Rapid Prototyping of Constructs in a Chloroplast Cell-Free System for Plastid Engineering

Chloroplasts are attractive targets of plant engineering with high foreign protein production capacity and genetic control that enables the expression of several genes stacked into operons. Transferring multi-genic functions requires the characterization of genetic parts and fine-tuning of protein expression, but the time-consuming transformation of chloroplasts prevents engineers from carrying out this task in plants. To overcome this problem, a cell-free system – based on purified and subsequently lysed tobacco chloroplasts – was used which enables the expression of proteins from a template.

In order to tune protein expression, different transcription initiation rates were designed with the RBS Calculator to produce luciferase under T7 promoter. Luciferase expression correlated with the predicted RBS strengths, suggesting that RBS Calculator can be used to fine-tune protein expression not just in bacteria, but also in chloroplasts.

To demonstrate the prototyping capacity of the cell-free system, two enzymes of the capsaicin biosynthetic pathway were selected. The sequences were also codon-optimized and their RBS was tuned to cover a high range of protein expression levels. The design and testing of 55 constructs for this study only took us 4 weeks, which would have never been possible by transforming plants.

In order to test a multi-gene system, the Klebsiella ‘refactored’ v2.1 cluster codifying for 16 genes of nitrogenase was expressed in chloroplast and E. coli cell-free systems. RT-PCR analysis confirmed the presence of all open reading frames in both systems but revealed differences in mRNA levels.

The chloroplast cell-free system prevents mistakes like using wrong codon-optimization or RBS, and could allow for up to 1000 constructs to be prototyped weekly. Those producing the desired expression pattern could be selected for plastid transformation in the plant. In addition, the cell-free system could also serve as a test-bed to finely balance expression of multi-gene clusters.