Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Abstract

ABSTRACTAchieving balanced gene expression within synthetic operons requires a spectrum of expression levels. Here we investigate the expression ofgfpreporter gene in tobacco chloroplasts, guided by variants of the plastidatpH5’ UTR, which harbors a binding site for PPR10, a protein that activatesatpHat the post-transcriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maizeatpH5’ UTRs in different design contexts. Notably, high GFP expression was not coupled to stabilization of monocistronicgfptranscripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishesgfpmRNA (and GFP protein), resulting in a substantial reduction in GFP accumulation. When combined with a mutantatpH5’ UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning reporter gene expression across a wide range, spanning from 0.02% to 25% of the total soluble cellular protein (TSP). These findings highlight the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.