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.