Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

Summary In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over 30 years old. Here we show the design–build–test of a novel synthetic genome structure that does not disturb the native plastome: the ‘mini‐synplastome’. The mini‐synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini‐synplastome in plants was developed in vitro to meet the following criteria: (i) episomal replication in plastids; (ii) facile cloning; (iii) predictable transgene expression in plastids; (iv) non‐integration of vector sequences into the endogenous plastome; and (v) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini‐synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker‐free plastid engineering, and provide an unparalleled platform for one‐step metabolic engineering in plants.

Genome-editing has enabled rapid improvement for staple food crops, such as potato, a key beneficiary of the technology. In potato, starch contained within tubers represents the primary product for use in food and non-food industries. Starch granules are produced in the plastids of tubers with plastid size correlated with the size of starch grana. The division of plastids is controlled by proteins, including the tubulin-like GTPase FtsZ1. The altered expression of FtsZ1 has been shown to disrupt plastid division, leading to the production of “macro-plastid”-containing plants. These macro-chloroplast plants are characterized by cells containing fewer and enlarged plastids. In this work, we utilize CRISPR/Cas9 to generate FtsZ1 edited potato lines to demonstrate that genome-editing can be used to increase the size of starch granules in tubers. Altered plastid morphology was comparable to the overexpression of FtsZ1 in previous work in potato and other crops. Several lines were generated with up to a 1.98-fold increase in starch granule size that was otherwise phenotypically indistinguishable from wild-type plants. Further, starch paste from one of the most promising lines showed a 2.07-fold increase in final viscosity. The advantages of enlarged starch granules and the potential of CRISPR/Cas9-based technologies for food crop improvement are further discussed.