Biocatalytic synthesis of 2′‐deoxynucleotide 5′‐triphosphates from bacterial genomic DNA: Proof of principle

Abstract 2′‐deoxynucleoside 5′‐triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom‐up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low‐ and middle‐income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top‐down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in‐house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid‐state bioreactor for on‐demand dNTP production.


Enzyme Kinetics Dependence on Magnesium Concentration
Magnesium is a co-factor which is necessary for the activity of nucleotide kinase enzymes.The dependence of adenylate kinase reaction velocity on magnesium concentration was tested as described in the body of the text with the pyruvate kinase-lactate dehydrogenase coupled spectrophotometric assay.The final reaction buffer consisted of 50 mM potassium acetate, 20 mM Tris-acetate, 100 ug/mL BSA, 3 mM PEP, 2 mM ATP, 0.3 mM NADH, 1.25 U/mL pyruvate kinase, 2.25 U/mL lactate dehydrogenase, 0.0025 mg/mL adenylate kinase, 300 μM dAMP, and varied concentrations of magnesium acetate ranging from 1 -20 mM.Solutions were brought up to 37°C before adding the substrate.Absorbance at 340 nm was read immediately after addition of substrate and followed for 5-10 minutes 1 .
The Vmax, app in these reaction conditions at 300 μM dAMP was found to be 18.96 ± 3.52 μmole/min/mg protein, which is consistent with the Vo for 300 uM dAMP calculated using the experimentally determined Vmax and KM of adenylate kinase.The KM, app for Mg 2+ was found to be 5.26 ± 1.21 mM, which is strikingly similar to the dissociation constant of Mg 2+ •AK (Kd = 4.0 ± 1.5 mM) reported by Tan et al. 2 The working concentration of magnesium acetate in gDNA digestion reactions and in-house dNTP synthesis reactions was 10 μM, which is above the KM Mg2+ , and thus does not limit the reaction velocity.However, other ions, such as potassium, are also reported to have an effect on enzyme activation and activity.Oeschger found that K + enhances the Vmax of guanylate kinase while greatly reduced the KM, and therefore increased affinity of the protein for dGMP 3 .However, the buffer used in the dNTP synthesis reactions is optimized for 100% activity of the DNA digesting enzymes.Although the activity of these nucleotide kinases is reduced in this buffer relative to previously published values, it is still sufficient to complete the conversion of dNMPs to dNTPs.According to the NanoDrop user manual, the protein concentration can be calculated as

Buffer Composition of Enzyme Kinetics Reactions
Where C is the concentration in mg/mL, A280 is the absorbance at 280 nm, 10 is a correction factor to convert from g/100 mL to mg/mL, and ε1% is the percent extinction coefficient and is equal to where εmolar is the molar extinction coefficient.
Enzyme kinetics experiments were initially conducted using the assumption 1 absorbance at A280 = 1 mg/mL and was later corrected as detailed in Table S5.Where V is the maximum velocity, C is the concentration of the protein in the reaction solution in mg/mL, 0.2 is the volume of the reaction in mL, and 60 is the conversion factor between seconds and minutes.

Lineweaver Burk Plots -NMP Kinases
Enzyme kinetics parameters were calculated using both nonlinear regression curve fit with the Michaelis Menten equation and linear regression curve fit with Lineweaver Burk equation.The Lineweaver Burk plot often compound or distort errors in the data, and thus are deemed less accurate for calculating kinetic parameters than a direct curve fit with Michaelis Menten.For this reason, we have presented the parameters calculated from Michaelis Menten in the body of the text.However, we have also included a summary of the Lineweaver Burk analysis below, whose results are consistent with the nonlinear regression parameters.
Figure S3.Lineweaver-Burk plots of inter-batch variation of NMP kinase activity.
Kinetic parameters, maximum velocity V in μmol/s and affinity constant KM, are calculated from the slope and yintercept outputs from linear regression of Lineweaver-Burk plots as described in the equations below.The specific activity (Vmax) is calculated as described previously.The protein concentrations for these experiments were quantified using NanoDrop A280 nm readings.

Kinetic Parameter Comparison
As expected, inter-batch variation is smaller than batch to batch variation.Regardless of protein quantitation with NanoDrop or Bradford assay and protein batch, kinetic parameters are on the same order of magnitude.Kinetic parameters calculated by Lineweaver Burk or by Michaelis-Menten give similar values.Values reported in the body of the text are from nonlinear regression with the Michaelis-Menten curve, as it is deemed more accurate due to the high weighting Lineweaver-Burk places on substrate concentrations below the KM.
Figure S6.Comparison of specific activity and KM values calculated for the proteins using different regression methods.Single batch refers to one batch of protein performed in triplicate.Single batch protein concentration was quantified using A280 nm and protein coextinction coefficients.Batch to batch refers to three independent batches of protein each performed in a single replicate.Batch to batch protein concentrations were quantified using Bradford assay.The difference between the single batch specific activity reported in the body of the text and the batch-to-batch specific activity presented here is largely due to the difference in protein quantitation method.The single batch protein concentration was quantified using A280 nm and coextinction coefficients, and the batch-to-batch protein concentration was quantified using Bradford assay.While the A280 nm and Bradford concentrations were consistent for the NMP kinases, there appears to be a discrepancy between the quantitation methods for NDK.The pure protein specific activity is paired with the corresponding batch of unpurified lysate from which it was purified.The pure protein specific activity is calculated from the nonlinear regression curve fits in Figure 4, and the lysate specific activity is calculated from the nonlinear regression curve fits in Figure S9.

Kinetics of Unpurified Kinase Lysate
Negative Control BL21(DE3) Lysate Kinetics The kinetics assay follows the absorbance of NADH at 340 nm over time.The double coupled pyruvate kinaselactate dehydrogenase consumes NADH at a 1:1 molar ratio with the amount of product produced by the nucleotide kinase.A calibration curve of molar NADH standards is used to calculate the moles of NADH at each time point, then the moles of NADH consumed, and finally the reaction velocity for each substrate concentration.Given that the BL21(DE3) negative control samples showed minimal variation in reaction velocity for different substrate concentrations (apart from dADP substrate), no kinetics analysis was performed on the negative control samples.

SDS-PAGE Image Analysis
To quantify protein purity from SDS-PAGE images, the image was imported into ImageJ and the image was black/white inverted.A rectangular region of interest (ROI) was drawn around the entirety of the induced protein lane (Figure 4).The lane intensity was quantified with the ImageJ Integrated Density function.An ROI of the same size was drawn around the purified protein lane, and the integrated density was again measured.This process was repeated with a smaller ROI to quantify the intensity of the kinase band in both the induced and purified lanes.The relative purity was calculated by dividing the integrated density ratio of kinase band : total lane for the purified lane by the integrated density ratio of kinase band : total lane for the induced lane.Unlike Fehlau et al., the dCTP concentration cannot be deduced from the remaining concentration of dCMP and dCDP because the dCDP peak is also not well resolved from other contaminants and nucleotides in our reaction mix.

HPLC ADP and ATP Elution Time
In some cases, the ADP and dCTP peaks could be resolved.In other cases, a distinct shoulder was observed on the ADP peak, and the dCTP concentration was calculated using OriginLab peak splitting algorithms as shown in Figure S14.The algorithm identifies hidden peaks by calculating the second derivative to determine local extrema in the data.
Figure S16.Second derivative hidden peak finder algorithm applied to HPLC spectrum of in-house dNTPs.In-house dNTPs synthesized from a 60 μg gDNA digest with Benzonase and Exonuclease III, followed by incubation with kinase enzymes and ATP at 37°C for 30 min.In-house dNTPs precipitated with EtOH and resuspended in 562.5 uL water (30x dilution factor) before loading on HPLC.
Lambda DNA PCR

Effect of EtOH Precipitation on Synthesized dNTPs
Precipitation of dNTPs with ethanol (EtOH) allows the dNTPs to resuspended in a smaller volume of water, and thus are more concentrated, as indicated by the increased band brightness in lane 3, relative to lanes 1 and 2 in Figure S18.

PCR Yield
A PCR reaction was performed with 3 kb lambda genome primers using either 200 µM commercial dNTPs, 150 µM commercial dNTPs, or in-house dNTPs (estimated to have a concentration of approximately 150 µM), following the protocols described in the main body of the text.After thermocycling, the PCR reaction was diluted 1:400 and the yield was quantified with PicoGreen.The concentration of DNA was determined from a calibration curve with Lambda DNA.In-house dNTP samples were quantified with PicoGreen prior to the PCR reaction and blank subtracted from the final PCR concentration to account for the contribution of undigested DNA fragments toward background fluorescence.There were no statistically significant differences observed between any of the groups when using a Kruskal-Wallis test with multiple comparisons.

E. coli genome PCR
A PCR was performed using primers to detect fragments of the E. coli genome to determine if contaminating DNA fragments in the in-house dNTPs produce false positive results.A PCR reaction mix was prepared as described in Table S9, using the adk forward and reverse primers listed in Table S2. 1 µL of 10 ng/µL adk-His-pET24a plasmid DNA was used in place of Lambda DNA as the template.For negative controls, 1 µL of water was added in place of the template.30 cycles of PCR were performed with a 50°C annealing temperature and 40 s extension time.
To remove undigested DNA fragments, the dNTP reaction solution was added to a PCR clean-up column (NEB) with 350 µL of binding buffer immediately following incubation with kinases.The column was spun for 1 minute at 13000 rpm.The silica column was discarded, and the filtrate (total volume ~400 µL) was collected.The PCR cleanup kit used with a 7:1 binding buffer to sample ratio is optimized to remove any single stranded DNA larger than 200 basepairs.The silica column was thus expected to bind the undigested DNA fragments, while the dNTPs should pass into the flow through.The dNTPs were then recovered by ethanol precipitation.37 µL of 3 M NaCl and 3 vols (1200 µL) of ice cold 100% ethanol were added to the PCR clean-up column flow-through.The ethanol solution was incubated 1 hour at -80C, then centrifuged 20 min at 4C.The supernatant was discarded, and the formed pellet was air dried for 30 min.The pellet was then resuspended in 18.75 µL of water and used immediately in the PCR reaction.
PCR using in-house dNTPs to target the E. coli genome resulted in false positive results.Attempts to purify the dNTPs using a PCR clean-up column resulted in failed PCRs for the positive control samples.The dNTPs were thus unable to be recovered from the column flow through.

Increased Digestion Enzyme Concentration
Increasing the amount of digestion enzyme does not improve the yield of dNTPs or the ability to perform larger fragment PCR reactions.dNTP Longevity dNTPs are stable for one week when stored in precipitated form at 4C. dNTPs show moderate stability after one week stored in precipitated from at room temperature (RT).
Figure S22.PCR of 2 kb lambda DNA fragment with in-house dNTPs stored under different conditions.Following dNTP synthesis, the dNTPs were precipitated with ethanol, then air dried.Fresh samples were resuspended in 18.75 μL nuclease free water and used immediately in PCR.Other samples were precipitated and air dried, then stored for 1 week at either 4°C or at RT. Lanes 1 -3: dNTPs prepared fresh and applied immediately in PCR after resuspension in nuclease free water.Lanes 4 -6: dNTPs stored in precipitated form at 4C for 1 week before resuspension in nuclease free water and application in PCR.Lanes 7 -9: dNTPs stored in precipitate form at RT for 1 week before resuspension in nuclease free water and application in PCR.L: 1 kb plus ladder (NEB).

Figure S2 .
Figure S2.Adenylate kinase reaction velocity dependence on magnesium acetate concentration.Dashed line indicates the working concentration of magnesium used in all other enzyme kinetics experiments, gDNA digest, and dNTP synthesis reactions.

Figure
Figure S5.Lineweaver Burk plots of independent batches of NMP kinase proteins (n=3).Protein concentration quantified with Bradford assay.

Figure S7 .
Figure S7.Lineweaver Burk plots of inter-batch variation of NDK activity toward the four target dNDP substrates.Protein concentration quantified with NanoDrop.

Figure S12 .
Figure S12.Variation in elution time of dNMP standards on HPLC (n = 18).Box plot showing mean and standard deviation with whiskers at minimum and maximum values.

Figure S14 .
Figure S14.Variation in elution time of dNTP standards on HPLC (n = 18).Box plot showing mean and standard deviation with whiskers at minimum and maximum values.

Figure S15 .
Figure S15.Co-elution of ADP and dCTP peaks.As noted by Fehlau et al., ATP and dCTP elute at a similar time on this HPLC method and are often unable to be resolved 4 .Other methods including LC/MS and MS of HPLC fractions were attempted but also failed to resolve the compounds.Other LC-MS methods have been published but report coelution of ATP and dGTP 5 .Due to the similarity in molecular structure, charge, and weight of the nucleotides, they are difficult to separate chromatographically.

Figure S19 .
Figure S19.DNA concentration measured at the endpoint of PCR reactions with varied concentration of dNTPs.Error bars represent SEM.200 µM and 150 µM dNTP samples contain commercial dNTPs.In-house dNTPs were produced from 60 µg gDNA and concentrated with ethanol precipitation.

Figure S20 .
Figure S20.PCR with in-house dNTPs targeting the E. coli genome."+" samples contain 10 ng adk-His-pET24a plasmid as template."-" samples do not have any added template.A: in-house dNTPs synthesized from 60 ug gDNA.B: 200 uM commercial dNTPs.C: In-house dNTPs synthesized from 60 ug gDNA and purified with a PCR clean-up column.MW: 1 kb plus (NEB).

Table S3 .
Buffer composition of enzyme kinetics reactions in this study compared to previously published studies of nucleotide monophosphate kinases.

Table S4 .
Molecular properties of proteins calculated from the amino acid sequence using the Expasy Swiss Institute of Bioinformations ProtParam tool.

Table S5 .
Protein concentrations in enzyme kinetics reactions calculated from A280 nm and extinction coefficients.

Table S6 .
Specific activity values for purified protein and unpurified protein in cell lysate calculated from Michaelis-Menten nonlinear regression.

Table S7 .
Results of SDS-PAGE image analysis.

Table S8 .
Primers used to amplify lambda DNA genome in PCR ). adk -: no adenylate kinase added to dNTP reaction.cmk -: no cytidylate kinase added to dNTP reaction.gmk -: no guanylate kinase added to dNTP reaction.tmk -: no thymidylate kinase added to dNTP reaction.ndk -: no nucleotide diphosphate kinase added to dNTP reaction.pos ctrl: all NMP kinases and NDK included in dNTP reaction, 1% agarose.90V, 30 min.