Clustering fatty acid synthesis enzymes on the membrane accelerates products exportation
In addition, we analyzed the fatty acid products and found that anchoring the enzymes on the membrane remarkably changed the ratio between the products in the cell and in the medium. To further investigate this phenomenon, we constructed the clustered enzymes in the cytoplasm (CBF group) and compared the fatty acid product yields of the four groups (Fig. 4A). We found that the amount of the fatty acid products exported by the membrane groups (MBF group produced 431.42 mg/L extracellular fatty acids while MF group produced 476.78 mg/L) was higher than that obtained by the cytoplasm groups (78.12 mg/L and 26.38mg/L in CBF and CF groups, respectively) did (Fig. 4C). Meanwhile, the total fatty acids produced by clustering the enzymes in the cytoplasm (853.9 mg/L fatty acids in CBF group) was higher than that obtained by the CF group (771.53 mg/L) (Fig. 4C), which was consistent with previous protein scaffolding studies [3]. These results confirmed that membrane scaffold enhanced the product titer in the medium, indicating that high amounts of free fatty acids were secreted outside.

Discussion

Organisms naturally cluster related enzymes to improve the efficiency of the whole metabolism pathway and save energy. Enzymes fused with membrane anchors were directed toward the membrane as expected after these enzymes were translated. Thus, the enzyme distribution was restricted to the 2D membrane rather than randomly diffused throughout the cytoplasm. Each membrane anchor is inclined to interact with another anchor because of spatial restriction, and such interaction could be stabilized by phospholipids around the transmembrane domain. Therefore, a series of enzymes involved in sequential reactions could be swiftly and orderly organized on the membrane of our proposed device. Any suitable enzyme could be localized in the periplasm and could utilize the substrates from the medium to synthesize target products. Our work has demonstrated the feasibility of the membrane scaffold to cluster multiple enzymes and further enhance the involved metabolic flux. The efficiency of fatty acid synthesis and the yield of free fatty acids were improved by artificially engineering different scaffolding patterns. To our knowledge, this report is the first to utilize the inner cell membrane of E. coli as a novel protein scaffold to enhance fatty acid production. Although the number of clustered enzymes in our design was limited because of the available interacting proteins, the freely anchored enzymes could produce similar amounts of fatty acids, indicating that the introduction of interacting proteins was not relevant. Our findings expanded the capacity of scaffolding proteins and reduced the complexity of device construction.
The random diffusion of small molecules through the cell membrane was slow and consistent with the concentration difference across the membrane. Our results showed that the products accumulated near the cell membrane when enzymes were anchored on the membrane, resulting in an increased local concentration. Such an increase triggered the product molecules to diffuse outside through the cell membrane, thereby remarkably increasing the product titer in the medium. The yield produced by the system with anchored enzymes on the membrane was higher than that obtained by the clustered enzymes in the cytoplasm presumably because of the continuous secretion of products. A fundamental reaction model has shown that accumulated products inhibit or reverse catalytic reactions. Our membrane scaffold device facilitated the exportation of products, promoted the metabolic flux, and simplified the post-processing work of the desired products.
Together, our design of using cell membrane as scaffold to anchor target enzymes to enhance the metabolic flux is effective. The construction process is simplified as fusing target enzymes to the N-terminal or the C-terminal of the membrane anchor protein (Lgt), and the number of the enzymes are not limited (Fig. 5). Potentially, enzymes can be anchored in the periplasm and utilize substrates from medium to make target products. Our design not only shows similar enzymes clustering effects as other artificial scaffolds, but also enhance the products exportation, driving the whole metabolism flux to the positive direction and resulting in further increased final yield compared to the cytoplasm scaffold system.