FIGURE
5 Identifying the most productive enzyme homolog for each step in a
step-wise manner by using
the
normalized screening. (a) Schematic representation of the workflow for
step-wise identifying the most productive enzyme homologs. (b) The most
active Nampt homolog, SuNampt, was first identified. “Mr” represents
Nampt from Meiothermus ruber , and “Mr ” represents Nampt
from Meiothermus rufus . (c) By mixing with SuNampt, the most
productive Prs homolog was proved to be MjPrs. (d) By combining with
SuNampt and Mjprs, the most active Rbk homolog (i.e., OkRbk) was
identified. The left histograms in (b), (c), and (d) show the results of
the complementation fluorescence values for Nampt homologs, Prs
homologs, and Rbk homologs, respectively. Error bars represent the
standard deviation of three biological replicates. The (×) symbols
represent that the
RT/Fvalues of these homologs were not calculated. The right histograms in
(b), (c), and (d) show the results of RT/F values of
different NMN metabolic pathway enzymes. Error bars represent propagated
error. See Table S2 for more detailed information on NMN synthesis
reactions.
3.4 Testing and optimizing NMN
biosynthesis
To confirm the results obtained using the normalized screening
procedure, the 30
homologs
were expressed in E. coli BL21(DE3), purified (Figure S4), and
tested for their activities of NMN biosynthesis. It was noticed that the
performance of our normalized screening procedure in evaluating the
homologs with middle
activity,
such as CbNampt, PcPrs, and ThRbk, was unsatisfactory. However, there
was no occurrence of the false positives using this procedure. As shown
in Figure 6, the activity of SuNampt, MjPrs, and OkRbk was highest in
the respective reaction system. In addition, the combination of OkRbk,
MjPrs, and SuNampt
improved
NMN production by 2.9-fold to 295 mg/L from the best initial enzyme set,
which consisted of EcRbk, PcPrs and MrNampt (Figure 2c). These results
indicated that the normalized screening procedure provided a quick and
easy way to screen active enzyme homologs. Particularly, it was well
suitable for preliminary screening of dozens of enzyme
homologs to find candidate homologs
with high
catalytic
activities.