Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell‐free protein synthesis with split GFP

Abstract

Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cellfree protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.

FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems

“…Recently, an article was published in which the split-GFP system was used to monitor enzyme synthesis in CFPS (Yuan et al, 2023). However, there are several signi cant differences with the system we developed.…”

“…2) Our GFP is fused to the N-terminus of MBP. This provides two signi cant advantages: it avoids the need to purify the GFP1-10 fragment from inclusion bodies (Cabantous et al, 2005;Yuan et al, 2023) as it keeps the protein soluble, and it allows for a simple, rapid, and high-yield puri cation through a nity chromatography. 3) While the uorescent signal in our system is high and stabilizes after approximately 4 hours, in the system developed by Yuan et al, the uorescence is rather low in the early hours, to the extent that the authors made their measurements between 8 and 16 hours (times when uorescence had not yet stabilized.…”

Split Proteins and Reassembly Modules for Biological Applications

“…[21] The integration with cell-free protein synthesis, as shown by Yuan et al, facilitated the development and optimization of enzyme homologs, thereby enhancing productivity. [22] Lundqvist et al contributed to these advancements by introducing a pre-maturated GFP 1-10 variant, which allowed for faster signal generation, further enhancing the utility of split-GFP for real-time observations. [23] The development of a tripartite system by Cabantous et al represented a refined approach to studying protein interactions.…”