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

Abstract

SummaryPlastids (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.

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

“…Lysin protein accumulation in chloroplasts to up to 30% of the plant's TSP demonstrates the effectiveness of our strategy and suggests that the same approach should be applicable also to the production of other biopharmaceuticals that exhibit antimicrobial activity (21). The enormous lysin expression levels obtained here and in a previous study (3) are caused by high protein stability inside the chloroplast, which is consistent with the idea that phage lysins have evolved considerable resistance to the proteases of their host bacteria. Because plastids possess a prokaryotic-type proteolytic machinery (22), it seems conceivable that lysins are poor substrates also for chloroplast proteases.…”

“…1 A). To assess the toxicity of Pal and Cpl-1 to E. coli, the genes were cocloned in a 1:1 ratio with gfp into the pRB95-derived vector pMO24 (3,18). Plasmid DNA isolated from bacterial clones was digested with NcoI and XbaI to determine the identity of the insert.…”

“…To facilitate high-level expression of the Pal and Cpl-1 protein antibiotics in plastids, the 2 coding regions were optimized by adjusting their sequences to the codon usage preferred by higher plant plastids (3,9). However, when cloning of the synthetic genes into standard plastid expression cassettes (refs.…”

“…Transgene expression from the plastid genome represents a particularly promising strategy in molecular farming (2) because of the plastids' potential to accumulate foreign proteins to very high levels (up to Ͼ70% of the plant's total soluble protein; ref. 3) and the increased biosafety provided by the maternal mode of plastid inheritance, which greatly reduces transgene transmission via pollen.…”

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

“…Plastid-encoded gene expression has yielded extremely high levels of protein production, with transformants producing 45% (De Cosa et al, 2001) to 70% (Oey et al, 2009) of the transgeneencoded protein per unit of soluble leaf protein and up to 72% (Ruhlman et al, 2010) of the transgeneencoded protein per unit of total leaf protein in tobacco (Nicotiana tabacum). While these expression levels are too high for most metabolic engineering strategies for the production of chemicals, fuels, or materials without creating unwanted stress on the host plant, expression levels can be somewhat controlled by the choice of regulatory elements flanking the transgenes.…”