Articles
Nature ChemiCal Biology
NMNH concentration was measured by comparing absorbance at 340nm to a
were held at 250 and 260°C, respectively. The GC was operated in constant
pressure mode with a pressure of 3.66psi. Helium was used as the carrier gas. Air
standard curve of NADPH.
−1
and hydrogen were supplied to the FID at 350 and 40mlmin , respectively. All
gasses were purchased from Airgas and 5μl of sample was injected with a split ratio
of 2:1.
XenA activity assays. The enzymatic buffer contained 200mM phosphate
buffer (pH7.5), 5mM ketoisophorone, 0.2mM NADPH, NADH or NMNH,
−
1
and 8μgml purified XenA protein. The initial reaction rate was quantified by
absorbance variation at 340nm using a spectrophotometer at 37°C. Reactions with
no ketoisophorone added were performed to quantify the nonspecific reaction
rate. The net reaction rate was calculated by subtracting the nonspecific reaction
For analysis of citral and its reduction product citronellal, the oven was initially
−1
held at 150°C for 10min and then ramped at a rate of 45°Cmin to 240°C. Citral
and citronellal eluted at 9.32 and 4.50min, respectively. Octanol was used as an
internal standard.
rate from the total reaction rate. For generating NMNH, a microcentrifuge tube
For analysis of trans-2-hexen-1-al and its reduction product trans-2-hexan-1-al,
+
−1
(
2ml) containing 35mM Tris-HCl (pH8.0), 4mM NMN , 1M NaCl and 140mM
the oven was initially held at 50°C for 1min, the oven was ramped at 15°Cmin
−1
glucose, was incubated for ~3h at 30°C with purified Bs GDH I195R. An aliquot of
this reaction system was taken to measure the accumulation of NMNH by reading
absorbance at 340nm. The mixture was then filtered through an Amicon Ultra 3K
filter (Millipore) to remove the protein. The NMNH concentration was measured
by comparing absorbance at 340nm to a standard curve of NADPH. The solvent
was evaporated, and the residue was collected to give NMNH as a yellowish oil.
to 120°C, then ramped at 20°Cmin to 230°C and held for 3min. Trans-2-hexen-
1-al and trans-2-hexan-1-al eluted at 6.41 and 4.78min, respectively. Octanol was
used as an internal standard.
For analysis of 4-phenyl-3-butyn-2-one (containing C≡C triple bond) and its
fully reduced product 4-phenyl-2-butanone, as well as the intermediate 4-phenyl-
2-butene-2-one (containing a C=C double bond), the oven was initially held at
−1
2
00°C for 1min, then ramped at 5°Cmin to 230°C and held for 1min. Octanol
P450 BM3 assay conditions. Reaction mixtures (0.2ml) contained 0.2M
potassium phosphate buffer (pH7.5), 0.3M glucose, 1M NaCl, 2mM NMNH with
was used as an internal standard. In vitro ketoisophorone reduction to levodione
was analyzed using the same method. Elution times are as follows: 4-phenyl-
3-butyne-2-one (5.53min), 4-phenyl-2-butene-2-one (6.76min), 4-phenyl-2-
butanone (4.55min), ketoisophorone (3.65min), levodione (4.08min) and octanol
(2.80min).
5
0
0μM cytochrome c (C2506, Sigma). Reactions were initiated by the addition of
−1
.75mgml purified BM3 variants. Reduction of cytochrome c was monitored
spectroscopically at 550nm. Quantification was performed using an extinction
−
1
−1
22
coefficient ε550 of 21.1mM cm (ref. ). NMNH was generated as described
For analysis of in vivo ketoisophorone biostransformation, the oven was held at
200°C for 15min. Elution times are as follows: octanol (2.80min), ketoisophorone
3.76min), levodione (7.25min), phorenol (8.35min) and 4-hydroxyisophorone
above for XenA activity assays.
(
Coupled enzymatic biotransformation. All biotransformation reactions were
(12.90min).
performed in buffer A at 30°C for 24h. Buffer A, modified from a previous
1
9
system , contained 200mM potassium phosphate buffer (pH7.5), 1M NaCl,
GDH TTN determination. TTN (Fig. 1g) was determined by the number of
moles of product formed divided by the moles of purified GDH added. The
assays were performed in reaction buffer A, as shown above, at 30°C and 33mM
ketoisophorone was used as the substrate. The reaction was started by spiking
+
+
3
00mM ꢅ-glucose, 6mM NMN (or NADP as the positive control), and
substrates. All assays were performed in triplicate, with no proteins or no cofactors
added as negative controls. The protein loading for GDH variants was kept at
−
1
−1
−1
0
.33mgml or 11.7μM. The various enzymes for biotransformation reactions
purified proteins (0.0132mgml or 0.47μM for GDH, 0.75mgml for XenA).
Samples were taken every 12h for 96h. The extraction and GC–FID analysis were
performed using a similar method to that mentioned above.
−
1
were added at the concentration of 0.75mgml .
For XenA–Bs GDH coupled cycling assays, the substrates ketoisophorone, citral,
or trans-2-hexen-1-al were added at 33, 10 or 50mM, respectively. For the OYE3-
GDH coupled cycling assays, the substrate 4-phenyl-3-butyn-2-one was added at
23
+
+
Quantification of intracellular NMN and NAD levels. A plasmid containing
Ft nadE and nadV (pWB203) was transformed into E. coli strains BW25113,
5
mM. At various time points over 24h, 100μl samples were taken and extracted
+
with 100μl ethyl acetate. Conversion was determined via gas chromatography–
flame ionization detection (GC–FID) with octanol as an internal standard (see
below). For NfsB-Bs GDH coupled cycling assays, 2mM nitrofurazone was used as
JW2670-1 and MX101 to examine their effects on NMN generation. Overnight
cultures were grown at 30°C while shaking at 250r.p.m. in 2xYT media containing
0.1mM IPTG, 0.2% ꢅ-glucose and appropriate antibiotics for 12h. To cultivate
cells for nucleotide analysis, 10ml of 2xYT media containing 0.5mM IPTG, 1mM
nicotinamide and appropriate antibiotics in a 50ml conical tube was inoculated
with 1% v/v overnight culture. Tubes were incubated at 30°C at 250r.p.m. for 4h.
Before gathering cells, cell density was measured at 600nm and 1ml of
24
the substrate . Nitrofurazone conversion was measured spectroscopically at 400nm
24
and quantification was performed using a standard curve . The initial levels added
of the above-mentioned substrates were mainly determined by their solubility in
the assay buffer. For P450 BM3-Bs GDH coupled cycling assays, 50μM cytochrome
c (C2506, Sigma) was used as the substrate. The reduction of cytochrome c was
measured spectroscopically at 550nm, and the quantification was performed using
culture was pelleted in a 1.5ml microcentrifuge tube. Supernatant was removed
by pipetting. The cell pellet was washed once with 1ml of room temperature
deionized water, repelleted, and the supernatant was removed by pipetting. Cells
were lysed with 1ml of 95°C 1% formic acid with 1μM 1-methylnicotinamide as
an internal standard. Cells were incubated at 95°C for 2min while intermittently
vortexing to ensure complete lysis. Lysates were quenched in an ice water bath
before pelleting cell debris. Supernatant was run on an ultrahigh-performance–
tandem mass spectrometry (UPLC–MS/MS) system for analysis. The UPLC–MS/
MS method is detailed in the Supplementary Information. Values from liquid
chromatography (LC)–MS/MS were correlated back to intracellular concentration
−1
−1
22
extinction coefficient ε550 of 21.1mM cm (ref. ).
+
NMN -dependent whole-cell biotransformation. One biotransformation plasmid
expressing XenA, LVR or ADH with a GDH (selected from pLZ217-pLZ225)
and pSM104 containing the glucose transport facilitator were transformed into
strain MX102 by electroporation. Then, 4ml seed cultures of 2xYT media with
appropriate antibiotics, 0.1mM IPTG and 0.2% (w/v) glucose were cultured at
3
0°C while shaking at 250r.p.m. for 16h. Next, 0.5% (v/v) seed cultures were used
9
to inoculate 150ml of 2xYT media with appropriate antibiotics, A5 trace metals
with cobalt and 0.5mM IPTG in a 250ml baffled shake flask and cultured at
using the number of cells per OD of 1 in 1ml of culture=1×10 and the
6
00
−15
56
intracellular volume of an E. coli cell as 1×10 l per cell .
30°C at 250r.p.m. When an optical density (OD600) of ~0.4 was reached, protein
Liquid chromatography was performed on a Waters ACQUITY UPLC with a
Waters ACQUITY UPLC CSH C18 column (1.7μm×2.1mm×50mm). Mobile
phases used in the separation were (A) water with 2% acetonitrile and 0.2% acetic
acid and (B) acetonitrile with 0.2% acetic acid. The compounds were separated
with a linear gradient from 10 to 90% buffer B over 1min, held at 90% buffer B for
1min, then returned to 10% buffer B and held at 10% buffer B for 1min. Flow rate
was held constant at 0.3mlmin− and 10μl of sample was injected for analysis.
MS/MS detection was performed by a Waters Micromass Quattro Premier
XE Mass Spectrometer operating in positive ion, MRM mode. Capillary voltage
expression was induced with 0.1% (w/v) arabinose, and the cells were cultured
for an additional 8h at 30°C while shaking at 250r.p.m. Cells were collected by
centrifugation for 15min at 20°C at 3,750r.p.m. The supernatant was discarded.
Cells were washed three times with 50ml of 100mM potassium phosphate
(
pH7.5), followed by being resuspended to an OD600 of 100 in assay buffer
1
consisting of 100mM potassium phosphate buffer (pH7.5), 200mM ꢅ-glucose,
0
2
.5% arabinose, and 0.5mM IPTG. Then 1ml of resuspended cells were added to
0ml of identical assay buffer in a 250ml unbaffled, screw-cap shake flask. KIP was
−1
−1
spiked into the flask to 5gl to initiate the reaction. Flask caps were secured tightly
to prevent evaporative loss of substrate or products. Flasks were incubated at 30°C
while shaking at 250r.p.m for 48h. After 48h, 1ml of culture was pelleted, and
the supernatant was used for analysis. 200µl of supernatant was extracted with an
was set to 3.3kV. Desolvation gas flow rate was 800lh at 300°C. Cone gas flow
−1
rate was 50lh . The source was maintained at 120°C. Primary mass, fragment
mass, cone voltages and collision energies for each compound are listed in
Supplementary Table 5.
−1
equal volume of ethyl acetate containing 200mgl octanol as an internal standard,
and the samples were analyzed by GC (see detailed method below). For samples
expressing all three conversion enzymes (XenA, LVR and ADH) on the same vector
+
Supporting E. coli growth with NMN -dependent glycolysis. E. coli strain
with ΔpgiΔzwfΔgnd knockout (strain MX103, Supplementary Table 3) cannot
metabolize glucose. The strain also has ΔnadRΔpncC knockouts to potentially
(
pLZ226), the Bs GDH was expressed individually on a separate vector (pSM106,
+
29
pSM107, or pSM108). The Zm glf gene was also expressed in this system (pSM110).
preserve intracellular NMN (ref. ). The strain was transformed with a plasmid
(
pSM103) containing F. tularens nadE and nadV, Z. mobilis glf and R. eutropha
GC–FID analytical methods. All GC analysis was performed on an Agilent 6850
gntK in a synthetic operon. In addition, the strain was also transformed with one
of the three plasmids: pLZ214 (XenA alone), pSM106 (Bs GDH Ortho alone) or
pLZ215 (Bs GDH Ortho with XenA).
(
Agilent Technologies) equipped with an FID. An Agilent DB-WAXetr capillary
column (30m×0.56mm×1μm) was used for separation. The inlet and detector