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Introduction et al. 2020). Various designing principles and engineer- β-Nicotinamide mononucleotide (NMN) is an impor- ing methods were also developed to alter the preference tant natural compound which can be found in every liv- of enzymes toward NMN (King et al. 2020). Considering ing organism. NMN has attracted much attentions as the importance of NMN, efficient manufacture of NMN + + the direct precursor of NAD and NADP , which are at an acceptable cost is vital for the application of NMN natural coenzymes playing vital roles in in vivo redox in medical and biotechnological industries. reactions (Hong et al. 2020; Yoshino et al. 2018; Ziegler Chemical synthesis of NMN requires the synthe- and Nikiforov, 2020). It was suggested that NMN supple- sis of nicotinamide riboside (NR) first, usually starting mentation could compensate for the deficiency of NAD from ribose derivatives and nicotinic acid derivatives or NADP , and recent studies have shown that NMN (Makarov and Migaud 2019). Chemically synthesized has diverse therapeutic applications such as treatment product of nicotinamide riboside usually contains two of neurological disorders, diabetes, obesity and anti- stereochemical configurations, alpha and beta, while only aging (Kiss et al. 2019; Uddin et al. 2017; Yao et al. 2017; the beta-isomer has biological activity (Franchetti et al. Yoshino et al. 2011). 2004). The most efficient record is the two-step method In addition to its potential medical applications, NMN reported by Zhang group in 2017, where a stereoselec- is also very useful in biocatalysis and biotransformation, tive N-glycosylation reaction was mediated by trimethyl- since NMN can be directly converted into NAD by cou- silyl trifluoromethanesulfonate (TMSOTf) between the pling with ATP and adenylyl transferases, and further starting materials ethyl nicotinate and 1,2,3,5-tetra-O- into NADP after additional phosphorylation (Walt et al. acetyl-β-d-ribofuranose. After deprotection in ammonia, + + 1984). The addition of NAD /NADP to reaction sys- only beta-isomer was obtained with 85% yield (Zhang tem will significantly improve the catalytic performance and Sauve 2017). Excessive phosphine oxychloride and of in vitro enzymatic redox reactions (Wu et al. 2013; delicate temperature control (0–5 °C) were required for Mordhorst and Andexer 2020). Furthermore, recent the subsequent phosphorylation to yield β-NMN (Lee studies have shown that NMN can directly participate in et al. 1999). A series of enzymatic or fermentation meth- redox reactions as a “noncanonical cofactor”. An enoate ods were also developed for the preparation of NMN, reductase XenA from P. putida can directly use NMN as however, either the low product titers or relatively high a coenzyme. When coupled with a glucose dehydroge- operation costs hindered their applications (Black et al. nase variant, the total turnover number (TTN) of NMN 2020a, b; Jeck et al. 1974; Marinescu et al. 2018). In 2021, could reach 39,000 (Black et al. 2020a, b; Richardson using metabolic engineering methods, a recombinant Qian et al. Bioresources and Bioprocessing (2022) 9:26 Page 3 of 9 Escherichia coli strain was constructed by Shoji and co- A0A0K9HLE0, Nakajima et al. 1978) were optimized workers (Shoji et al. 2021), which could produce 6.79 g·L according to E. coli codon usage alphabet and chemi- of NMN extracellularly from glucose and nicotinic acid, cally synthesized by Genscript Co. Ltd. (Nanjing, representing the highest record reported so far among China). The Klm -nrk and Gbst-ack gene fragments were the biological methods. inserted between NdeI and XhoI of pET21a without stop Enzymatic phosphorylation reactions require a phos- codon, resulting in recombinant pET21a/Klm-nrk and phoryl donor, and ATP is the most commonly used one. pET21a/Gbst-ack, and the inserted genes were sequenced However, the cost of ATP is high, making it unacceptable by Sanger’s DNA sequencing method. E. coli BL21(DE3) to be directly added at stoichiometric amount to the reac- was used for expression of the recombinant proteins. tion system. Besides that, removal of coproduct ADP also Nicotinamide riboside chloride and nicotinamide mono- complicates the downstream isolation process. Hence, an nucleotide with a high purity (> 97%) were supplied by efficient and economic ATP regeneration system is pre - Furuipharma Co. Ltd. Acetyl phosphate (AcP) was chem- requisite for industrial application of ATP-dependent ically synthesized from 85% phosphoric acid and acetic enzymatic phosphorylation reactions. Several differ - anhydride (Crans and Whitesides 1983). All the other ent ATP regeneration systems with different phosphate chemicals of analytical grade were obtained from com- donors have been developed (Chen and Zhang 2021), mercial sources. –1 such as pyruvate kinase (PK; EC 188.8.131.52) with phos- Terrific Broth (TB) medium, consisted of 12 g·L yeast –1 –1 –1 phoenolpyruvate (PEP), creatine kinase (CK; EC 184.108.40.206) extract, 24 g·L tryptone, 4 mL·L glycerol, 9.4 g·L –1 with creatine phosphate (CP), acetate kinase (AcK; EC K HPO and 2.2 g·L KH PO , was used for the cell 2 4 2 4 220.127.116.11) with acetyl phosphate (AcP), and polyphosphate growth and protein expression. kinase (PPK; EC 18.104.22.168) with polyphosphate (polyP). However, the costs of PEP and CP are so high that limit Expression and purification of kinase Klm‑NRK the applications of PK and CK mediated ATP regenera- Plasmid pET21a/Klm-nrk was transformed into chemi- tion systems (Caschera and Noireaux 2015). With regard cally competent E. coli BL21(DE3) cells by heat shock to PPK/polyP regeneration system, although polyphos- method. Recombinant strains were grown at 37 °C in TB –1 phate can be simply obtained from commercial sources medium containing 100 μg·mL ampicillin. When OD or just prepared by heating of N aH PO /Na HPO mix- of the culture reached 0.6, the temperature was switched 2 4 2 4 ture in an electric furnace (Honda et al. 2016), the spe- to 25 °C, and IPTG was added to a final concentration of cific activity of PPKs is usually low (less than 30 U/mg), 0.25 mM for induction expression for 12 h. Cells were which hindered their practical applications (Achberger- harvested by centrifugation and stored at –20 °C for fur- ová and Nahálka 2014; Akiyama et al. 1992; Motomura ther use. et al. 2014). Besides that, the potentially inhibitory effect For the purpose of protein purification, recombinant of polyP should also be taken into consideration (Zhang cells were resuspended in buffer A (20 mM sodium et al. 2017). Consequently, AcK/AcP regeneration sys- phosphate buffer, pH 7.4, 0.5 M NaCl, 20 mM imida - tem, consisting of cheap substrate (Crans and Whitesides zole) and disrupted by ultrasonication. After centrifu- 1983) and highly efficient enzyme (Nakajima et al. 1978), gation (10,000 × g, 30 min) at 4 °C, the supernatant was is generally regarded as a promising candidate for practi- loaded onto a HisTrap Ni–NTA FF column (5 mL, GE cal applications. Healthcare Co.) pre-equilibrated with buffer A. Buffer B In this study, we identified a new and highly active nic - (20 mM sodium phosphate buffer, pH 7.4, 0.5 M NaCl, otinamide riboside kinase (EC 22.214.171.124) from Kluyvero- 500 mM imidazole) was used for gradient elution of myces marxianus. A biocatalytic phosphorylation of NR Klm-NRK. The purification was then carried out accord - was developed for the efficient synthesis of NMN. As ing to the protocol provided by GE Inc. The purity of the –1 much as 100 g·L of NR was smoothly converted into collected fractions was examined by SDS-PAGE. The NMN, by coupling with the economically viable AcK/ fractions containing the target protein were combined AcP regeneration system. This approach does not require and the elution buffer was replaced with storage buffer excessive phosphorus oxychloride, and achieves a higher (50 mM Tris–HCl, pH 7.4; 25 mM KCl, 0.1 mM EDTA, product titer than other biological methods, rendering it 2 mM DTT and 50% (v/v) glycerol). a promising method for the bioproduction of NMN. Enzyme assay and characterization of recombinant Experimental Klm‑NRK Plasmid, strain, reagents and media Enzyme assay The Klm-nrk gene (uniport accession number: W0TD38) The coproduct of enzymatic phosphorylation of NR with and Gbst-AcK gene (uniport accession number: ATP is ADP, which can be regenerated by PK/PEP with Qian et al. Bioresources and Bioprocessing (2022) 9:26 Page 4 of 9 the formation of pyruvate, then pyruvate can be reduced and disrupted by ultrasonication. Gbst-AcK was purified to lactate with the oxidation of NADH catalyzed by LDH. using the same method as Klm-NRK. Gbst-AcK activity The activity of Klm -NRK was assayed spectrophotomet- was assayed as the generation of ATP from ADP with a rically at 30 °C by monitoring the oxidation of NADH sufficient amount of AcP. The 1-mL reaction mixture at 340 nm (Dölle and Ziegler, 2009). The standard assay contained 6 mM ADP, 30 mM AcP, 5 mM M gCl , 50 mM mixture (1 mL) composed of 100 mM potassium phos- pH 7.0 KPB and 20 μL enzyme solution. The solution was phate buffer (pH 7.0), 0.5 mM NR, 0.5 mM ATP, 2 mM incubated in water bath at 30 °C for 5 min, and then 200 MgCl , 5 mM phosphoenolpyruvate, 0.15 mM NADH, μL 1.0 M HCl was added for enzyme deactivation, 2.8 mL 3.5 U PK /5 U LDH (pyruvate kinase/lactate dehydro- 100 mM pH 7.0 KPB was then added for dilution before genase mix, Sigma) and appropriate amount of purified high-performance liquid chromatography analysis. The Klm-NRK. One unit of Klm-NRK activity was defined as ATP and ADP concentrations were measured with an the amount of enzyme catalyzing the oxidation of 1 μmol HPLC (LC2010A, Shimadzu) equipped with a Chrom- NADH per minute under above conditions. Core C18 column (4.6 mm × 250 mm, 5-μm particle size, Nanomicrotech Co.). The mobile phase consisted of 75% Kinetic analysis solvent A (40 mM KH PO and 5 mM tetramethylammo- 2 4 The kinetic parameters of the purified Klm -NRK were nium hydrogen sulfate, and pH was adjusted to 6.2 with determined at 30 °C in 50 mM pH 7.5 Tris–HCl contain- 1.0 M KOH) and 25% of solvent B (methanol) at a flow –1 ing 50 mM NaCl, 50 mM KCl, 12 mM M gCl and 0.01% rate of 0.5 mL·min and column temperature of 30 °C. bovine serum albumin, by assaying the initial reaction The injection volume was 10 μL and individual peak rates (in triplicate) with varied concentrations of NR areas were detected at wavelength of 254 nm. One unit of (0–0.5 mM, final concentration) and 0.5 mM ATP. For AcK was defined as the amount of the enzyme required determination of the apparent K value toward ATP, a for catalyzing the formation of 1 μmol of ATP per minute fixed concentration of NR (0.5 mM) was used (Dölle and under the standard assay conditions. Ziegler 2009). For each 1-mL reaction, 6.18 μg of purified Klm-NRK was added. The maximal reaction rate (V ) Enzymatic synthesis of NMN max and apparent Michaelis–Menten constant (K ) of the The typical phosphorylation of NR at 20-mL scale was –1 purified Klm-NRK were calculated by GraphPad Prism 7. performed as follows: after 50–100 g·L NR and 1.4 The protein concentration was measured by the standard equiv. of ATP were dissolved into 12 mL ddH O, and Bradford assay (Bradford, 1976). the pH was adjusted to 7.0 with 2.0 M NaOH. Further- more, 2 mL of Klm-NRK supernatant (from a lysate of pH and temperature optima and thermostability50 g /L cells suspended in pH 7.0 KPB, totally 64 U) wcw The optimum pH of Klm-NRK was determined at 30 °C and 2 mM MgCl (final concentration) were added, and in the following buffers (0.1 M): sodium citrate (pH 4.0–finally ddH O was supplemented to a total volume of 6.0), sodium phosphate (pH 6.0–8.5), and glycine–NaOH 20 mL. The system was stirred by magnetic agitation in a (pH 8.5–1.0). The temperature optimum was determined reactor with a water thermostat jacket set at 30 °C. Con- under the standard condition except for incubated at trol experiments were conducted without adding ATP or various temperatures (25–65 °C) for a period of 2 min. by replacing the Klm-NRK supernatant with lysate of E. Thermal stability was determined by pre-incubating the coli/pET21a. –1 purified enzyme (2 mg·mL ) at desired inactivation tem- When the exogenous ATP was replaced by an ATP peratures (30, 40, 50 or 60 °C) for a proper period of time regeneration system (AcK/AcP), 0.2–2.0 mM ATP (final followed by measuring the residual activity. The residual concentration), 1.4 equiv. of AcP, and 8300 U Gbst-AcK activity was expressed as a percentage of the initial activ- cell-free extract (2 mL lysate of 50 g /L cells suspended wcw –1 ity (V·V ). The inactivation rate constants (k ) were in pH 7.0 KPB) were added to the 20-mL reaction system. 0 D calculated from the slopes of semi-logarithmic plot of The pH was adjusted to 7.0 by automatically titrating –1 residual activity versus time (Ln (V·V ) =—k ·t). And 1.0 M NaOH. Samples (each 0.1 mL) were taken periodi- 0 D the half-lives (t ) of the enzyme were calculated from cally for HPLC analysis. 1/2 –1 the equation t = 0.693·k . After the reaction was completed, the reaction mixture 1/2 D was lyophilized and the yield of NMN was determined. Expression, purification and enzyme assay of GbstAc ‑ K Gbst-AcK was expressed using the same method as Klm- HPLC analysis NRK. After centrifugation, 0.5 g of recombinant Gbst- The NR and NMN concentrations were measured with AcK cells were resuspended in 10 mL KPB (0.1 M, pH 7.0) HPLC method as described above. Qian et al. Bioresources and Bioprocessing (2022) 9:26 Page 5 of 9 Purification of NMN and NMR analysis Table 1 Comparison of nicotinamide riboside kinases (NRKs) The reaction mixture was first passed through a mem - from different sources brane of 3,000 molecular weight cut-off (MWCO). Then Entry NRK Specific activity K K M, ATP M, NR –1 the reaction mixture was loaded on a 500-mL column of (U·mg ) (μM) (μM) macro-porous adsorption resin (HZ-801, donated from a b b 1 Human NRK1 0.275 ± 0.177 4.8 ± 0.3 3.4 ± 0.5 Huazhen Company, China) and eluted with water. The a b b 2 Human NRK2 2.32 ± 0.20 250 ± 12 46 ± 8 fractions containing NMN were pooled and concentrated 3 Sc‑NRK1 0.535 ± 0.600 NA NA via rotary evaporation at 37 °C under vacuum, and fur- c c c 4 Klm‑NRK 7.90 ± 0.42 70 ± 6 45 ± 11 ther lyophilized to obtain NMN as white solid powder. NA not available, Sc Saccharomyces cerevisiae, Klm Kluyveromyces marxianus The product structure was verified by H NMR analysis. Bieganowski and Brenner, 2004 Dölle and Ziegler, 2009 Results and discussion This work Identification of nicotinamide riboside kinase Two bioinformatic approaches were employed to iden- tify potentially active NRKs, including pBLAST search seen that Klm-NRK lost half of its activity in 21.2 h even in NCBI database with the protein sequence of ScNRK1 incubated at temperature as low as 30 °C, suggesting as a probe and protein BLAST in Uniprot database with Klm-NRK was vulnerable. Bovine serum albumin (BSA) NRK as the keyword. Five genes were chosen from 750 has been proved to be helpful for stabilization of human candidates and chemically synthesized by Genscript Co. NRK1 at 4 °C (Sasiak and Saunders 1996). However, when Ltd. (Nanjing, China). All the five genes were success - –1 0.4 mg·mL BSA (final concentration) was added to the fully expressed in E. coli BL21(DE3). Then their activity solution of purified Klm -NRK, no obvious improvement in phosphorylation of NR was determined. Among them, on thermostability was observed. It is unacceptable to Klm-NRK (uniport accession number: W0TD38) from add potential enzyme stabilizer to NR phosphorylation Kluyveromyces marxianus, Ct-NRK (uniport accession reaction systems, and protein engineering and immobili- number: G0RZA1) from thermophilic fungus Chaeto- zation techniques will be more promising to improve the mium thermophilum, and Lt-NRK (uniport accession thermal stability of Klm-NRK. number: C5DCS5) from Lachancea thermotolerans, Kinetic parameters were also determined. The K showed relatively higher NRK activities. Especially, Klm- and V values of purified Klm -NRK measured toward max NRK, sharing 55.1% amino acid identity with ScNRK1, −1 −1 ATP were 0.07 mM and 8.77 µmol·min ·mg , respec- –1 showed the highest activity of 632 U·g wet cell, which tively, while those measured toward NR were 0.045 mM –1 was much higher than 81.7 U·g of Ct-NRK and 77.8 −1 −1 and 8.48 µmol·min ·mg . The catalytic efficiency –1 U·g of Lt-NRK. Therefore, Klm -NRK was chosen for −1 −1 (k /K ) toward ATP was 57.4 s ·mM (Fig. 2), while cat M further study. −1 −1 (k /K ) toward NR was 84.4 s ·mM . The cata - cat M NR lytic efficiency of human NRK1 reported toward ATP −1 −1 Biochemical characterization of Klm‑NRK was 6.8 s ·mM while that of human NRK2 toward −1 −1 The kinase Klm-NRK was purified to homogeneity using ATP was 3.9 s ·mM (Tempel W et al. 2007). There - a Ni -column (Additional file 1: Fig. S5A). Klm-NRK fore, the catalytic efficiency of Klm -NRK was 8.4-fold and migrated at around 27 kDa, corresponding to its theoreti- 14.7-fold higher than those of human NRK1 and NRK2, cal molecular weight. The specific activity of the purified respectively. All the above proved that this newly identi- enzyme measured under the standard condition was 7.9 fied Klm-NRK is a robust enzyme with high catalytic effi - –1 U·mg protein. The specific activities of human NRK1, ciency in the phosphorylation of NR. human NRK2 and Saccharomyces cerevisiae NRK1 were –1 –1 –1 0.275 U·mg , 2.32 U·mg and 0.535 U·mg (Bieganow- ski and Brenner 2004), respectively (Table 1). Among the Enzymatic synthesis of NMN reported NR kinases, Klm-NRK showed the highest spe- To utilize Klm-NRK for practical synthesis of NMN, the cific activity. reaction conditions were optimized. An excess amount Effect of pH on the activity of Klm-NRK revealed (1.4 eq.) of ATP was first used as the phosphoryl donor. that the optimal pH of Klm-NRK was at pH 7.5 in KPB As shown in Table 2, 99.2% conversion of NR was (Fig. 1A). Klm-NRK displayed the highest activity at achieved within 10 h in the presence of 64 U Klm-NRK –1 –1 55 °C according to the temperature profile (Fig. 1B). at substrate loads of 50 g·L NR and 141 g·L ATP The purified Klm -NRK retained 50% of the initial activ- (Table 2, entry 1). When the NR load was increased up to –1 ity after incubation at 30 °C for 21.2 h, 40 °C for 14.5 h, 75 g·L , it could also be almost completely transformed 50 °C for 7.29 h and 60 °C for 0.29 h (Fig. 1C). It can be under the same condition within 27 h (Table 2, entry Qian et al. Bioresources and Bioprocessing (2022) 9:26 Page 6 of 9 Fig. 1 Characterization of the nicotinamide riboside kinase from Kluyveromyces marxianus. A pH optima of the purified Klm‑NRK. The activity was measured in the potassium phosphate buffers (pH 6.0–8.5). Relative activity was expressed as a percentage of maximum activity under the –1 experimental conditions. The maximum activity of Klm‑NRK at pH 7.5 was 8.9 U·mg (100%). B Activity–temperature profile. It was determined –1 at various temperatures (30–65 °C) in potassium phosphate buffer (100 mM, pH 7.0). The maximum activity of Klm‑NRK at 55 °C was 30.3 U·mg –1 (100%). C Thermal inactivation of Klm‑NRK. Purified Klm‑NRK (2 mg·mL ) was preincubated in potassium phosphate buffer (100 mM, pH 7.0) at –1 30 °C (●), 40 °C (▲), 50 °C (◆) or 60 °C (■), then the residual activity was measured. The initial activity of Klm‑NRK was 7.9 U·mg at 30 °C (100%) Fig. 2 Michaelis–Menten kinetics of Klm‑NRK for the substrates NR and ATP. Apparent parameters were determined by nonlinear regression using GraphPad Prism 7 2). However, when the NR load was further elevated concentration. Consequently, even if only 0.2 mM ATP –1 up to 100 g·L , only 82.1% conversion was achieved at was added exogenously, an almost complete conversion –1 27 h (Table 2, entry 3). This might be attributed to the of 50 g·L NR was easily achieved within 6 h in the pres- high loads of ATP that might influence the activity of ence of 64 U Klm-NRK and 8300 U Gbst-AcK (Table 2, Klm-NRK. entry 4). Because the K values of Gbst-AcK measured The main drawbacks of using high concentration towards ATP and ADP were 1.2 mM and 0.8 mM (Naka- of externally added ATP in the reaction include both jima et al. 1978), a higher concentration (2 mM) of ini- the high cost of ATP and the downstream process- tially loaded ATP was then investigated, resulting in a –1 ing issue caused by the formation of an equal mole of complete conversion of 50 g·L NR into NMN within byproduct ADP (Additional file 1: Fig. S7). Therefore, only 2 h (Table 2, entry 5). Finally, when the NR loading –1 an ATP regeneration system (AcK/AcP) was then intro- was further escalated to 100 g·L , 98.3% conversion was duced to replace the exogenously added ATP in high achieved in 8 h (Table 2, entry 6), affording 1.87 g NMN Qian et al. Bioresources and Bioprocessing (2022) 9:26 Page 7 of 9 Table 2 Enzymatic phosphorylation of nicotinamide riboside with Klm‑NRK –1 –1 Entry NR (g·L ) ATP (g·L ) P donor Time (h) Conv. (%) 1 50 141 ATP 8 99.2 2 75 211 ATP 27 99.2 3 100 282 ATP 27 82.1 4 50 0.114 AcP/AcK 6 99.7 5 50 1.14 AcP/AcK 2 99.7 6 100 1.14 AcP/AcK 8 98.3 (84.2% molar yield) which was isolated from the lyophi- Totally 750 mL water was used for the purification of lized crude product (ca. 3.77 g), representing a space– NMN during the column chromatography with a macro- −1 −1 time yield of 281 g L d . porous adsorption resin (bed volume: 500 mL), among ATP was recycled approximately 144.8 times in the sys- which 250 mL water was used to pre-wash the impuri- tem since only 2 mM ATP was initially added, avoiding ties before elution. Subsequently, the NMN-containing inhibitory effect of the coproduct ADP. The extremely eluent (ca. 500 mL) was concentrated to a final volume –1 high specific activity of Gbst-AcK (1876 U·mg ), rela- of about 50 mL. Furthermore, the concentrate was lyo- tively low molecular weight (43.4 kDa, Additional file 1: philized, affording 1.24 g NMN in > 97% purity. H-NMR Fig. S5B) and the high substrate loading contribute to a (400 MHz, D O), δ/ppm: 9.47 (s, 1H), 9.29 (d, J = 6.2 Hz, very high total turnover number of 56,876 mol product 1H), 8.99 (d, J = 8.1 Hz, 1H), 8.35–8.26 (m, 1H), 6.22 (d, per mol Gbst-AcK, indicating its great potential for ATP J = 5.4 Hz, 1H), 4.65 (s, 1H), 4.57 (t, J = 5.2 Hz, 1H), 4.45 regeneration. (dd, J = 4.9, 2.5 Hz, 1H), 4.31 (d, J = 12.0 Hz, 1H), 4.15 The result of control experiments with neither ATP (d, J = 9.1 Hz, 1H); C-NMR (100 MHz, D O), δ/ppm: nor the enzyme (Klm-NRK) proved that no NR was 165.84, 145.99, 142.50, 139.87, 133.95, 128.53, 99.98, converted. However, even when the lysate of blank E. 87.50, 87.42, 77.75, 71.04, 64.19, 64.14. coli/pET21a cells was added, approximately 3.5% of NR decomposition was still monitored in 8 h, suggesting a detectable activity of nucleotide phosphorylase inside the Conclusions E. coli BL21(DE3) cells. Therefore, a better host with less In summary, Klm-NRK, a nicotinamide riboside kinase nucleotide phosphorylase activity may further increase with the highest activity reported so far was discovered the product yield. from Kluyveromyces marxianus. For the first time, the Qian et al. Bioresources and Bioprocessing (2022) 9:26 Page 8 of 9 Author details enzymatic synthesis of NMN was achieved in high- State Key Laboratory of Bioreactor Engineering, East China University of Sci‑ –1 titer (93.5 g·L ) by employing the new and highly ence and Technology, Shanghai 200237, People’s Republic of China. Shanghai active NRK, and by adopting an efficient and cost- Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s effective ATP regeneration system, which resulted in a Republic of China. Suzhou Bioforany EnzyTech Co. Ltd, No. 8 Yanjiuyuan Road, −1 −1 space–time yield of 281 g·L ·day . Further studies to Economic Development Zone, Changshu, Jiangsu 215512, People’s Republic improve the catalytic performance of Klm-NRK by pro- of China. tein engineering and immobilization are now ongoing, Received: 15 September 2021 Accepted: 7 March 2022 which are expected to make Klm-NRK a more efficient tool for the large-scale manufacturing of NMN. Abbreviations References NMN: Nicotinamide mononucleotide; NR: Nicotinamide riboside; NAD / Achbergerová L, Nahálka J (2014) PPK1 and PPK2‑ which polyphosphate kinase NADP : Nicotinamide adenine dinucleotide/nicotinamide adenine dinucleo‑ is older? Biologia 69:263–269. https:// doi. org/ 10. 2478/ s11756‑ 013‑ 0324‑x tide phosphate; TMSOTf: Trimethylsilyl trifluoromethanesulfonate; Klm‑NRK: Akiyama M, Crooke E, Kornberg A (1992) The polyphosphate kinase gene of Nicotinamide riboside kinase from Kluyveromyces marxianus; Gbst‑AcK: Acetate Escherichia coli. isolation and sequence of the ppk gene and membrane kinase from Bacillus stearothermophilus; AcP: Acetyl phosphate; ADP: Adeno‑ location of the protein. J Biol Chem 267:22556–22561. https:// doi. org/ 10. sine diphosphate; ATP: Adenosine triphosphate. 1016/ S0021‑ 9258(18) 41708‑5 Bieganowski P, Brenner C (2004) Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss‑Handler independ‑ Supplementary Information ent route to NAD in fungi and humans. Cell 117:495–502. https:// doi. The online version contains supplementary material available at https:// doi. org/ 10. 1016/ S0092‑ 8674(04) 00416‑7 org/ 10. 1186/ s40643‑ 022‑ 00514‑6. Black WB, Aspacio D, Bever D, King E, Zhang L, Li H (2020a) Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotina‑ mide mononucleotide, a noncanonical redox cofactor. Microb Cell Fact Additional file 1: Figure S1. Un‑ optimized Klm‑nrk gene analysis. Figure 19:1–10. https:// doi. org/ 10. 1186/ s12934‑ 020‑ 01415‑z S2. Optimized Klm‑nrk gene analysis. Figure S3. Un‑ optimized Gbst‑ack Black WB, Zhang LY, Mak WS, Maxel S, Cui Y T, King E, Fong B, Martinez AS, gene analysis. Figure S4. Optimized Gbst‑ack gene analysis. Figure S5. Siegel JB, Li H (2020b) Engineering a nicotinamide mononucleotide Analysis of the purified Klm‑NRK and Gbst ‑AcK by SDS‑PAGE (12%). Figure redox cofactor system for biocatalysis. Nat Chem Biol 16:87–94. https:// S6. HPLC spectra of NR, NMN, ADP and ATP standards. Figure S7. Repre‑ doi. org/ 10. 1038/ s41589‑ 019‑ 0402‑7 sentative HPLC spectra for enzymatic phosphorylation of NR. Figure S8. Bradford MM (1976) A rapid and sensitive method for the quantitation of NMR spectra of enzymatically synthesized NMN. microgram quantities of protein utilizing the principle of protein‑ dye binding. Anal Biochem 72:248–254. https:// doi. org/ 10. 1016/ 0003‑ 2697(76) 90527‑3 Acknowledgements Caschera F, Noireaux V (2015) A cost‑ effective polyphosphate ‑based metabo ‑ We are grateful to Mr. Ming Wang from Jiangxi Biotechnology (Suzhou) Co. lism fuels an all E. coli cell‑free expression system. Metab Eng 27:29–37. Ltd. for his kind help with the development of HPLC analysis method. https:// doi. org/ 10. 1016/j. ymben. 2014. 10. 007 Chen H, Zhang YHPJ (2021) Enzymatic regeneration and conservation of ATP: Authors’ contributions challenges and opportunities. Crit Rev Biotechnol 41:16–33. https:// doi. XLQ performed the experiments, analyzed the data and drafted the manu‑ org/ 10. 1080/ 07388 551. 2020. 18264 03 script; YSD assisted in the experiments; prof. 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This work was financially supported by the National Key R&D Program of org/ 10. 1016/j. ab. 2008. 10. 033 China (2019YFA09005000), the National Natural Science Foundation of China Franchetti P, Pasqualini M, Petrelli R, Ricciutelli M, Vita P, Cappellacci L (2004) (21776085, 21871085 and 31971380), and the Fundamental Research Funds Stereoselective synthesis of nicotinamide β‑riboside and nucleoside for the Central Universities (22221818014). analogs. Bioorg Med Chem Lett 14:4655–4658. https:// doi. org/ 10. 1016/j. bmcl. 2004. 06. 093 Availability of data and materials Honda K, Hara N, Cheng M, Nakamura A, Mandai K, Okano K, Ohtake H (2016) All data generated or analyzed during this study are included in this article In vitro metabolic engineering for the salvage synthesis of NAD . Metab and its supplementary information file. 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Bioresources and Bioprocessing – Springer Journals
Published: Mar 21, 2022
Keywords: Enzymatic phosphorylation; Nicotinamide riboside; Nicotinamide riboside kinase; β-Nicotinamide mononucleotide (NMN); ATP regeneration system
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