Clustering fatty acid synthetic enzymes on the membrane
Considering the successful construction of the membrane scaffold, we next recruited FabI, FabZ, FabG, TesAʹ, the four crucial fatty acid synthases, in our membrane scaffold system to demonstrate the applicability and efficiency of our proposed device. Four groups of fatty-acid-overproducing strains with different scaffolding patterns were developed. The MBF group (Fig. 4A) had four cascaded FAS enzymes on the inner membrane with the help of interacting proteins, whereas MF group only comprised four FAS enzymes anchored on the membrane without the aid of interacting proteins. The cells harboring these four groups were cultured, and the total fatty acids were extracted and measured through GC-MS (Fig. 4B). The total fatty acid produced by anchoring the enzymes onto the membrane in the MBF (1021.92 mg/L) and MF (1056.43 mg/L) groups was more than that obtained by simply overexpressing the enzymes in the cytoplasm (897.91 mg/L). However, the total fatty acids obtained by simply anchoring the enzymes on the membrane in MF group was similar to that produced by clustering enzymes in MBF group, which indicated that the inner membrane, in contrast to the cytoplasm, likely retained the membrane-anchored proteins in a relatively restrained zone, resulting in effects similar to those of clustering proteins. Thus, we performed fluorescence complementation or fluorescence resonance energy transfer experiments to verify whether simply anchoring enzymes on the membrane can cluster proteins. However, no positive results were observed (Fig. 3C), which is possibly due to the enzymes anchored on the membrane were sufficiently close with one another to generate cascading effects and enhance the metabolic flux, but not close enough to be detected through fluorescence complementation or fluorescence resonance energy transfer experiments. Nonetheless, our results demonstrated that simply anchoring the target enzymes on the membrane without the introduction of interacting proteins can enhance the metabolic flux as described in other artificial scaffold systems 5, 6.