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.