ИСТИНА

The increasing spread of antibiotic-resistant microorganisms is a growing concern for modern animal production, agriculture and medicine. Phage-encoded endolysins have acquired significant attention as antimicrobials due to their high efficiency and mechanism of action. To produce a functionally active Gram-negative bacterium bacteriophage CP933 endolysin in plants, a combination of transient expression and vacuole targeting strategies was employed. Cytoplasmic expression of the cp933 gene in Nicotiana benthamiana using Potato virus X - based transient expression vectors (pP2C2S and pGR107) resulted in death of the apical portion of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its N-terminus to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933 to the delta type vacuoles. Plants producing the CP933 fusion protein did not exhibit the severe toxic effect and the level of expression was 0.16 mg/g of plant tissue. Antimicrobial assays revealed that the Gram-positive plant pathogenic bacterium Clavibacter michiganensis was the most susceptible to the plant-produced CP933 showing 18% growth inhibition, whereas the Gram-negative bacterium Escherichia coli (BL 21 (DE3)) was not affected significantly. Using vacuole protein targeting is a promising approach for the production of functionally active proteins that exhibit toxicity when expressed in plant cells.