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.