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.