Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins.

Plastid transformation has become an attractive tool in biotechnology. Because of the prokaryotic nature of the plastid's gene expression machinery, expression elements (promoters and untranslated regions) that trigger high-level foreign protein accumulation in plastids usually also confer high expression in bacterial cloning hosts. This can cause problems, for example, when production of antimicrobial compounds is attempted. Their bactericidal activity can make the cloning of the corresponding genes in plastid transformation vectors impossible. Here, we report a general solution to this problem. We have designed a strategy (referred to as toxin shuttle) that allows the expression in plastids of proteins that are toxic to Escherichia coli. The strategy is based on blocking transcription in E. coli by bacterial transcription terminators upstream of the gene of interest, which subsequently are excised in planta by site-specific recombination. We demonstrate the applicability of the strategy by the high-level expression in plastids (to up to 30% of the plant's total soluble protein) of 2 phage-derived protein antibiotics that are toxic to E. coli. We also show that the plastid-produced antibiotics efficiently kill pathogenic strains of Streptococcus pneumoniae, the causative agent of pneumonia, thus providing a promising strategy for the production of next-generation antibiotics in plants.

Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic.

Plastids (chloroplasts) possess an enormous capacity to synthesize and accumulate foreign proteins. Here we have maximized chloroplast protein production by over-expressing a proteinaceous antibiotic against pathogenic group A and group B streptococci from the plastid genome. The antibiotic, a phage lytic protein, accumulated to enormously high levels (>70% of the plant's total soluble protein), and proved to be extremely stable in chloroplasts. This massive over-expression exhausted the protein synthesis capacity of the chloroplast such that the production of endogenous plastid-encoded proteins was severely compromised. Our data suggest that this is due to translational rather than transcriptional limitation of gene expression. We also show that the chloroplast-produced protein antibiotic efficiently kills the target bacteria. These unrivaled expression levels, together with the chloroplast's insensitivity to enzymes that degrade bacterial cell walls and the elimination of the need to remove bacterial endotoxins by costly purification procedures, indicate that this is an effective plant-based production platform for next-generation antibiotics, which are urgently required to keep pace with rapidly emerging bacterial resistance.