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Engineering and Purification of Microcin C7 Variants Resistant to Trypsin and Analysis of Their Biological Activity
Guangxin Yang1, Lijun Shang1, Lu Liu1,2
1State Key Laboratory of Animal Nutrition and Feeding, Ministry of Agriculture Rural Affairs Feed Industry Centre, China Agricultural University, Beijing Bio-Feed Additives Key Laboratory, Beijing 100193, China.
Abstract:
Microcin C7 (McC) as a viable form of antimicrobial has gained substantial attention due to its distinctive antimicrobial activity, by targeting aspartyl tRNA synthetase. McC can be a potential solution against pathogenic microbial infections in the postantibiotic era. However, considering that degradation by digestive enzymes can disrupt the function of this peptide in the gastrointestinal tract, in this study, we attempt to design McC variants to overcome several barriers that may affect its stability and biological activity. The mccA gene encoding the McC peptide precursor was mutated and 12 new McC variants with trypsin resistance were found. The Yej+rimL- strain was used as an indicator to determine the minimum inhibitory concentrations (MICs). The results showed that three variants, including R2A, R2T and R2Q, among 12 variants formed by the replacement of the second arginine of the McC peptide with different amino acids, were resistant to trypsin and had an outstanding antimicrobial ability, with MIC values of 12.5, 25, and 25 μg/mL, respectively. Taken together, our findings show that the engineering of the site-directed mutagenesis of McC significantly enhances McC trypsin resistance and maintains a great antimicrobial activity.
Insights
Engineered Microcin C7 (McC) variants show enhanced resistance to digestive enzymes and potent antimicrobial activity. Site-directed mutagenesis improved stability, offering a promising solution for postantibiotic era infections.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Microcin C7 (McC) exhibits unique antimicrobial activity by inhibiting aspartyl tRNA synthetase.
- McC presents a potential therapeutic agent against microbial infections in the postantibiotic era.
- Digestive enzyme degradation poses a challenge to McC's stability and efficacy in the gastrointestinal tract.
Purpose of the Study:
- To design and engineer Microcin C7 variants with improved stability against digestive enzymes.
- To enhance the biological activity and therapeutic potential of Microcin C7.
Main Methods:
- Site-directed mutagenesis of the mccA gene to create McC variants.
- Assessment of trypsin resistance in the generated McC variants.
- Determination of minimum inhibitory concentrations (MICs) using a Yej+rimL- indicator strain.
Main Results:
- Twelve new McC variants with enhanced trypsin resistance were successfully generated.
- Three variants (R2A, R2T, R2Q) demonstrated significant trypsin resistance and potent antimicrobial activity.
- MIC values for R2A, R2T, and R2Q were 12.5, 25, and 25 μg/mL, respectively.
Conclusions:
- Site-directed mutagenesis of McC effectively enhances its resistance to trypsin degradation.
- Engineered McC variants maintain significant antimicrobial efficacy, highlighting their therapeutic potential.
