Biosynthesis of Antimicrobial Peptides
Antimicrobial peptides (AMP) are a large class of active oligopeptides resistant to harmful organisms (or pathogens) such as bacteria, fungi, parasites, and viruses. Because it generally has a sufficient positive charge and is often accompanied by hydrophobicity, it can be combined with the biofilm containing a negative charge under the action of static electricity, penetrating and destroying the membrane structure, resulting in cell death. Different from the single-target bactericidal principle of traditional antibiotics, antimicrobial peptides can damage pathogens at multiple targets and greatly reduce the generation of drug-resistant bacteria. They also have broad-spectrum antibacterial activity.
Biosynthesis and expression system for antimicrobial peptides
In addition to the chemical synthesis of peptides according to a specific sequence of amino acids, more and more antimicrobial peptides are obtained through biosynthesis. Compared with the natural extraction methods of antimicrobial peptides directly separated and purified from animals, plants, and microorganisms, the genetic engineering technologies and the upgraded synthetic biology methods all have the advantages of simple operation procedure, low cost, and small pollution, which have become the most important ways to obtain antimicrobial peptides. With the rapid development of synthetic biology, researchers are trying to use new chassis cells and a variety of synthetic biology tools to improve the microbial synthesis of antimicrobial peptides.
Prokaryotic expression system for antimicrobial peptides
Because Escherichia coli has the advantages of a fast growth rate, low culture cost, and clear biological background, it was first used in the biosynthesis of antimicrobial peptides by genetic engineering method. The most commonly used E. coli strain is E. coli BL21 (DE3), and the commonly used expression vectors include pET and pGEX.
Additionally, some antimicrobial peptides are synthesized by the expression system of Bacillus subtilis. Compared with E. coli, the B. subtilis expression system can directly secrete antimicrobial peptides outside the cell, which is conducive to the collection, separation, and purification of target proteins.
However, all prokaryotic expression systems also face some challenges. For example, most antimicrobial peptides with biological activity have broad-spectrum killing ability against prokaryotic host strains. Besides, antimicrobial peptides are easy to be degraded by endogenous proteases due to their positive net charge, but this problem can be properly overcome by the fusion protein strategy.
Eukaryotic expression system for antimicrobial peptides
The representative eukaryotic expression system is the expression system of yeast, including S. cerevisiae, P. pastoris, C. reinhardtii, etc. The expression vectors encompass pPICZa, pPIC9K, pGAPZa, etc. Compared with the prokaryotic expression system, the yeast expression system has the advantages of low toxicity, high activity, and easy separation of antimicrobial peptides, and can promote the extracellular expression of antimicrobial peptides and post-translation modification (e.g. facilitating disulfide bond formation, o-glycosylation, and n-glycosylation, etc.). But there are also some shortcomings, such as slow cell growth, low yield, and some antimicrobial peptides (such as melittin) that could harm the host strain.
Molecular design and synthetic biology modification
As mentioned above, the natural antimicrobial peptides produced by microbial expression systems sometimes have a low yield, excessive host toxicity, and insufficient resistance activity. Some antimicrobial peptides are hemolytic and difficult to promote. To solve the above problems, researchers tried to modify the natural antimicrobial peptide gene sequence version 1.0 by using genetic engineering techniques such as intercepting effective sequences, amino acid replacement, and modifying structural parameters. For example, the resistance of gram-negative bacteria was enhanced by replacing Gly with Trp in the protein sequence or increasing the proportion of Trp of the natural antimicrobial peptide Chensinin-1. Redesigning the hydrophilic and hydrophobic region of the antimicrobial peptide LGR16 can regulate its hemolysis activity and resistance.
With the popularity of synthetic biology and the rise of artificial intelligence (AI), a prediction algorithm can be established to analyze and learn the data of natural antimicrobial peptide sequences, and relevant characteristics and rules can be found by using this algorithm, which is expected to be extended to the prediction and accurate design of unknown antimicrobial peptides and achieve version 2.0 of the antimicrobial peptide modification. Currently, a support vector machine (SVM) platform has been used to study the functional similarity and sequence homology of α-helical antimicrobial peptides, which can be used to design novel α-helical antimicrobial peptides based on the physicochemical properties of antimicrobial peptides.
Depending upon the platform, the data of 286 α-helix antimicrobial peptides and 286 non-antimicrobial peptides were trained to identify the key characteristics that determine the activity of antimicrobial peptides. The accuracy was up to 91.9%, and the specificity and sensitivity were 93.0% and 90.7%, respectively. This model has been used to identify several α-helical antimicrobial peptides that are difficult to produce through natural evolution or point mutation. In 2021, Das et al. used a variational autoencoder based on variational inference to learn about 1.7 million peptides reported in all universal protein resources, isolated antimicrobial peptides and divided them into two categories of antibacterial activity. Through the establishment of a "classifier" for the antimicrobial ability of peptides, new high-activity antimicrobial peptides were obtained through "rejection sampling".
The CRISPR/Cas9 gene editing system is one of the latest gene-editing technologies in the field of synthetic biology. CRISPR/ Cas9-mediated gene silencing, gene knockout, or manipulation of specific DNA sequences can also increase the production of allogenic antimicrobial peptides, leading to faster folding or appropriate post-translational modification of them. Park et al. used the CRISPR/Cas system to introduce Cas9 nuclease and specific RNA into BM-N cells of silkworm ovary, which weakened the function of endogenous genes and negatively regulated the Toll pathway of anti-infection. They successfully edited the Cactus gene of silkworms and activated the immune response signal pathway of the silkworm to secrete antimicrobial peptides such as Moricin and Lebocin. At present, there are only a few researches using CRISPR/Cas9 and other gene editing techniques in synthetic biology to guide the synthesis and sterilization of antimicrobial peptides, but its potential is huge.
Fig 1. The CRISPR/Cas9 gene editing system facilitates cells to secrete antimicrobial peptides1
Conclusion of antimicrobial peptides
Currently, antimicrobial peptides have been used in the clinical treatment of pathogenic bacteria infection, wound healing, cancer, and other aspects. Nevertheless, the overall promotion of natural antimicrobial peptides is inevitably plagued by sources, production costs, biosafety, and other factors. With the development of cross-disciplines such as synthetic biology and medical tissue engineering, new ideas and technical means will be provided for the biological design and synthesis of antimicrobial peptides.
Reference
- WEI Daixu, GONG Hailun, ZHANG Xuwei. Biosynthesis of antimicrobial peptides and its medical application[J]. Synthetic Biology Journal, 2022, 3(4): 709-727
