Figure legend
Figure 1.
Fatty
acid metabolism pathway in E. coli . FabA, FabB, FabD, FabF, FabG,
FabH, FabI, FabZ are the main enzymes of the fatty acid biosynthesis
pathway in E. coli . FabB, FabF, Fab G, FabI, FabZ/FabA are mainly
responsible for fatty acid elongation. TesA is the enzyme that is able
to release free fatty acid by hydrolysis of acyl-ACP species. FabG,
FabZ, FabI and TesA were picked as targets for manipulation in these
experiments based on previous studies.
Figure 2. Design and verification of the membrane localization of the
engineered Lgt. (A) Schematic of the engineered membrane protein. Lgt is
used as a scaffold to carry functional groups (β-lactamase and EGFP as
examples) to the membrane. (B) Membrane localization is verified by
confocal microscopy.
Figure 3.
Design
and verification of the artificial membrane clustering. (A) Schematic of
the engineered membrane protein. Protein interaction domains are fused
with the ends of Lgt. (B) Schematic of the four groups of engineered
membrane protein. The latter three groups use SH3, PDZ, and GBD
interactions, respectively. The No binding group has no interaction
domains. Split EGFP is fused with the N-terminus of the membrane protein
to verify the protein interactions. (C) The protein interactions in
different groups are verified by confocal microscopy. The detected EGFP
fluorescence indicates the interactions between designed proteins.
Figure 4.
Clustering
fatty acid metabolism enzymes on the membrane enhances the product yield
and secretion. (A) Schematic of the four strategies of clustering
enzymes. The MBF Group uses protein interaction domains to cluster
target enzymes on the membrane. The MF Group directly localizes target
enzymes on the membrane. The CBF Group utilizes protein interaction
domains to cluster target enzymes in the cytoplasm. The CF Group
overexpressing cytoplasmic target enzymes is taken as the control. B)
Total fatty acid extracted from the cell expressing each groups of
engineered enzymes. (C) The intracellular and extracellular fatty acid
titer produced by different groups.
Figure 5. Summary of applications of membrane scaffold system.
Potentially, unlimited number of target enzymes can be fused to Lgt and
clustered on the membrane. The enzymes can be either presented in the
cytoplasm site or the periplasm site. In the cytoplasm site, enzymes can
utilize substrates produced in the cell, and the products could pass the
cell membrane by diffusion. In the periplasm site, enzymes can utilize
the substrates added in the culture and directly release products into
the culture.