FIGURE 7 Optimization of NMN production. Effects of temperature
from 25−45°C (a), pH from 6.0−9.0 (b), and enzyme ratios
(c)
on NMN production. (d) NMN synthesis was improved by stepping through a
series of optimizations. Error bars represent the standard deviation of
three biological replicates. See Table S3 for more detailed information
on NMN synthesis reactions.
4
Conclusion
In this work, a novel strategy that combined CFPS with split GFP for
rapidly prototyping enzyme homologs was developed. To demonstrate the
potential of this strategy, it was
applied
to optimize a
three-step
biosynthetic
pathway for the production of NMN.
By
applying this strategy, the time to characterize 10 enzyme homologs of
each catalytic step had been reduced from a few weeks to 24 hours.
Finally, the three-step NMN biosynthetic pathway was further optimized
to reach a 12-fold improvement over our initial setup.
A key feature of our work was that the expression of pathway enzymes in
CFPS was monitored by using the split GFP assay
instead
of 14C-leucine incorporation, relative to previous
works which
applied
CFPS-ME
approach to prototype biosynthetic pathways (Dudley et al., 2020; Grubbe
et al., 2020; Karim et al., 2020; Karim & Jewett, 2016; Rasor et al.,
2022). Compared with 14C-leucine incorporation, the
split GFP assay provided a fast and technically simple way to monitor
proteins produced by
CFPS
in a time as fast as 2 h (Figure S2). In addition, the split GFP signal
correlated well (R2>0.99)
(Figure
3c) with the amount of GFP11-tagged proteins, and the amount of protein
to be detected could reach the picomolar level (Figure 3c) (Cabantous et
al., 2005). Another important feature of our work was that the high
activity enzyme homologs were identified using a normalized screening
procedure. In
this
procedure, the enzyme homologs were rapidly produced by CFPS in
parallel, and then these proteins were
mixed
with essential substrates and GFP 1-10 detector
fragment
to perform target molecule production and protein content
quantification, respectively.
At
last, the most productive enzyme homolog could be easily selected by
choosing the homolog with the highest RT/F value. This
normalized screening procedure offers a rapid and
facile
way for characterizing enzyme homologs,
especially for the primary
screening of a large amount of enzyme homologs to
identify
potential enzyme hits. Taken together, it is anticipated that the
strategy combining CFPS with split GFP will facilitate rapid
design-build-test cycles for identifying the most productive enzyme
homologs to produce desired products. In addition, this strategy is
expected to expand
the
application space and reduce the application difficulty of CFPS-ME,
which has been proved to be a powerful strategy for prototyping the
synthesis of high-value commodity chemicals.
ACKNOWLEDGMENTS
This study was supported by the National Key R&D Program of China (No.
2018YFA0901700) and the National Natural Science Foundation of China
(No. 31871739).