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).