Prototyping Gene Expression in a Chloroplast Cell-Free System for Plant Engineering

Plants are difficult to engineer due to their complex eukaryotic genomes, slow generation time and long transformation. As a result, the same genetic parts are frequently reused and very few parts are available. In addition, mistakes in design significantly impede progress because they take a long time to discover. The risk increases when multi-genic systems, which require careful tuning of gene expression, are transferred into plants. Here we apply a chloroplast cell-free system, purified from tobacco leaves, to prototype single and multi-genic expression for plastid engineering.

Plastids are plant subcellular compartments of prokaryotic origin with ribosomes that recognize Shine-Dalgarno sequences. We used RBS Calculator to design several RBSs to express GFP under the T7 promoter. Protein expression correlated with the predicted RBS strengths, suggesting that the RBS Calculator can be used to fine-tune expression not just in bacteria, but also in plastids. In addition, codon-optimized and non-codon optimized versions of two enzymes from the capsaicin biosynthetic pathway were assayed in the chloroplast extract. Errors in codon-optimization led to low protein expression. Being able to prototype in the chloroplast cell-free system saved valuable time and effort before plastid transformation.

In this work we use chloroplast cell-free extract to assay synthetic nitrogenase clusters at mRNA level, demonstrating the utility of the system for prototyping multi-genic constructs. The synthetic clusters were transformed into tobacco plastids to correlate cell-free and in vivo expression. RNA-seq data obtained from these plants shows that the chloroplast cell-free reflects in vivo gene expression.

Our chloroplast cell-free system enables precise characterization of genetic parts and the testing of 10³-10⁴ construct in a week. Constructs producing the desired expression pattern in vitro can be selected for transformation which will reduce the amount of work related to plant regeneration and increase the success rate of engineering complex functions in plants.