Plasmids and strains
E. coli strains DH5α and BL21 (DE3) were used for cloning and protein expression, respectively. The vectors used for construction included pETDuet1, pACYCDuet1, pRSF-Duet1, and pBAD18. All of the plasmids used in this study are shown in Table 1.
The arabinose operon from pBAD18 was amplified and cloned twice into pETDuet1, pACYCDuet1, and pRSF-Duet1 to replace the original T7 operon, thereby producing pET-Ara, pACYC-Ara, and pRSF-Ara. Each vector contained two copies of arabinose operons. For the verification of membrane localization, the DNA fragment containing the N-terminal DsbA signal sequence, followed by the genes of β-lactamase, phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), and GFP was cloned into pET-Ara (Fig. 2A). For the verification of artificial clustering, the DNA fragment comprising a DsbA signal sequence (ssDsbA), one type of interacting protein, Lgt, and a split EGFP was cloned into arabinose operon (Figs. 3A and 3B). A flexible linker FL3 was introduced between crucial protein parts to ensure their proper functioning.
Four groups of engineered fatty acid-related enzymes were used to verify our design (Fig. 3A). The genes involved in the membrane binding FAS (MBF), membrane FAS (MF), and cytoplasmic binding FAS (CBF) groups were cloned into pET-Ara, pACYC-Ara, and pRSF-Ara, respectively. The genes in the cytoplasmic FAS (CF) group were cloned into pET-Ara. The MBF group contained cytoplasmic and periplasmic protein interaction domains to cluster engineered proteins. The enzymes in the MF group were directly fused with the C-terminus of Lgt. The enzymes in the CBF group were directly fused with the protein interaction domains and expressed in the cytoplasm. The CF group comprised the enzymes expressed in the cytoplasm. In each group, the FabI and FabZ proteins or the fusion proteins were cloned into two arabinose operons in pET-Ara. The FabG and TesAʹ proteins or the fusion proteins were cloned into two arabinose operons in pRSF-Ara.