Mini-synplastomes for plastid genetic engineering.

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.

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology.

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.

Chemoenzymatic Synthesis of Starting Materials and Characterization of Halogenases Requiring Acyl Carrier Protein-Tethered Substrates.

Flavin-adenine dinucleotide (FAD)-dependent halogenases are widespread in natural product biosynthetic gene clusters and have been demonstrated to employ small organic molecules as substrates for halogenation, as well as substrates that are tethered to carrier proteins (CPs). Despite numerous reports of FAD-dependent halogenases utilizing CP-tethered substrates, only a few have been biochemically characterized due to limited accessibility to the physiological substrates. Here, we describe a method for the preparation of acyl-S-CP substrates and their use in biochemical assays to query the activity of FAD-dependent halogenases. Furthermore, we describe a mass spectrometry-based method for the characterization of acyl-S-CP substrates and the corresponding halogenated products generated by the halogenases. Finally, we test the substrate specificity of a physiological chlorinase and a physiological brominase from marine bacteria, and, for the first time, demonstrate the distinct halide specificity of halogenases. The methodology described here will enable characterization of new halogenases employing CP-tethered substrates.