Prototyping Nitrogenase Expression in Plastids with a Chloroplast Cell-Free System

Plastids are plant subcellular compartments that are attractive targets of plant engineering because they are amenable to transformation, have high protein expression capacity and are maternally inherited. In order to transfer complex functions, like nitrogen fixing capacity, into plastids, gene expression has to be finely tuned. This requires several design iterations, but the time-consuming transformation of plants keeps engineers from executing this process in vivo.

In this work, we used a chloroplast cell-free system to prototype synthetic nitrogenase clusters for plastid expression. The cell-free system, purified from tobacco chloroplasts, has the transcription and translation machinery of this organelle. The clusters were restructured by using well-defined parts, codon-optimizing genes and putting them under the control of the T7 RNA polymerase (T7RNAP) which allows the transfer between species. We show that the chloroplast cell-free system is able to express these synthetic clusters composed of 16-18 genes at mRNA level.

In order to correlate expression in the chloroplast cell-free system with in vivo data, we transformed these clusters into tobacco chloroplasts along with the T7RNAP, which was put under the control of an inducible riboswitch that allows translation upon theophylline induction. RNA-seq data of these plants showed that T7RNAP responds to the chemical induction and all clusters are expressed at the mRNA level. In vivo data obtained from these plants correlated well with our chloroplast cell-free data demonstrating that the chloroplast cell-free system can serve to prototype multi-genic constructs before plant transformation.

Our chloroplast cell-free system could allow the precise characterization of genetic parts and the testing of 10³-10⁴ construct in a week. Those constructs producing the desired expression pattern can be selected for plastid transformation which would reduce the amount of work related to plant transformation and increase the success rate of engineering complex proteins in plants.