Engineering Success

Overview:

The goal of this project was to enable E.coli capable of co-express ing  the nisin (or bicereucin) and darobactin. Nisin, bicereucin, and darobactin require precursor peptides and post-translational modifying (PTM) enzymes to gain antimicrobial activity. We utilized two approaches to produce bsjA-darA and nisA-darA fusion peptides, respectively.

In Cycle 1, the precursor proteins and PTM enzymes were expressed separately on two plasmids, i.e., pRSFduet-DarL-BsjL-His-DarA-BsjA and pETduet-DarE-BsjM.

In Cycle 2, the precursor proteins and PTM enzymes were co-expressed on the same plasmid, i.e., pRSFduet-DarL-NisL-His-DarA-NisA-DarE and pETduet-NisB-NisC.

 

Figure 1. Schematic diagram of cycle 1 and cycle 2.

 

Cycle 1:

Design:

To achieve separate expression of the precursor proteins and PTM enzymes, we designed the expression frame of DarL-BsjL-His-DarA-BsjA and DarE-BsjM, where DarL and BsjL are leaders of the core peptides (DarA and BsjA) for recognition and binding by the PTM enzymes (DarE and BsjM). Based on the literature, E. coli endogenous peptidases can cleave the darobactin leader, so a His-tag was included between the leader and core peptide, to enable purification of the mature product after leader removal.

 

 

Figure 2. Expression frame of the plasmids constructed in cycle 1 .

 

Build:

We used the restriction enzymes to create complementary sticky ends on the vector (pRSFduet and pETduet) and fragments (DarL-BsjL-His-DarA-BsjA and DarE-BsjM). T4 DNA ligase was then used to ligate the vector with the fragments, generating plasmid A (pRSFduet-DarL-BsjL-His-DarA-BsjA) and plasmid C (pETduet-DarE-BsjM).

 

Figure 3. The generation of the plasmids constructed in cycle 1 .

 

After constructing the recombinant plasmids A and C, we needed to co-transform them into the same bacterial strain. Plasmid A was first transformed, and positive transformants were selected to prepare competent cells containing plasmid A, and plasmid C was then transformed into it. We performed gel electrophoresis and sequencing to verify the successful transformation o f two plasmids into E.coli.

 

 

Figure 4. Colony PCR and sequencing results of transformants containing plasmids A and C.

 

Test:

We inoculated positive clones and induced protein expression with IPTG. After induction, bacterial cells were lysed by sonication to release cellular contents and proteins. Nickel column purification was then performed on the lysate supernatant to obtain the target fusion peptides at higher purity (Figure 5).

 

 

Figure 5. SDS-PAGE result of the protein expression and purification.

 

The purified proteins were subjected to in vitro cleavage with lysyl endopeptidase to release the core peptides. Finally, agar diffusion growth inhibition assays demonstrated that the cleaved DarA-BsjA fusion peptide have little inhibitory effect on Escherichia coli (Gram-negative), while they have antibacterial effect on Bacillus subtilis (Gram-positive), but the antibacterial effect is not very significant (Figure 6).

 

 

 

Figure 6. Antibacterial effect of DarA-BsjA fusion peptide antimicrobial peptides on Escherichia coli (top) and Bacillus subtilis (below)

 

Learn:

Perhaps it is because after expressing the fusion peptide, in vitro cleavage experiments are required, and the cleavage results need to be detected by mass spectrometry. The fusion peptide was not completely cleaved, resulting in weak activity and no antibacterial effect. In the future, we will improve the experiment to ensure the cutting effect and the antibacterial activity of the antibacterial peptide.

Cycle 2:

Design:

To achieve co-expression of the precursors and PTM enzymes, we designed two expression frames. One was T7-DarL-NisL-His-DarA-NisA-T7-DarE, where the first  MCS contained DarL-NisL-His-DarA-NisA, and the second  MCS contained the darobactin PTM enzyme DarE. DarL and NisL are leaders of DarA and NisA for recognition and binding by the PTM enzymes. The second expression frame was NisB-NisC, the PTM enzyme of  NisA.

 

 

Figure 7. Expression frame of the plasmids constructed in cycle 2.

 

Build:

Through enzymatic digestion and ligation, we ligated the vector backbones pRSFduet and pETduet with the fragments DarL-NisL-His-DarA-NisA-DarE and NisB-NisC to construct plasmid B (pRSFduet-DarL-NisL-His-DarA-NisA-DarE) and D (pETduet-NisB-NisC).

 

 

Figure 8. The generation of the plasmids constructed in cycle 2.

After constructing the recombinant plasmids B and D, we needed to co-transform them into the same bacterial strain. Plasmid B was first transformed, and positive transformants were selected to prepare competent cells containing plasmid B, and plasmid D was then transformed into it. We performed gel electrophoresis and sequencing to verify the successful transformation o f two plasmids into E.coli.

 

 

Figure 9. Colony PCR and sequencing results of transformants containing plasmids B and D.

 

Test:

We inoculated the positive transformant and induced protein expression with IPTG. The bacterial cells were then lysed by sonication, and nickel purification was performed to obtain the fusion peptides at higher purity (Figure 10).

 

Figure 10. SDS-PAGE result of the protein expression and purification.

 

 

Figure 11. Antibacterial effect of DarA-NisA fusion antimicrobial peptide on Bacillus subtilis

 

In vitro lysyl endopeptidase cleavage released the core peptides from the purified proteins. Finally, agar diffusion assays showed the cleaved DarA-NisA fusion peptide inhibited Bacillus subtilis (Gram-positive), but the antibacterial effect is not very significant (Figure 11).

Learn:

Co-expressing the precursors and PTM enzymes on the same plasmid enabled the modification of DarA and NisA to confer antimicrobial activity. At the molecular level, we fused DarA and NisA to generate a peptide with complementary anti-Gram-positive and anti-Gram-negative effects.Perhaps it is because after expressing the fusion peptide, in vitro cleavage experiments are required, and the fusion peptide is not completely cleaved, resulting in weak activity and no antibacterial effect. In the future, we will use mass spectrometry to detect the cutting effect and ensure the formation of fusion peptides for antibacterial purposes. In addition, in the future, we will also conduct antibacterial tests on other common Gram negative and Gram positive bacteria.