The Plant Journal

doi: 10.1111/tpj.12980pmid: N/A

Benning, Christoph; Sweetlove, Lee

doi: 10.1111/tpj.13245pmid: 27483204

Puchta, Holger

doi: 10.1111/tpj.13100pmid: 26677816

It is possible to target individual sequence motives within genomes by using synthetic DNA‐binding domains. This one‐dimensional approach has been used successfully in plants to induce mutations or for the transcriptional regulation of single genes. When the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system was discovered, a tool became available allowing the extension of this approach from one to three dimensions and to construct at least partly synthetic entities on the genome, epigenome and transcriptome levels. The second dimension can be obtained by targeting the Cas9 protein to multiple unique genomic sites by applying multiple different single guiding (sg) RNAs, each defining a different DNA‐binding site. Finally, the simultaneous use of phylogenetically different Cas9 proteins or sgRNAs that harbour different types of protein binding motives, allows for a third dimension of control. Thus, different types of enzyme activities – fused either to one type of Cas9 orthologue or to one type of RNA‐binding domain specific to one type of sgRNA – can be targeted to multiple different genomic sites simultaneously. Thus, it should be possible to induce quantitatively different levels of expression of certain sets of genes and at the same time to repress other genes, redefining the nuclear transcriptome. Likewise, by the use of different types of histone‐modifying and/or DNA (de)methylating activities, the epigenome of plants should be reprogrammable. On our way to synthetic plant genomes, the next steps will be to use complex genome engineering approaches within or between species borders to restructure and recombine natural or artificial chromosomes.

Arendt, Philipp; Pollier, Jacob; Callewaert, Nico; Goossens, Alain

doi: 10.1111/tpj.13138pmid: 26867713

With tens of thousands of characterized members, terpenoids constitute the largest class of natural compounds that are synthesized by all living organisms. Several terpenoids play primary roles in the maintenance of cell membrane fluidity, as pigments or as phytohormones, but most of them function as specialized metabolites that are involved in plant resistance to herbivores or plant–environment interactions. Terpenoids are an essential component of human nutrition, and many are economically important pharmaceuticals, aromatics and potential next‐generation biofuels. Because of the often low abundance in their natural source, as well as the demand for novel terpenoid structures with new or improved bioactivities, terpenoid biosynthesis has become a prime target for metabolic engineering and synthetic biology projects. In this review we focus on the creation of new‐to‐nature or tailor‐made plant‐derived terpenoids in photosynthetic organisms, in particular by means of combinatorial biosynthesis and the activation of silent metabolism. We reflect on the characteristics of different potential photosynthetic host organisms and recent advances in synthetic biology and discuss their utility for the (heterologous) production of (novel) terpenoids.

Hanson, Maureen R.; Lin, Myat T.; Carmo‐Silva, A. Elizabete; Parry, Martin A.J.

doi: 10.1111/tpj.13139pmid: 26867858

Photosynthesis in C3 plants is limited by features of the carbon‐fixing enzyme Rubisco, which exhibits a low turnover rate and can react with O2 instead of CO2, leading to photorespiration. In cyanobacteria, bacterial microcompartments, known as carboxysomes, improve the efficiency of photosynthesis by concentrating CO2 near the enzyme Rubisco. Cyanobacterial Rubisco enzymes are faster than those of C3 plants, though they have lower specificity toward CO2 than the land plant enzyme. Replacement of land plant Rubisco by faster bacterial variants with lower CO2 specificity will improve photosynthesis only if a microcompartment capable of concentrating CO2 can also be installed into the chloroplast. We review current information about cyanobacterial microcompartments and carbon‐concentrating mechanisms, plant transformation strategies, replacement of Rubisco in a model C3 plant with cyanobacterial Rubisco and progress toward synthesizing a carboxysome in chloroplasts.

Schuler, Mara L.; Mantegazza, Otho; Weber, Andreas P.M.

doi: 10.1111/tpj.13155pmid: 26945781

C4 photosynthetic plants outperform C3 plants in hot and arid climates. By concentrating carbon dioxide around Rubisco C4 plants drastically reduce photorespiration. The frequency with which plants evolved C4 photosynthesis independently challenges researchers to unravel the genetic mechanisms underlying this convergent evolutionary switch. The conversion of C3 crops, such as rice, towards C4 photosynthesis is a long‐standing goal. Nevertheless, at the present time, in the age of synthetic biology, this still remains a monumental task, partially because the C4 carbon‐concentrating biochemical cycle spans two cell types and thus requires specialized anatomy. Here we review the advances in understanding the molecular basis and the evolution of the C4 trait, advances in the last decades that were driven by systems biology methods. In this review we emphasise essential genetic engineering tools needed to translate our theoretical knowledge into engineering approaches. With our current molecular understanding of the biochemical C4 pathway, we propose a simplified rational engineering model exclusively built with known C4 metabolic components. Moreover, we discuss an alternative approach to the progressing international engineering attempts that would combine targeted mutagenesis and directed evolution.

Gonzalez‐Esquer, C. Raul; Newnham, Sarah E.; Kerfeld, Cheryl A.

doi: 10.1111/tpj.13166pmid: 26991644

Bacterial microcompartments (BMCs) are megadalton‐sized protein assemblies that enclose segments of metabolic pathways within cells. They increase the catalytic efficiency of the encapsulated enzymes while sequestering volatile or toxic intermediates from the bulk cytosol. The first BMCs discovered were the carboxysomes of cyanobacteria. Carboxysomes compartmentalize the enzyme ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) with carbonic anhydrase. They enhance the carboxylase activity of RuBisCO by increasing the local concentration of CO2 in the vicinity of the enzyme's active site. As a metabolic module for carbon fixation, carboxysomes could be transferred to eukaryotic organisms (e.g. plants) to increase photosynthetic efficiency. Within the scope of synthetic biology, carboxysomes and other BMCs hold even greater potential when considered a source of building blocks for the development of nanoreactors or three‐dimensional scaffolds to increase the efficiency of either native or heterologously expressed enzymes. The carboxysome serves as an ideal model system for testing approaches to engineering BMCs because their expression in cyanobacteria provides a sensitive screen for form (appearance of polyhedral bodies) and function (ability to grow on air). We recount recent progress in the re‐engineering of the carboxysome shell and core to offer a conceptual framework for the development of BMC‐based architectures for applications in plant synthetic biology.

Haslam, Richard P.; Sayanova, Olga; Kim, Hae Jin; Cahoon, Edgar B.; Napier, Johnathan A.

doi: 10.1111/tpj.13172pmid: 27483205

Plant seed lipid metabolism is an area of intensive research, including many examples of transgenic events in which oil composition has been modified. In the selected examples described in this review, progress towards the predictive manipulation of metabolism and the reconstitution of desired traits in a non‐native host is considered. The advantages of a particular oilseed crop, Camelina sativa, as a flexible and utilitarian chassis for advanced metabolic engineering and applied synthetic biology are considered, as are the issues that still represent gaps in our ability to predictably alter plant lipid biosynthesis. Opportunities to deliver useful bio‐based products via transgenic plants are described, some of which represent the most complex genetic engineering in plants to date. Future prospects are considered, with a focus on the desire to transition to more (computationally) directed manipulations of metabolism.

Nielsen, Agnieszka Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos; Wlodarczyk, Artur Jacek; Gnanasekaran, Thiyagarajan; Perestrello Ramos H de Jesus, Maria; King, Brian Christopher; Bakowski, Kamil; Jensen, Poul Erik

doi: 10.1111/tpj.13173pmid: 27005523

Shih, Patrick M.; Liang, Yan; Loqué, Dominique

doi: 10.1111/tpj.13176pmid: 27030440

The Green Revolution has fuelled an exponential growth in human population since the mid‐20th century. Due to population growth, food and energy demands will soon surpass supply capabilities. To overcome these impending problems, significant improvements in genetic engineering will be needed to complement breeding efforts in order to accelerate the improvement of agronomical traits. The new field of plant synthetic biology has emerged in recent years and is expected to support rapid, precise, and robust engineering of plants. In this review, we present recent advances made in the field of plant synthetic biology, specifically in genome editing, transgene expression regulation, and bioenergy crop engineering, with a focus on traits related to lignocellulose, oil, and soluble sugars. Ultimately, progress and innovation in these fields may facilitate the development of beneficial traits in crop plants to meet society's bioenergy needs.