The plant enzyme Rubisco is the main enzyme converting atmospheric carbon dioxide into biological compounds, however, this enzymatic process is inefficient in vascular plants; this study demonstrates that tobacco plants can be engineered to fix carbon with a faster cyanobacterial Rubisco, thus potentially improving plant photosynthesis. Rubisco — a major enzyme assimilating atmospheric CO2 into the biosphere — is an important target for efforts to improve the photosynthetic efficiency of plants. These authors successfully engineered tobacco plants containing a functioning Rubisco from a cyanobacterium. The cyanobacterial (photosynthetic blue–green algae) enzyme has a greater catalytic rate than any 'C3' plant. The lines generated here pave the way for future addition of the remaining components of the cyanobacterial CO2-concentrating mechanism, an important step towards enhancing photosynthetic efficiency and improving crop yields. In photosynthetic organisms, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere1. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants2,3. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield4,5,6. However, the complex nature of Rubisco’s assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful7,8. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains9,10. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes11,12. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins4,5,6.

The antiquity and global abundance of the enzyme, RuBisCO, attests to the crucial and longstanding role it has played in the biogeochemical cycles of Earth over billions of years. The counterproductive oxygenase activity of RuBisCO has persisted over billions of years of evolution, despite its competition with the carboxylase activity necessary for carbon fixation, yet hypotheses regarding the selective pressures governing RuBisCO evolution have been limited to speculation. Here we report the resurrection and biochemical characterization of ancestral RuBisCOs, dating back to over one billion years ago (Gyr ago). Our findings provide an ancient point of reference revealing divergent evolutionary paths taken by eukaryotic homologues towards improved specificity for CO₂, versus the evolutionary emphasis on increased rates of carboxylation observed in bacterial homologues. Consistent with these distinctions, in vivo analysis reveals the propensity of ancestral RuBisCO to be encapsulated into modern-day carboxysomes, bacterial organelles central to the cyanobacterial CO₂ concentrating mechanism.

Summary: The photosynthetic efficiency of C3 plants suffers from the reaction of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) with O₂ instead of CO₂, leading to the costly process of photorespiration. Increasing the concentration of CO₂ around Rubisco is a strategy used by photosynthetic prokaryotes such as cyanobacteria for more efficient incorporation of inorganic carbon. Engineering the cyanobacterial CO₂‐concentrating mechanism, the carboxysome, into chloroplasts is an approach to enhance photosynthesis or to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. We have chosen to explore the possibility of producing β‐carboxysomes from Synechococcus elongatus PCC7942, a model freshwater cyanobacterium. Using the agroinfiltration technique, we have transiently expressed multiple β‐carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO and CcmN) in Nicotiana benthamiana with fusions that target these proteins into chloroplasts, and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the β‐carboxysome are able to assemble in plant chloroplasts into highly organized assemblies resembling empty microcompartments. We demonstrate that a foreign protein can be targeted with a 17‐amino‐acid CcmN peptide to the shell proteins inside chloroplasts. Our experiments establish the feasibility of introducing carboxysomes into chloroplasts for the potential compartmentalization of Rubisco or other proteins.

Summary: Introducing a carbon‐concentrating mechanism and a faster Rubisco enzyme from cyanobacteria into higher plant chloroplasts may improve photosynthetic performance by increasing the rate of CO₂ fixation while decreasing losses caused by photorespiration. We previously demonstrated that tobacco plants grow photoautotrophically using Rubisco from Synechococcus elongatus, although the plants exhibited considerably slower growth than wild‐type and required supplementary CO₂. Because of concerns that vascular plant assembly factors may not be adequate for assembly of a cyanobacterial Rubisco, prior transgenic plants included the cyanobacterial chaperone RbcX or the carboxysomal protein CcmM35. Here we show that neither RbcX nor CcmM35 is needed for assembly of active cyanobacterial Rubisco. Furthermore, by altering the gene regulatory sequences on the Rubisco transgenes, cyanobacterial Rubisco expression was enhanced and the transgenic plants grew at near wild‐type growth rates, although still requiring elevated CO₂. We performed detailed kinetic characterization of the enzymes produced with and without the RbcX and CcmM35 cyanobacterial proteins. These transgenic plants exhibit photosynthetic characteristics that confirm the predicted benefits of introduction of non‐native forms of Rubisco with higher carboxylation rate constants in vascular plants and the potential nitrogen‐use efficiency that may be achieved provided that adequate CO₂ is available near the enzyme.

Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5ʹ untranslated regions [5ʹUTRs], nine promoter:5ʹUTR fusions, 10 3ʹUTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.

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.