mass, NAD, 662.0941; NCD, 638.0850. Calculated molecular mass, NAD, 662.1018; NCD, 638.0906. Mass error, NAD, 11.6 ppm; NCD, 8.7 ppm. d Cofactor concentrations in DH5α strains with inducible expression of different enzymes. e Cofactor concentrations of engineered strains harboring different copy of the inducible NCD biosynthesis module. XYC5016, BW25113(ΔldhA, dld::cat) with single copy of NCD biosynthesis module; XYC5017, BW25113(ΔldhA, dld::cat) with the NCD biosynthesis module in low-copy plasmid; XYC5008, BW25113(ΔldhA, dld::cat) with the NCD biosynthesis module in high copy plasmid; XYC2017, DH5α with the NCD biosynthesis module in low-copy plasmid; XYC8017, DH10B with the NCD biosynthesis module in low-copy plasmid. Experiments were conducted in triplicates (n = 3), data are presented as mean values ± SD. The experiments were performed independently at least twice, with similar results. Source data are available in the Source Data file.

Fig. 5. Application of NCD self-sufficient cells.

a NCD self-sufficient cells implanted with an NCD-linked metabolic circuit converting L-malate into D-lactate. b Intracellular cofactor concentrations. c Crude enzyme activities of Mae and Ldh with different cofactors. Ldh*-NAD assayed with NAD and D-lactate, Ldh*-NCD with NCD and D-lactate, Mae*-NAD with NAD and L-malate, Mae*-NCD with NCD and L-malate. d Profiles of L-malate consumption and D-lactate production. About 1.0 × 10¹⁰ cells were incubated in MOPS buffer (pH 7.5) in the presence of 10 mM L-malate at 37 °C for 4 h. XYD042, BW25113 (ΔldhA, Δdld, arsB::Para-FtNadE-c-his-NcdS-2-CtCTPS*)/pUC-Plac-Ldh*-rbs-Mae*; XYD046, BW25113 (ΔldhA, dld::cat)/pUC-Plac-Ldh*-rbs-Mae*. Data are presented as mean values ± SD, error bars represent the standard deviation of data from three different cultures. The experiments were performed independently twice, with similar results. Source data are available in the Source Data file.