Designing Chloroplast Expression Systems with Predictable Protein Output

Transgenes in leaves from native expression signals are expressed at such high levels that gene expression compromises growth, while in non-green plastids protein levels are very low. We developed a two-part regulatory system that relies on (1) translation control sequences of plastid transgene mRNAs that respond to (2) nuclear genes that selectively stabilize and facilitate translation of the plastid mRNAs. The engineered atpH mRNA translation control sequences are upstream of the AUG in plastid mRNAs. The nuclear genes encode engineered PPR10 proteins that recognize the cognate site. In potato, GFP from wild-type signals accumulates up to 25% of total soluble cellular protein (TSP) in leaves that seriously compromises plant development. The engineered binding site reduces GFP accumulation to levels that do not impact growth, while expression of the engineered PPR10 from a tuber-specific promoter boosts GFP accumulation 20x. Based on studies in the model plant tobacco, dependent on the tissue type, the two-part system enables regulated expression of plastid genes in the range of 0.05% to 25% of TSP. The two-part system enables regulated plastid transgene expression without compromising yield.