FIGURE 3 Monitoring
protein production in CFPS by
the
split GFP
assay.
(a) Schematic representation of the workflow for monitoring protein
production in CFPS by the split GFP assay. (b) Producing six NMN pathway
enzymes by CFPS and monitoring their expression levels by the split GFP
assay. BC: Blank CFPS reaction; NW: Nanopure water. Statistical
significance was examined using a two-tailed T-test analysis. ***:P
< 0.001.
Error
bars represent the standard deviation of three biological replicates.
(c) Sensitivity of the split GFP assay. The correlation coefficients
between
complementation
fluorescence value and the amount of HsRbk-LG were
0.9986,
0.9991, 0.9992, 0.9987, and 0.9977 for incubating for a period of 8 h,
10 h, 12 h, 14 h, and 16 h, respectively. Error bars represent the
standard deviation of three biological replicates.
3.3
Prototyping enzyme homologs for NMN biosynthesis by using a normalized
screening procedure
After designing a feasible biosynthetic pathway for NMN production and
demonstrating
the
ability to monitor CFPS via split GFP assay, a normalized screening
procedure, which incorporated these two technologies, was employed to
rapidly prototype
enzyme
homologs (Figure 1b). Briefly, enzyme homologs were parallelly expressed
by CFPS. For each homolog, its CFPS reaction mixture was divided into
two
parts: one part was used to catalyze the conversion of the desired
substrate to
product,
and the other part was mixed with GFP 1-10 detector to regenerate the
fluorescent GFP signal for monitoring its expression
level.
The ratio
(RT/F)
between the final titer of the
product
and the fluorescence
value
was calculated for each individual homolog. Then the normalized
screening was performed by comparing the RT/F values
based on a criterion, in which the homolog with the highest
RT/F value was regarded as the most productive homolog.
To identify the putative enzyme homologs in NMN biosynthetic pathway,
the amino acid sequences of HsNampt, MrNampt, HsPrs, PcPrs, HsRbk and
EcRbk were used as
query
sequences to perform BLAST analysis against the Universal Protein
Resource (UniProt) or National Center for Biotechnology Information
database (NCBI) database. For a particular query sequence, some
resulting fallacious sequences were
discarded, and then the left sequences were used to generate a
phylogenetic tree. Thus, six phylogenetic trees were obtained in total
(Figure 4). Notably, since the prokaryotic
NMN
pathway enzymes were found more likely to be
productive
(Figure 3c), we had a
bias
to select more sequences from the phylogenetic trees that were
constructed based on the enzymes from prokaryotes (i.e., EcRbk, PcPrs
and
MrNampt).
As a result, two sequences from each
phylogenetic
tree constructed based on HsRbk (Figure 4a), HsPrs (Figure 4c), or
HsNampt (Figure 4e), and six sequences from each phylogenetic tree
generated from EcRbk (Figure 4b), PcPrs (Figure 4d), or MrNampt (Figure
4f) were selected. In summary, there were 10 homologs for each enzyme of
the three-enzyme NMN pathway, which included two query homologs and
eight homologs chosen from phylogenetic trees. The 30 enzyme sequences
were all cloned into the pET28a-derived expression plasmid (Figure S1)
for adding the linker and GFP11 tag.