Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP.

Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cell-free protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.

Biosynthesis of a Therapeutically Important Nicotinamide Mononucleotide through a Phosphoribosyl Pyrophosphate Synthetase 1 and 2 Engineered Strain of Escherichia coli.

Nicotinamide mononucleotide (NMN), a precursor of NAD+, can be synthesized by the conversion of nicotinamide with the help of nicotinamide phosphoribosyl transferase (NAMPT) via the salvage pathway. NMN has recently gained great attention as an excellent therapeutic option due to its long-term effective pharmacological activities. In this study, we constructed a recombinant strain of Escherichia coli by inserting NAMPT and phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and PRPS2 (from Homo sapiens) genes to investigate the effect of PRPS1 and PRPS2 on NMN synthesis. The metabolically engineered strain of E. coli BL21 (DE3) exhibited 1.57 mM NMN production in the presence of Mg2+ and phosphates in batch fermentation studies. For further improvement in NMN production levels, effects of different variables were studied using a response surface methodology approach. A significant increment was achieved with a maximum of 2.31 mM NMN production when supplemented with 1% ribose, 1 mM Mg2+ and phosphate, and 0.5% nicotinamide in the presence of a lactose (1%) inducer. Additionally, insertion of the PRPS1 and PRPS2 genes in the phosphoribosyl pyrophosphate synthesis pathway and individual gene expression studies facilitated a higher NMN production at the intracellular level than the reported studies. The strain exhibited intracellular production of NMN from cheap substrates such as glucose, lactose, and nicotinamide. Hence, the overall optimized process can be further scaled up for the economical production of NMN using a recombinant strain of E. coli BL21 (DE3), which is the future perspective of the current study.

Biological synthesis of nicotinamide mononucleotide.

Nicotinamide mononucleotide (NMN) or Nicotinamide-1-ium-1-ß-D-ribofuranoside 5'-phosphate is a nucleotide that can be converted into nicotinamide adenine dinucleotide (NAD) in human cells. NMN has recently attracted great attention because of its potential as an anti-aging drug, leading to great efforts for its effective manufacture. The chemical synthesis of NMN is a challenging task since it is an isomeric compound with a complicated structure. The majority of biological synthetic routes for NMN is through the intermediate phosphoribosyl diphosphate (PRPP), which is further converted to NMN by nicotinamide phosphoribosyltransferase (Nampt). There are various routes for the synthesis of PRPP from simple starting materials such as ribose, adenosine, and xylose, but all of these require the expensive phosphate donor adenosine triphosphate (ATP). Thus, an ATP regeneration system can be included, leading to diminished ATP consumption during the catalytic process. The regulations of enzymes that are not directly involved in the synthesis of NMN are also critical for the production of NMN. The aim of this review is to present an overview of the biological production of NMN with respect to the critical enzymes, reaction conditions, and productivity.

Nicotinamide mononucleotide production by fructophilic lactic acid bacteria.

Nicotinamide mononucleotide (NMN), an intermediate in nicotinamide adenine dinucleotide biosynthesis, is recently attracting much attention for its pharmacological and anti-aging efficacies. However, current commercial products containing NMN are very high-priced because efficient and facile methods for industrial NMN production are limited. In this study, aiming for its nutraceutical application, we attempted to screen lactic acid bacteria for intracellular and/or extracellular NMN production. Using a bioassay system with an auxotrophic yeast that requires nicotinamide riboside (NR; dephosphorylated NMN), three candidates were obtained from a library of 174 strains of facultative anaerobic lactic acid bacteria. All three candidates belonged to the genus Fructobacillus and produced NR in the culture media (0.8-1.5 mg/l). Lactic acid bacteria of the genus Fructobacillus are known to use D-fructose as an electron acceptor in anaerobic lactic acid fermentation; addition of D-fructose to the medium caused intracellular accumulation of NMN and NR, but no extracellular production of these compounds was observed. Draft genome sequencing for one of the candidates suggested that nicotinamide phosphoribosyltransferase, which exists commonly in mammals but is less reported in microorganisms, is a key enzyme for NMN and NR production in the fructophilic bacteria.

Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor.

BACKGROUND: Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes. RESULTS: In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN+, a 130-fold increase over the cell's basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor. CONCLUSION: These results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.

ß-nicotinamide mononucleotide (NMN) production in Escherichia coli.

Diabetes is a chronic and progressive disease with continuously increasing prevalence, rising financial pressure on the worldwide healthcare systems. Recently, the insulin resistance, hallmark of type 2 diabetes, was cured in mice treated with NAD+ precursor ß-nicotinamide mononucleotide (NMN), no toxic effects being reported. However, NMN has a high price tag, more cost effective production methods are needed. This study proposes a biotechnological NMN production method in Escherichia coli. We show that bicistronic expression of recombinant nicotinamide phosphoribosyl transferase (Nampt) and phosphoribosyl pyrophosphate (PRPP) synthetase in the presence of nicotinamide (NAM) and lactose may be a successful strategy for cost effective NMN production. Protein expression vectors carrying NAMPT gene from Haemophilus ducreyi and PRPP synthetase from Bacillus amyloliquefaciens with L135I mutation were transformed in Escherichia coli BL21(DE3)pLysS. NMN production reached a maximum of 15.42 mg per L of bacterial culture (or 17.26 mg per gram of protein) in these cells grown in PYA8 medium supplemented with 0.1% NAM and 1% lactose.

Discovery of a novel nicotinamide phosphoribosyl transferase (NAMPT) inhibitor via in silico screening.

Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis, catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide (Nam). The diverse functions of NAD suggest that NAMPT inhibitors are potential drug candidates as anticancer agents, immunomodulators, or other agents. However, difficulty in conducting high-throughput NAMPT assay with good sensitivity has hampered the discovery of novel anti-NAMPT drugs with improved profiles. We combined an in silico screening strategy with a radioisotope (RI)-based enzyme assay and rationally identified promising NAMPT inhibitors with novel structures. AS1604498 was the most potent inhibitor, with an IC50 of 44 nM, and inhibited THP-1 and K562 cell line growth with the IC50 of 198 nM and 673 nM, respectively. The mode of action was found to reduce intracellular NAD following apoptosis, suggesting that these compounds inhibit NAMPT in cell-based assay. This strategy can be used to discover new drug candidates with targets which are difficult to assess through high-throughput screening. Our hit compounds may be used as seed compounds for developing new therapeutics with NAMPT.

CD73 protein as a source of extracellular precursors for sustained NAD+ biosynthesis in FK866-treated tumor cells.

NAD(+) is mainly synthesized in human cells via the "salvage" pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the "salvage" pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD(+) or NAD(+) precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD(+) precursors for NAD(+) biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD(+) biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.

Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis.

Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-A resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN --> NMN --> NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN --> NaAD --> NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis.

NAD(P) synthesis and pyridine nucleotide cycling in plants and their potential importance in stress conditions.

Pyridine nucleotides are key redox carriers in the soluble phase of all living cells, and both NAD and NADP play crucial roles in pro-oxidant and antioxidant metabolism. Recent data also suggest a number of non-redox mechanisms by which these nucleotides could influence cell function. In cases where these mechanisms involve NAD(P) consumption, resynthesis must occur to maintain nucleotide pools. Important information on the pathways involved in NAD synthesis in plants is beginning to appear, but many outstanding questions remain. This work provides an overview of the current state of knowledge on NAD synthesis pathways in plants and other organisms, analyses plant sequences for the first two enzymes of the de novo synthesis of NAD, proposes a preliminary model for the intracellular distribution of NAD synthesis, presents plant homologues of recently identified yeast mitochondrial NAD transporters, and discusses factors likely to be important in the regulation of NAD synthesis and contents in plants, with particular reference to stress conditions.

Kinetic mechanism of nicotinic acid phosphoribosyltransferase: implications for energy coupling.

Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) is a facultative ATPase that uses the energy of ATP hydrolysis to drive the synthesis of nicotinate mononucleotide and pyrophosphate from nicotinic acid (NA) and phosphoribosyl pyrophosphate (PRPP). To learn how NAPRTase uses this hydrolytic energy, we have further delineated the kinetic mechanism using steady-state and pre-steady-state kinetics, equilibrium binding, and isotope trapping. NAPRTase undergoes covalent phosphorylation by bound ATP at a rate of 30 s-1. The phosphoenzyme (E-P) binds PRPP with a KD of 0.6 microM, a value 2000-fold lower than that measured for the nonphosphorylated enzyme. The minimal rate constant for PRPP binding to E-P is 0.72 x 10(5) M-1 s-1. Isotope trapping shows that greater than 90% of bound PRPP partitions toward product upon addition of NA. Binding of NA to E-P.PRPP is rapid, kon >/= 7.0 x 10(6) M-1 s-1, and is followed by rapid formation of NAMN and PPi, k >/= 500 s-1. After product formation, E-P undergoes hydrolytic cleavage, k = 6.3 s-1, and products NAMN, PPi, and Pi are released. Quenching from the steady state under Vmax conditions indicates that slightly less than half the enzyme is in phosphorylated forms. To account for this finding, we propose that one step in the release of products is as slow as 5.2 s-1 and, together with the E-P cleavage step, codetermines the overall kcat of 2.3 s-1 at 22 degrees C. Energy coupling by NAPRTase involves two strategies frequently proposed for ATPases of macromolecular recognition and processing. First, E-P has a 10(3)-fold higher affinity for substrates than does nonphosphorylated enzyme, allowing the E-P to bind substrate from low concentration and nonphosphorylated enzyme to expel products against a high concentration. Second, the kinetic pathway follows "rules" [Jencks, W. P. (1989) J. Biol. Chem. 264, 18855-18858] that minimize unproductive alternative reaction pathways. However, an analysis of reaction schemes based on these strategies suggests that such nonvectorial reactions are intrinsically inefficient in ATP use.

Conversion of a cosubstrate to an inhibitor: phosphorylation mutants of nicotinic acid phosphoribosyltransferase.

Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) forms nicotinic acid mononucleotide (NAMN) and PPi from 5-phosphoribosyl 1-pyrophosphate (PRPP) and nicotinic acid (NA). The Vmax NAMN synthesis activity of the Salmonella typhimurium enzyme is stimulated about 10-fold by ATP, which, when present, is hydrolyzed to ADP and Pi in 1:1 stoichiometry with NAMN formed. The overall NAPRTase reaction involves phosphorylation of a low-affinity form of the enzyme by ATP, followed by generation of a high-affinity form of the enzyme, which then binds substrates and produces NAMN. Hydrolysis of E-P then regenerates the low-affinity form of the enzyme with subsequent release of products. Our earlier studies [Gross, J., Rajavel, M., Segura, E., and Grubmeyer, C. (1996) Biochemistry 35, 3917-3924] have shown that His-219 becomes phosphorylated in the N1 (pi) position by ATP. Here, we have mutated His-219 to glutamate and asparagine and determined the properties of the purified mutant enzymes. The mutant NAPRTases fail to carry out ATPase, autophosphorylation, or ADP/ATP exchanges seen with wild-type (WT) enzyme. The mutants do catalyze the slow formation of NAMN in the absence of ATP with rates and KM values similar to those of WT. In striking contrast to WT, NAMN formation by the mutant enzymes is competitively inhibited by ATP. Thus, the NAMN synthesis reaction may occur at a site overlapping that for ATP. Previous studies suggest that the yeast NAPRTase does not catalyze NAMN synthesis in the absence of ATP. We have cloned, overexpressed, and purified the yeast enzyme and report its kinetic properties, which are similar to those of the bacterial enzyme.

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis.

A number of Bacillus subtilis genes involved in NAD biosynthesis have been cloned and sequenced. One of the genes encodes a polypeptide homologous to Escherichia coli L-aspartate oxidase, and its mutation resulted in a nicotinic acid (Nic)-dependent phenotype; this gene was termed nadB. A second open reading frame (orf2) was found downstream of nadB, and an insertional plasmid separating orf2 and nadB also gave a Nic-dependent phenotype. This result suggests that orf2 may also be involved in NAD biosynthesis and that nadB and orf2 are in the same operon. Upstream of nadB was a third gene, transcribed in the opposite direction to that of nadB-orf2. The amino acid sequence derived from the third gene was quite similar to those derived from nifS genes of various nitrogen-fixing bacteria; therefore, the third gene was termed nifS. As with nadB and orf2, mutations in nifS also resulted in a Nic-dependent phenotype. The promoter regions of nadB and nifS overlapped each other and both contained -10 and -35 sequences which resemble those of E sigma A-type promoters. Transcription from both the nifS and nadB promoters, as well as expression of a nadB-lacZ fusion, was repressed by Nic. However, nadB transcription and nadB-lacZ expression were decreased, at most, only slightly by a deletion in nifS. The possible role of the nifS gene product in NAD biosynthesis is discussed.

Inhibition of quinolinate phosphoribosyl transferase by pyridine analogs of quinolinic acid.

The enzyme quinolinate phosphoribosyl transferase was purified from ATCC strain 23269. An HPLC method was developed for the analysis of the product of the enzyme reaction, nicotinate mononucleotide. Steady state kinetics in the forward reaction demonstrated a sequential mechanism for the enzyme. In order to gain more information on the mechanism of the enzyme reaction, a series of 2 substituted nicotinic acids and 2 substituted 3-nitropyridines were investigated as inhibitors of the reaction. The results indicate that potent inhibition results when the quinolinic acid analogs possessed a negatively charged group at the 2 position of the pyridine ring.

Purine and pyridine nucleotide production in human erythrocytes.

Human erythrocyte adenyl and pyridine nucleotide production has been tested in cell-free lysates and in intact cells. The main products obtained in cells incubated with adenine and nicotinic acid are adenosine triphosphate and nicotinate mononucleotide, respectively, under any experimental condition used (incubation time, base concentration). Adenine-phosphoribosyltransferase activity determined in crude lysates is about 100 times higher than nicotinate-phosphoribosyltransferase activity, while cellular adenyl nucleotide production is only three times higher than that of pyridine nucleotide. A strong intracellular regulation for the former, but not latter, synthetic process is thus suggested. Intact erythrocyte nicotinate nucleotide production is inhibited by adenine, while nicotinate-phosphoribosyltransferase activity is not. The possible regulation by adenyl nucleotides is discussed in light of the modulating action of ATP on nicotinate-phosphoribosyltransferase activity. The kinetic characteristics of both adenine- and nicotinate-phosphoribosyltransferases, determined on crude lysates, are reported.

The metabolism of [carboxyl-14C]anthranilic acid. I. The incorporation of radioactivity into NAD+ and NADP+.

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 X 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.