2.1 Strains and plasmids
Escherichia coli Top10 was used for plasmid preparation, E. coliBL21(DE3) was used for in vivo protein overexpression, and E. coliRosetta(DE3) was used to prepare cell extracts for CFPS. The amino acid sequences of HsNampt (nicotinamide phosphoribosyltransferase, Nampt fom Homo sapiens ), MrNampt (Nampt fom Meiothermus ruber ), HsPrs (phosphoribosyl pyrophosphate synthetase, Prs fom Homo sapiens ), PcPrs (Prs fomPyrobaculum calidifontis ), HsRbk (ribokinase, Rbk fom Homo sapiens ), and EcRbk (Rbk fom Escherichia coli ) were obtained from the UniProt or NCBI. To obtain additional NMN biosynthetic pathway enzyme sequences, these 6 sequences were used as query sequences to perform BLASTP search against the UniProt or NCBI database. For a particular query sequence, the resulting amino acid sequences with a percent identity value in the range of 30-99% were collected. Duplicate and incomplete sequences were discarded. The remaining sequences were aligned using ClustalW2 program (Larkin et al., 2007). The phylogenetic tree was generated using Molecular Evolutionary Genetics Analysis (MEGA 5) program (K. Tamura et al., 2011) with a Jones-Taylor-Thornton model and a maximal likelihood method. Two sequences from each phylogenetic tree constructed based on HsRbk, HsPrs or HsNampt were randomly chosen, and six sequences from each phylogenetic tree generated from EcRbk, PcPrs or MrNampt were randomly selected. Thus, there were 30 NMN pathway enzyme sequences in total. In addition, a 12-amino acid linker and a 16-amino acid “GFP11” tag were added to the end of all the 30 enzymes. To do this, the following encoded amino acid sequence “GSDGGSGGGSTSRDHMVLHEYVNAAGIT” was added directly at the end of the coding gene sequence before the stop codon. These constructed sequences were then codon-optimized for expression inE. coli , synthesized, and cloned into pET-28a vector at the Nde I and Xho I sites by the synthesis company (Generay, Shanghai, China) to generate expression plasmids (Figure S1). These expression plasmids were used for both in vivo and in vitroexpression of proteins.
For the preparation of the GFP1-10 fragment, the DNA fragment coding for a superfolder GFP variant with additional mutations (Cabantous & Waldo, 2006) was synthesized and cloned into pET-28a vector at the Nde I and BamH I sites by the synthesis company (Generay, Shanghai, China) to generate plasmid pET28a-GFP1-10. To prepare pyrophosphatase from E. coli (EcPPase), a DNA fragment encoding EcPPase was amplified from the genomic DNA ofE. coli BL21(DE3) using primers 5’-GGTGCCGCGCGGCAGCCATATGAGCTTACTCAACGTCCC-3’ and 5’-TGGTGGTGGTGGTGGTGCTCGAGTTATTTATTCTTTGCGCGCT-3’. The PCR fragment was then mixed with pET28a backbone digested with the restriction enzymesNde I and Xho I along with reagents for Gibson assembly to yield plasmid pET28a-EcPPase. All of the plasmids used in this study are listed in Table S1. Amino acid sequences of all the proteins used in this study are available in Online Supporting Information.
2.2 Cell extract preparation
Cell extracts were prepared using previously described methods with modifications (Karim & Jewett, 2016; Levine et al., 2019). E. coli Rosetta(DE3) cells were grown in 2 × YTPG media (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 7 g/L potassium phosphate monobasic, 3 g/L potassium phosphate dibasic, 18 g/L glucose). These cells were firstly cultured at the 50 mL scale in 250 mL shake flasks overnight, and then an appropriate amount of overnight culture was inoculated into 1 L of 2 × YTPG media to begin the 1 L culture at a 0.1 OD600. The inoculated 1 L culture was placed into 37 °C incubator with shaking at 200 rpm. When cells reached OD600 = 0.6–0.8, the cultures were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). When induction cultures were grown to OD600 = 3.0, the cells were harvested by centrifugation at 5,000 g at 4 °C for 10 min and were washed three times with cold S30 buffer (10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate and 2 mM dithiothreitol (DTT) ). After final wash and centrifugation, the pelleted wet cells were weighed, flash frozen in liquid nitrogen, and stored at −80 °C. To generate crude extracts, cell pellets were thawed on ice, suspended in S30 buffer (1 mL per gram cell pellet), and lysed at 20,000 psi (homogenizing pressure) using an OS Cell Disrupter (Constant Systems Limited, Northants, UK). The lysate was then centrifuged twice at 12,000 g at 4 °C for 30 min. The supernatant (i.e., lysate) was transferred to a new container without disturbing the pellet and flash-frozen in liquid nitrogen for storage at −80 °C.
2.3 Cell-free protein synthesis reactions
All CFPS reactions used a modified PANOx-SP formula described in previous pubications with modifications (Jewett & Swartz, 2004; Levine et al., 2019). A 15 μL CFPS reaction in a 1.5 mL microcentrifuge tube was prepared by mixing the following components: ATP (1.8 mM); GTP, UTP, and CTP (1.3 mM each); folinic acid (0.1mM); oxalic acid (4 mM),E. colitRNA mixture (260 μg/mL); 20 standard amino acids (2 mM each); NAD (0.4 mM); coenzyme A (0.27 mM); phosphoenolpyruvate (PEP; 33 mM); spermidine (1.5 mM); putrescine (1 mM); potassium glutamate (130 mM); magnesium glutamate (10 mM); HEPES (57 mM), and cell extract (10 μL). For each reaction plasmid was added at 4 nM. Reactions were incubated at 30 °C for 16 h.
2.4 Preparation of GFP1-10 detector fragment
GFP1-10 detector fragment was produced and purified from the inclusion body fraction as previously described with some modifications (Knapp et al., 2017). Briefly, E. coli BL21(DE3) harboring pET28a-GFP1-10 was grown in 1 L of Luria-Bertani (LB) media at 37 °C (200 rpm). Expression of GFP1-10 detector fragment was induced at OD600 = 0.6 by adding of 1 mM IPTG, and cells were harvested by centrifugation after an additional 5 h of cultivation. The cell pellets were suspended in 15 mL of TNG buffer (100 mM Tris-HCl pH 7.4, 100 mM NaCl, 10% (v/v) glycerol), lysed via pressure homogenization with one pass at 20,000 psi, and centrifuged at 12,000 g for 30 min. The supernatant was discarded and pellets were resuspended in 10 mL of TNG buffer, sonicated for 15 min and again centrifuged to sediment cell debris and inclusion bodies containing GFP1-10 detector fragment. This procedure was repeated twice. The resulting pellet, which contained mainly inclusion bodies pellet, was weighed and dissolved in 9 M urea solution (1 mL for each 75 mg of inclusion bodies). After a centrifugation step at 12,000 g for 30 min , the resulting supernatant was divided into 1 mL aliquots, and each aliquot was diluted by adding 25 mL of TNG buffer. The final GFP1–10 detector fragment solution was stored at −80 °C until use.
2.5 Monitoring proteinproduction in CFPS by the split GFP assay
Unless otherwise noted, expression of GFP11 fusion enzymes in CFPS was monitored by mixing 5 μL of sample (enzyme-enriched CFPS cell lysates) with 195 μLof detector solution in a 96-well plate and incubation at 4 °C for 8 h to support the formation of fluorescent GFP protein. To investigate the sensitivity and accuracy of the assay, HsRbk-LG was expressed in E. coli BL21(DE3) and purified as described below. The purified HsRbk-LG was diluted in TNG buffer at several different concentrations between the range of 0.5 μM and 8 μM. 5 μL of the respective HsRbk-LG dilution was mixed with 195 μLof detector solution in a 96-well plate. Once both solutions were mixed, the 96-well plate was stored at 4 °C, and the fluorescence signals were measured at different time points. In all cases, fluorescence was measured in the microplate reader (Infinite M200, Tecan Austria GmbH) with the wavelength of excitation at 488 nm and emission at 520 nm. The complementation fluorescence (∆F) was calculated using Equation (1) described in Online Supporting Information.
2.6 Protein in vivoexpression and purification
The plasmids used to express the desired enzyme genes were transformed into E. coli BL21(DE3) by the chemical thermal shock method. Single colonies were picked from agar plates containing 50 μg/mL kanamycin and then were inoculated into 10 mL of LB medium containing the same antibiotic to produce the first culture. The first culture was incubated at 37 °C in a shaker at 200 rpm overnight for 13−16 h. The appropriate amount of overnight culture was inoculated into LB media containing the same antibiotic to begin the 100 mL culture at a 0.1 OD600. The inoculated 100 mL culture was grown under the same culture conditions. When the OD600 reached 0.6−0.8, enzyme expression was induced by adding 0.2 mM IPTG and the culture was then incubated at 16 °C and 180 rpm for 16 h. The cells were collected by centrifugation at 5,000 g at room temperature for 10 min and were washed two times with binding buffer (20 mM Tris–HCl, 0.1 M NaCl, pH 7.5). After final wash and centrifugation, the pelleted wet cells were suspended in 10 mL of binding buffer and disrupted by sonication. The lysate was then centrifuged at 10,000 g and 4 °C for 30 min, and the supernatant, which contained the crude protein, was loaded onto a Ni-NTA His-Bind Resin. The protein was eluted with elution buffer (20 mM Tris–HCl, 0.1 M NaCl, 0.25 M imidazole, pH 8.0), and then the desired protein was collected. The protein concentration was measured with the Bio-Rad Bradford protein kit with bovine serum albumin (BSA) as the standard.
2.7 NMN synthesis in CFPS-ME reactions andpurified enzyme systemreactions
All NMN synthesis reactions were carried out at a volume of 100 μL in 96-well plates. To produce NMN in CFPS-ME reactions, NMN pathway enzymes were expressed in CFPS reactions as described in Section 2.3 . When CFPS reactions were complete, the enzyme-enriched CFPS cell lysates were mixed with desired substrates and cofactors to activate NMN synthesis. “Blank” CFPS reactions with no DNA added were used as control. See Table S2 for a more detailed description of the reaction components. To produce NMN in purified enzyme system reactions, purified NMN pathway enzymes were obtained as described in Section 2.6 and then mixed with desired substrates and cofactors to activate NMN synthesis. See Table S3 for a more detailed description of the reaction conditions. Unless otherwise noted, NMN synthesis reactions were incubated at 40 °C for 3 h. After reactions were finished, the concentrations of NMN in samples were analyzed immediately.
2.8 NMN measurement
The concentrations of NMN in samples were analyzed using a validated fluorometric assay method with some modifications (Marinescu et al., 2018; Shoji et al., 2021; Zhang et al., 2011). Assays were performed in 96-well plates with a 90 µL final volume per well, consisting of 25 µL of sample, 10 µL of 20% (v/v) acetophenone in dimethyl sulfoxide (DMSO), and 10 µL of 2 M KOH. The mixture was incubated on ice for 2 min before adding 45 µL of 88% formic acid to each well. After incubation at 37 °C for 10 min, 60 µL of the mixture in each well was transferred into a flat-bottom 96-well black plate. The fluorescence was measured on a microplate reader (Infinite M200, Tecan Austria GmbH) with the following settings: excitation wavelength 382nm and emission wavelength 445nm. The concentration of NMN was calculated from the fluorometric assay standard curve (Figure S2), which was created from the fluorescence data of standard NMN (Sigma N3501-25MG) samples in series concentrations.