Expanding the palette of cis-elements to tune the expression of chloroplast transgenes in tobacco.

The capacity of the chloroplast genetic system to express proteins from polycistronic mRNAs offers advantages for applications that require the expression of multiple transgenes. Optimal outcomes in such applications often require the production of transgene products in particular stoichiometries. A current limitation in this regard is the paucity of characterized cis-elements that program transgene expression in increments over a wide dynamic range. We leveraged advances in understanding chloroplast gene expression to design cis-elements that modulate the expression of a transgene within a polycistronic transcript. In one approach, we tuned down the activating effects of cis-elements that bind the pentatricopeptide repeat proteins PPR10 and HCF152 by introducing mutations that modestly decrease binding affinity in vitro. In a second approach, we used translational efficiencies determined by ribosome profiling to select 5'UTRs anticipated to program varying translational activities. Elements were evaluated in dicistronic reporters in transplastomic tobacco. The results showed that mutations that decrease PPR10 binding affinity caused a corresponding decrease in GFP abundance and that the suite of native 5'UTRs programmed a wide range of GFP abundance in incremental steps and with various developmental dynamics. Our results demonstrate that binding affinities of PPR proteins for modified cis-elements in vitro can be used as predictive tools to design cis-elements for transgene tuning and that translational efficiencies programmed by endogenous 5'UTRs approximate relative transgene output in synthetic chloroplast operons. These findings add to the options for cis-elements to optimize protein balance while minimizing effects on host plant vigor.