FIGURE 2 Design and selection of NMN biosynthetic pathways. (a)
Design of three pathways for
NMN
biosynthesis. (b) Standard Gibbs free energy changes for NMN
biosynthesis pathways. (c) Using 8 pathway combinations to produce NMN.
HPM, HsRbk + PcPrs + MrNampt; EPM,
EcRbk
+ PcPrs + MrNampt; HHM, HsRbk + HsPrs + MrNampt; EHM, EcRbk + HsPrs +
MrNampt; HPH,
HsRbk
+ PcPrs + HsNampt; EPH, EcRbk + PcPrs + HsNampt; HHH, HsRbk + HsPrs +
HsNampt; EHH, EcRbk + HsPrs + HsNampt. See Table S2 for details. Error
bars represent the standard deviation of three biological replicates.
Adk, adenosine kinase; Apt, adenine phosphoribosyltransferase; Rbk,
ribokinase; Prs, phosphoribosyl pyrophosphate synthetase; Nampt,
nicotinamide phosphoribosyltransferase. PPi, pyrophosphate.
3.2 Adaptation of split GFP assay to monitor protein expression
in CFPS
The protein production of CFPS was usually monitored and quantified by
using radioactive amino acid incorporation (Jewett & Swartz, 2004;
Karim & Jewett, 2018), which allowed researchers to control and assess
pathway performance in a precise manner in the CFPS-ME framework (Grubbe
et al., 2020; Karim et al., 2020; Rasor et al., 2022). However,
radioactive
incorporation was unavailable for many laboratories and its procedure
was laborious. To overcome the limitations of radioactive incorporation,
we
adapted
a split GFP assay to monitor CFPS protein production . In brief, a
12-amino acid linker and a 16-amino acid GFP-derived peptide
tag
(GFP11) were cloned to the C terminus of the gene encoding the target
protein. The fusion protein was produced via CFPS, while a truncated,
non-fluorescing detector GFP protein (GFP1-10) was produced separately.
Combining the CFPS cell lysates which
contained
the GFP11-tagged protein with the detector for a short time would elicit
a quantifiable fluorescent signal. Hence, protein production of CFPS
could be monitored simply by fluorescence
determination
(Figure 3a).
As a proof-of-principle, the split GFP assay was applied to monitor the
expression levels of 6 enzymes included in the initial enzyme sets for
NMN synthesis. An 84 nucleotide sequence encoding the 12-amino acid
linker and 16-amino acid “GFP11” tag was added directly at the end of
the coding sequence of Hs/MrNampt, Hs/PcPrs, and Hs/EcRbk before the
stop codon. The constructed sequences were cloned into the pET-28a
vector at the Nde I and Xho I sites (Figure S1) by the
synthesis company. CFPS reactions were performed by using these enzyme
expression plasmids as DNA templates and incubated for 16 h at 30°C.
When the CFPS reactions were complete, the enzyme-enriched CFPS cell
lysates were mixed
and
incubated with a GFP1-10
detector
solution.
Compared
to the “Blank” CFPS reaction (using water instead of expression
template), a combination of six
enzyme-enriched
cell lysates and the GFP1-10 detector all emitted significant
fluorescence as expected. More importantly, the
fluorescence
was directly proportional to the amount of added enzyme-enriched cell
lysates (Figure 3b).
To
further assess the sensitivity and accuracy of the assay,
serial
dilutions of purified GFP11-tagged HsRbk (HsRbk-LG) were prepared, and
each dilution was then added to the GFP1-10 detector solution to
regenerate GFP fluorescence. As shown in Figure
3c,
there always was a strong correlation
(R2>0.99) between the complementation
fluorescence and the amount of HsRbk at different incubation time points
(ranging from 8 to 16h). In addition, the amount of detected protein
could reach the picomolar level, which indicated that this approach was
highly sensitive and
accurate.
Taken together, these results suggested that the split GFP assay allowed
to
monitor protein expression in CFPS in a convenient and sensitive way.