DescriptionPlastid genome engineering holds great promise in many biotechnological applications, including production of vaccines and antibodies, conferring insect tolerance by expressing dsRNAs, implanting novel metabolic pathways and improving photosynthetic efficiency and so on. However, the plastid transformation technology is not yet available in most crop plants.
I was part of the group that developed a system for plastid transformation in Arabidopsis thaliana, a Brassicaceae species, that is potentially applicable to the important oilseed crop canola (Brassica napus) and vegetable brassicas such as broccoli, cauliflower and cabbage (Brassica oleracea). Efficient plastid transformation is enabled by eliminating a duplicate fatty acid biosynthetic pathway in chloroplasts.
High level expression of recombinant proteins is desirable, but it may cripple the plant growth. To achieve on demand expression of plastid transgenes, I was involved in a project that exploited the use of an engineered PPR (pentatricopeptide) RNA-binding protein for post-transcriptional regulation of chloroplast genes. One application is ethanol-inducible expression of chloroplast proteins by activating mRNA translation when the protein is needed. Thus, the transplastomic plants can be grown full size in the absence of the inducer and expression is turned on during the protein production phase. The second application is tuber-specific expression of plastid genes where plastid gene expression is normally very law. When the engineered PPR variant is expressed from a tuber-specific patatin promoter, a 60-fold increase over 0.02%, the maximum protein yield achieved to date, has been obtained in the potato tuber.
Plastids represent an appealing target for synthetic biology applications, where predictable protein output is of paramount importance. I designed synthetic operons from which reporter gene expression could be obtained in tobacco chloroplasts over a large dynamic range by post-transcriptional regulation, using the right combination of RNA binding proteins, RNA-binding protein binding sites and RNA processing elements.