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.