Contribution

Overview

Today, the issue of pathogen drug resistance has severely threatened human health. To address this problem, it is necessary to develop drug-lead compounds with novel structures and mechanisms of action. Microbial natural products have proven to be an important source of bioactive natural products. Peptide compounds in particular feature low adverse reactions, broad target binding capabilities, high specificity, and low potential for resistance.

This project focused on peptides like nisin, bicereucin, and darobactin, which have good antimicrobial effects. They belong to the class of ribosomally synthesized and post-translationally modified peptide natural products, following the same biosynthetic logic. This project combines these peptide molecules through combinatorial biosynthesis to obtain fusion peptides with broad-spectrum antimicrobial activity. The new parts we designed are:

 

 

1.  Create new composite parts: BBa_K4846018 (pETduet-DarE-BsjM) and BBa_K4846020 (pRSFduet-DarL-BsjL-His-DarA-BsjA).

DarA is the core sequence of darobactin that requires modification by the post-translational modifying (PTM) enzyme DarE to generate the mature product. DarL is the sequence recognized and bound by DarE for modification. BsjA is the amino acid sequence of bicereucin that requires modification by the PTM enzyme BsjM to form the mature product. BsjL is the sequence recognized and bound by BsjM for modification. In plasmid  pRSFduet-DarL-BsjL-His-DarA-BsjA, we concatenated the l eaders (DarL and BsjL) and c ore peptides (DarA and BsjA) of darobactin and bicereucin via a His-tag, so that their respective PTM enzymes can modify the core peptides. In pETduet-DarE-BsjM, we co-expressed DarE and BsjM for modifying the DarA and BsjA core peptides.

We successfully constructed pRSFduet-DarL-BsjL-His-DarA-BsjA and pETduet-DarE-BsjM by enzymatic digestion and ligation, then co-transformed both plasmids into the same strain. We induced expression in the positive transformant, then lysed the cells by sonication to release contents and proteins. To obtain the target fusion peptides at higher purity, we performed nickel column purification on the lysate supernatant. The purified proteins were cleaved in vitro by lysyl endopeptidase to release the core peptides.

1）After obtaining the purified proteins, we performed in vitro cleavage experiments using lysyl endopeptidase to obtain the core peptide for subsequent testing in inhibition experiments.

 

Figure 1  Antibacterial effect of DarA-BsjA fusion peptide antimicrobial peptides on Escherichia coli

 

Figure 2  Antibacterial effect of two antimicrobial peptides on Bacillus subtilis

We used Gram negative bacteria Escherichia coli and Gram positive bacteria Bacillus subtilis as indicator bacteria to verify the antibacterial effect of antimicrobial peptides. From the Figure 1and2, it can be seen that antimicrobial peptides have little inhibitory effect on Escherichia coli, while they have antibacterial effect on Bacillus subtilis, but the antibacterial effect is not very significant. This result is not very ideal, possibly 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 use mass spectrometry to detect the cutting effect and ensure the formation of fusion peptides for antibacterial purposes. In addition, we will also conduct antibacterial tests on other common Gram negative and Gram positive bacteria in the future.

2.  Create a new composite part BBa_K4846019: pRSFduet-DarL-NisL-His-DarA-NisA-DarE

NisA is the amino acid sequence of nisin that requires modification by the enzymes NisB and NisC to generate the mature product. The pRSFduet-DarL-NisL-His-DarA-NisA-DarE plasmid has two multiple cloning sites for co-expressing precursor peptides and PTM enzymes. In the first site, we concatenated the leaders (DarL and NisL) and core peptides (DarA and NisA) of darobactin and nisin via a His-tag, so their respective enzymes (DarE and NisB-NisC) can modify the cores. The second site contains the darobactin PTM enzyme DarE.

We successfully constructed pRSFduet-DarL-NisL-His-DarA-NisA-DarE and pETduet-NisB-NisC by enzymatic digestion and ligation, then co-transformed both plasmids into the same strain. We induced expression in the positive transformant, then lysed the cells by sonication to release contents and proteins. To obtain the target fusion peptides at higher purity, we performed nickel column purification on the lysate supernatant. The purified proteins were cleaved in vitro by lysyl endopeptidase to release the core peptides. Finally, agar diffusion growth inhibition assays showed the cleaved DarA-NisA fusion peptide inhibited Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative), demonstrating broad-spectrum antimicrobial activity (Figure 2).

 

Figure 3 SDS-PAGE result of the protein expression and purification