Overview
We plan to improve FPP precursor synthesis as well as downstream product production by exogenously expressing the MVA pathway, incorporating an artificially designed IUP pathway, and through a modular strategy of increasing the intracellular hydrophobic environment.
We characterized the supply of FPP precursors by lycopene synthesis. Lycopene is inherently colorful and can be used for rapid characterization, with a good linear relationship between its concentration and absorbance. We tested the feasibility as well as the efficiency of our strategy in E. coli DH5a. First, we tested the effect of adding the MVA pathway on lycopene production. We found that the MVA pathway significantly enhanced the production of lycopene. Next, we added the artificially designed IUP pathway, selecting two enzymes previously reported in the literature, PhoN and IPK, and added DMAOH and IOH to the medium, which significantly increased lycopene production at appropriate concentrations. Finally, we tested the strategy of increasing the intracellular hydrophobic environment, which again increased the lycopene production. The lycopene production was increased approximately 13-fold compared to our most primitive strain.
FPP sensing system based on split fluorescent protein
Although we used lycopene synthesis to characterize FPP supply, given the complex FPP metabolic network within bacteria, we further developed a sensing system that directly binds FPP and emits fluorescence (FPR) and validated the feasibility of this system in E. coli DH5α as well as the optimal detection time. Limited by the lack of available FPP monomers, we used DH5α that had been characterized with the MVA pathway transferred as a positive control and DH5α with only the MVA pathway as a negative control. As shown in the result graph, FPPR has only a small background fluorescence intensity in wild-type DH5α, which is due to the fact that E. coli naturally contains the MEP pathway for FPP synthesis and low levels of FPP concentration exist in the bacteria. In contrast, the addition of the MVA pathway greatly increased the fluorescence signal intensity of FPPR (~3.6-fold), demonstrating the responsiveness of our reporter system to the concentration of FPP.
We next further validated the delay of the system by testing the variation of the fluorescence intensity of DH5α containing MVA as well as FPPR with bacterial growth, and the results showed that the fluorescence intensity started to increase at approximately K/2, with a significant fluorescence signal appearing after the bacterial growth entered a stable phase, and eventually stabilized as the bacteria entered a plateau phase. This is similar to the concentration change of FPP secondary metabolism, further demonstrating the usability and stability of our sensor in the secondary metabolism phase
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