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Background: The CO 2 -concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO 2 availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C 3 and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence., Results: We identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C 3 orchids and between strong CAM and C 3 bromeliads suggested Rubisco had evolved in response to differing CO 2 concentration., Conclusions: The results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ 13 C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-β4 loop, pore loop 1, and the presensor 1-β hairpin (PS1-βH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO 2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures., Competing Interests: The authors declare no competing interest.

The rising global population is forcing the need for adapting alternative sustainable technologies for enhanced crop productivity. The CO 2 Concentration Mechanisms (CCMs) evolved in algae to counter the inefficient CO 2 fixing enzyme, RuBisCo and slower diffusion of CO 2 in water offers good scope for the above purpose. The CCMs are single-celled CO 2 supply mechanisms that depend on multiple CO 2 /HCO 3 transporters and acclimation states and accumulate 100-fold more CO 2 than low CO 2 environments. Although some insights have been obtained regarding the CCMs of blue-green algae and green algae like Chlamydomonas reinhardtii , further progress needs to take place to understand the molecular and biochemical basis for intracellular transport of CO 2 . In this review, complete information pertaining to the core CCM is presented and discussed in light of the available literature. In addition to this, information on CO 2 /HCO 3 sensing, photo-acclimation in low CO 2 , liquid-like nature of pyrenoid, untapped potential of high CO 2 responses and high CO 2 requiring mutants, and prospects of engineering CCM components into higher plants are presented and discussed.

During photosynthesis the AAA+ protein and essential molecular chaperone Rubisco activase (Rca) constantly remodels inhibited active sites of the CO 2 -fixing enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) to release tightly bound sugar phosphates. Higher plant Rca is a crop improvement target, but its mechanism remains poorly understood. Here we used structure-guided mutagenesis to probe the Rubisco-interacting surface of rice Rca. Mutations in Ser-23, Lys-148, and Arg-321 uncoupled adenosine triphosphatase and Rca activity, implicating them in the Rubisco interaction. Mutant doping experiments were used to evaluate a suite of known Rubisco-interacting residues for relative importance in the context of the functional hexamer. Hexamers containing some subunits that lack the Rubisco-interacting N-terminal domain displayed a ∼2-fold increase in Rca function. Overall Rubisco-interacting residues located toward the rim of the hexamer were found to be less critical to Rca function than those positioned toward the axial pore. Rca is a key regulator of the rate-limiting CO 2 -fixing reactions of photosynthesis. A detailed functional understanding will assist the ongoing endeavors to enhance crop CO 2 assimilation rate, growth, and yield., Competing Interests: The authors declare no competing interest.

Allopolyploids are often faced with the challenge of maintaining well-coordination between nuclear and cytoplasmic genes inherited from different species. The synthetic allotetraploid Cucumis × hytivus is a useful model to explore cytonuclear coevolution. In this study, the sequences and expression of cytonuclear enzyme complex RuBisCO as well as its content and activity in C. × hytivus were compared to its parents to explore plastid-nuclear coevolution. The plastome-coded rbcL gene sequence was confirmed to be stable maternal inheritance, and parental copy of nuclear rbcS genes were both preserved in C. × hytivus. Thus, the maternal plastid may interact with the biparentally inherited rbcS alleles. The expression of the rbcS gene of C-homoeologs (paternal) was significantly higher than that of H-homoeologs (maternal) in C. × hytivus (HHCC). Protein interaction prediction analysis showed that the rbcL protein has stronger binding affinity to the paternal copy of rbcS protein than that of maternal copy in C. × hytivus , which might explain the transcriptional bias of the rbcS homoeologs. Moreover, both the activity and content of RuBisCO in C. × hytivus showed mid-parent heterosis. In summary, our results indicate a paternal transcriptional bias of the rbcS genes in C. × hytivus , and we found new nuclear-cytoplasmic combination may be one of the reasons for allopolyploids heterosis.

Diatoms (Bacillariophyta) are ubiquitous microalgae which produce a siliceous exoskeleton and which make a major contribution to the productivity of oceans and freshwaters. They display a huge diversity, which makes them excellent ecological indicators of aquatic ecosystems. Usually, diatoms are identified using characteristics of their exoskeleton morphology. DNA-barcoding is an alternative to this and the use of High-Throughput-Sequencing enables the rapid analysis of many environmental samples at a lower cost than analyses under microscope. However, to identify environmental sequences correctly, an expertly curated reference library is needed. Several curated libraries for protists exists; none, however are dedicated to diatoms. Diat.barcode is an open-access library dedicated to diatoms which has been maintained since 2012. Data come from two sources (1) the NCBI nucleotide database and (2) unpublished sequencing data of culture collections. Since 2017, several experts have collaborated to curate this library for rbcL, a chloroplast marker suitable for species-level identification of diatoms. For the latest version of the database (version 7), 605 of the 3482 taxonomical names originally assigned by the authors of the rbcL sequences were modified after curation. The database is accessible at https://www6.inra.fr/carrtel-collection_eng/Barcoding-database .

Background: The genus Ilex (Aquifoliaceae) has a near-cosmopolitan distribution in mesic habitats from tropical to temperate lowlands and in alpine forests. It has a high rate of hybridization and plastid capture, and comprises four geographically structured plastid groups. A previous study showed that the plastid rbcL gene, coding for the large subunit of Rubisco, has a particularly high rate of non-synonymous substitutions in Ilex, when compared with other plant lineages. This suggests a strong positive selection on rbcL, involved in yet unknown adaptations. We therefore investigated positive selection on rbcL in 240 Ilex sequences from across the global range., Results: The rbcL gene shows a much higher rate of positive selection in Ilex than in any other plant lineage studied so far (> 3000 species) by tests in both PAML and SLR. Most positively selected residues are on the surface of the folded large subunit, suggesting interaction with other subunits and associated chaperones, and coevolution between positively selected residues is prevalent, indicating compensatory mutations to recover molecular stability. Coevolution between positively selected sites to restore global stability is common., Conclusions: This study has confirmed the predicted high incidence of positively selected residues in rbcL in Ilex, and shown that this is higher than in any other plant lineage studied so far. The causes and consequences of this high incidence are unclear, but it is probably associated with the similarly high incidence of hybridization and introgression in Ilex, even between distantly related lineages, resulting in large cytonuclear discordance in the phylogenies.

Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Inaccuracies in biochemically characterizing the amount and CO 2 -fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco ( Rs Rubisco) mutant with improved CO 2 -fixing properties. The Rs Rubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate ( k cat C , up 27%), carboxylation efficiency ( k cat C / K m for CO 2 , increased 17%), unchanged CO 2 /O 2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of Rs Rubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L 8 S 8 ) assembly. An N-terminal Val-11-Ile substitution did not affect Rs Rubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on Rs Rubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).

Almost all the inorganic carbon on Earth is converted into biomass via the Calvin-Benson-Bassham (CBB) cycle. Here, the central carboxylation reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which can be found in numerous primary producers including plants, algae, cyanobacteria, and many autotrophic bacteria. Although RubisCO possesses a crucial role in global biomass production, it is not a perfect catalyst. Therefore, research interest persists on accessing the full potential of yet unexplored RubisCOs. We recently developed an activity-based screen suited to seek active recombinant RubisCOs from the environment-independent of the native host's culturability. Here, we applied this screen to twenty pre-selected genomic fosmid clones from six cultured proteobacteria to demonstrate that a broad range of phylogenetically distinct RubisCOs can be targeted. We then screened 12,500 metagenomic fosmid clones from six distinct hydrothermal vents and identified forty active RubisCOs. Additional sequence-based screening uncovered eight further RubisCOs, which could then also be detected by a modified version of the screen. Seven were active form III RubisCOs from yet uncultured Archaea. This indicates the potential of the activity-based screen to detect RubisCO enzymes even from organisms that would not be expected to be targeted.

Rubisco activase (Rca) is a catalytic chaperone that remodels the active site, promotes the release of inhibitors and restores catalytic competence to Rubisco. Rca activity and its consequent effect on Rubisco activation and photosynthesis are modulated by changes to the chloroplast environment induced by fluctuations in light levels that reach the leaf, including redox status and adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio. The Triticum aestivum (wheat) genome encodes for three Rca protein isoforms: 1β (42.7 kDa), 2β (42.2 kDa) and 2α (46.0 kDa). The regulatory properties of these isoforms were characterised by measuring rates of Rubisco activation and ATP hydrolysis by purified recombinant Rca proteins in the presence of physiological ADP/ATP ratios. ATP hydrolysis by all three isoforms was sensitive to inhibition by increasing amounts of ADP in the assay. In contrast, Rubisco activation activity of Rca 2β was insensitive to ADP inhibition, while Rca 1β and 2α were inhibited. Two double and one quadruple site-directed mutants were designed to elucidate if differences in the amino acid sequences between Rca 1β and 2β could explain the differences in ADP sensitivity. Changing two amino acids in Rca 2β to the corresponding residues in 1β (T358K & Q362E) resulted in significant inhibition of Rubisco activation in presence of ADP. The results show that the wheat Rca isoforms differ in their regulatory properties and that amino acid changes in the C domain influence ADP sensitivity. Advances in the understanding of Rubisco regulation will aid efforts to improve the efficiency of photosynthetic CO 2 assimilation., (© 2019 The Author(s).)

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO 2 via the Calvin-Benson-Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris , a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP), greatly facilitating the structure-function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure-function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO 2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1 Ps STQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (ΔglcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E. coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1 Ps STQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1 mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R. rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E. coli but also in photosynthetic organisms., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

C4 photosynthetic enzymes are present in C3 plants and participate in non-photosynthetic metabolism. Wheat spike bracts had a higher drought tolerance, photosynthesis and senesced later compared to the flag leaves under water deficit. This research was conducted to investigate the different response of primary carbon metabolism induced by C4 photosynthetic enzymes in wheat flag leaves and spike bracts including glumes and lemmas under water deficit. The activities of C4 photosynthetic enzymes and Ribulose bisphosphate carboxylase oxygenase (Rubisco), the expression of related genes and primary carbon metabolism contents were demonstrated in wheat flag leaves and spike bracts exposed to water deficit. Results showed that drought stress strongly inhibited wheat photosynthetic metabolism by decreasing Rubisco activity in flag leaves. The activities of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), phosphate dikinase (PPDK) and NADP- malic dehydrogenase (NADP-MDH) increased in wheat spike bracts under water deficit. Transcript levels of C4 photosynthetic genes in wheat spike bracts were higher under water deficit than that of control. Furthermore, the results indicated that drought stress induced changes in the contents of primary carbon metabolism including malate, oxaloacetic acid (OAA), citric, fumaric acid were organ-specific. In conclusion, the functions of C4 photosynthetic enzymes appear to be important for wheat spike bracts primary carbon metabolism and defence response under drought stress., (Copyright © 2019. Published by Elsevier Masson SAS.)

The central enzyme of photosynthesis, Rubisco, is regulated by Rubisco activase (Rca). Photosynthesis is impaired during heat stress, and this limitation is often attributed to the heat-labile nature of Rca. We characterized gene expression and protein thermostability for the three Rca isoforms present in wheat ( Triticum aestivum ), namely TaRca1-β, TaRca2-α, and TaRca2-β. Furthermore, we compared wheat Rca with one of the two Rca isoforms from rice ( Oryza sativa ; OsRca-β) and Rca from other species adapted to warm environments. The TaRca1 gene was induced, whereas TaRca2 was suppressed by heat stress. The TaRca2 isoforms were sensitive to heat degradation, with thermal midpoints of 35°C ± 0.3°C, the temperature at which Rubisco activation velocity by Rca was halved. By contrast, TaRca1-β was more thermotolerant, with a thermal midpoint of 42°C, matching that of rice OsRca-β. Mutations of the TaRca2-β isoform based on sequence alignment of the thermostable TaRca1-β from wheat, OsRca-β from rice, and a consensus sequence representing Rca from warm-adapted species enabled the identification of 11 amino acid substitutions that improved its thermostability by greater than 7°C without a reduction in catalytic velocity at a standard 25°C. Protein structure modeling and mutational analysis suggested that the thermostability of these mutational variants arises from monomeric and not oligomeric thermal stabilization. These results provide a mechanism for improving the heat stress tolerance of photosynthesis in wheat and potentially other species, which is a desirable outcome considering the likelihood that crops will face more frequent heat stress conditions over the coming decades., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

The development of DNA barcoding method has given rise to a promising way of studying genetic taxonomy. Our previous study showed that pyrosequencing profile of 18S rDNA V7 hypervariable region can be used for identifying water sources without resolving the exact components of diatom colonies in water samples. In this continued study, we aimed to improve the established analysis method and to provide scientific evidence for forensic practices. A drowning animal model was set up by injecting mimic drowning fluid into the respiratory tract of the rabbit. In order to minimize the interference of animal DNA, the hypervariable region of chloroplast ribulose-1,5-bisphosphate carboxylase large unit gene (rbcL) was used as the pyrosequencing target region for the consistency analysis of plankton populations in tissues and water samples. After decoding the pyrosequencing profile of the targeted rbcL gene with the AdvISER-M-PYRO algorithm, the plankton colony that was inhaled into drowning animal lung tissue could be successfully traced back to the source of drowning fluid. Our data suggest that this method could be a reliable tool assisting forensic drowning site inference.

Evidence is compiled to demonstrate a redox scale within Earth's photosynthesisers that correlates the specificity of their RuBisCO with organismal metabolic tolerance to anoxia, and ecological selection by dissolved O 2 /CO 2 and nutrients. The Form 1B RuBisCO found in the chlorophyte green algae, has a poor selectivity between the two dissolved substrates, O 2 and CO 2 , at the active site. This enzyme appears adapted to lower O 2 /CO 2 ratios, or more "anoxic" conditions and therefore requires additional energetic or nutrient investment in a carbon concentrating mechanism (CCM) to boost the intracellular CO 2 /O 2 ratio and maintain competitive carboxylation rates under increasingly high O 2 /CO 2 conditions in the environment. By contrast the coccolithophores and diatoms evolved containing the more selective Rhodophyte Form 1D RuBisCO, better adapted to a higher O 2 /CO 2 ratio, or more oxic conditions. This Form 1D RuBisCO requires lesser energetic or nutrient investment in a CCM to attain high carboxylation rates under environmentally high O 2 /CO 2 ratios. Such a physiological relationship may underpin the succession of phytoplankton in the Phanerozoic oceans: the coccolithophores and diatoms took over the oceanic realm from the incumbent cyanobacteria and green algae when the upper ocean became persistently oxygenated, alkaline and more oligotrophic. The facultatively anaerobic green algae, able to tolerate the anoxic conditions of the water column and a periodically inundated soil, were better poised to adapt to the fluctuating anoxia associated with periods of submergence and emergence and transition onto the land. The induction of a CCM may exert a natural limit to the improvement of RuBisCO efficiency over Earth history. Rubisco specificity appears to adapt on the timescale of ∼100 Myrs. So persistent elevation of CO 2 /O 2 ratios in the intracellular environment around the enzyme, may induce a relaxation in RuBisCO selectivity for CO 2 relative to O 2 . The most efficient RuBisCO for net carboxylation is likely to be found in CCM-lacking algae that have been exposed to hyperoxic conditions for at least 100 Myrs, such as intertidal brown seaweeds., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Photosynthesis in the seagrass Zostera muelleri remains poorly understood. We investigated the effect of reduced irradiance on the incorporation of 13 C, gene expression of photosynthetic, photorespiratory and intermediates recycling genes as well as the enzymatic content and activity of Rubisco and PEPC within Z. muelleri. Following 48 h of reduced irradiance, we found that i) there was a ∼7 fold reduction in 13 C incorporation in above ground tissue, ii) a significant down regulation of photosynthetic, photorespiratory and intermediates recycling genes and iii) no significant difference in enzyme activity and content. We propose that Z. muelleri is able to alter its physiology in order to reduce the amount of C lost through photorespiration to compensate for the reduced carbon assimilation as a result of reduced irradiance. In addition, the first estimated rate constant (K cat ) and maximum rates of carboxylation (V cmax ) of Rubisco is reported for the first time for Z. muelleri., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a ubiquitous enzyme that catalyzes the conversion of atmospheric CO 2 into organic carbon in primary producers. All naturally occurring RubisCOs have low catalytic turnover rates and are inhibited by oxygen. Evolutionary adaptations of the enzyme and its host organisms to changing atmospheric oxygen concentrations provide an impetus to artificially evolve RubisCO variants under unnatural selective conditions. A RubisCO deletion strain of the nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus was previously used as a heterologous host for directed evolution and suppressor selection studies that led to the identification of a conserved hydrophobic region near the active site where amino acid substitutions selectively impacted the enzyme's sensitivity to O 2 In this study, structural alignments, mutagenesis, suppressor selection, and growth complementation with R. capsulatus under anoxic or oxygenic conditions were used to analyze the importance of semiconserved residues in this region of Synechococcus RubisCO. RubisCO mutant substitutions were identified that provided superior CO 2 -dependent growth capabilities relative to the wild-type enzyme. Kinetic analyses of the mutant enzymes indicated that enhanced growth performance was traceable to differential interactions of the enzymes with CO 2 and O 2 Effective residue substitutions also appeared to be localized to two other conserved hydrophobic regions of the holoenzyme. Structural comparisons and similarities indicated that regions identified in this study may be targeted for improvement in RubisCOs from other sources, including crop plants. IMPORTANCE RubisCO catalysis has a significant impact on mitigating greenhouse gas accumulation and CO 2 conversion to food, fuel, and other organic compounds required to sustain life. Because RubisCO-dependent CO 2 fixation is severely compromised by oxygen inhibition and other physiological constraints, improving RubisCO's kinetic properties to enhance growth in the presence of atmospheric O 2 levels has been a longstanding goal. In this study, RubisCO variants with superior structure-functional properties were selected which resulted in enhanced growth of an autotrophic host organism ( R. capsulatus ), indicating that RubisCO function was indeed growth limiting. It is evident from these results that genetically engineered RubisCO with kinetically enhanced properties can positively impact growth rates in primary producers., (Copyright © 2019 Satagopan et al.)

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.

Cyanobacterial carboxysomes encapsulate carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Genetic deletion of the major structural proteins encoded within the ccm operon in Synechococcus sp. PCC 7002 (ΔccmKLMN) disrupts carboxysome formation and significantly affects cellular physiology. Here we employed both metabolite pool size analysis and isotopically nonstationary metabolic flux analysis (INST-MFA) to examine metabolic regulation in cells lacking carboxysomes. Under high CO 2 environments (1%), the ΔccmKLMN mutant could recover growth and had a similar central flux distribution as the control strain, with the exceptions of moderately decreased photosynthesis and elevated biomass protein content and photorespiration activity. Metabolite analyses indicated that the ΔccmKLMN strain had significantly larger pool sizes of pyruvate (>18 folds), UDPG (uridine diphosphate glucose), and aspartate as well as higher levels of secreted organic acids (e.g., malate and succinate). These results suggest that the ΔccmKLMN mutant is able to largely maintain a fluxome similar to the control strain by changing in intracellular metabolite concentrations and metabolite overflows under optimal growth conditions. When CO 2 was insufficient (0.2%), provision of acetate moderately promoted mutant growth. Interestingly, the removal of microcompartments may loosen the flux network and promote RuBisCO side-reactions, facilitating redirection of central metabolites to competing pathways (i.e., pyruvate to heterologous lactate production). This study provides important insights into metabolic regulation via enzyme compartmentation and cyanobacterial compensatory responses., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Functions of domains or motifs, which are encoded by the transit peptide (TP) of the precursor of the small subunit of Rubisco (prSSU), have been investigated intensively in dicots. Functional characterization of the prSSU TP, however, is still understudied in maize. In this study, we found that the TP of maize prSSU1 did not function fully in chloroplast targeting in Arabidopsis or vice versa, indicating the divergent function of TPs in chloroplast targeting between maize and Arabidopsis. Through deletion or substitution assays, we found that the N-terminal region of maize or Arabidopsis prSSU1 was necessary and sufficient for importing specifically the fused-green fluorescent protein (GFP) into each corresponding chloroplast. Finally, we found that the first-five amino acids and MM motif in the N-terminal domain of the maize TP played an essential role in maize chloroplast targeting. Thus, our analyses demonstrate that the N-terminal domain of the prSSU1 TP is the key determinant in chloroplast targeting between maize and Arabidopsis. Our study highlights the unique properties of the maize prSSU1 TP in chloroplast targeting, thus helping to understand the role of N-terminal domain in chloroplast targeting across species. It will help to manipulate chloroplast transit peptides (cTPs) for crop bioengineering., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The cyanobacterium Synechococcus elongatus PCC 7942 is a potential photosynthetic cell-factory. In this study, two native promoters from S. elongatus PCC 7942 driving the expression of abundant cyanobacterial proteins phycocyanin (P cpcB7942 ) and RuBisCO (P rbc7942 ) were characterized in relation to their sequence features, expression levels, diurnal behavior, and regulation by light and CO 2 , major abiotic factors important for cyanobacterial growth. P cpcB7942 was repressed under high light intensity, but cultivation at higher CO 2 concentration was able to recover promoter activity. On the other hand, P rbc7942 was repressed by elevated CO 2 with a negative regulatory region between 300 and 225 bp. Removal of this region flipped the effect of CO 2 with Rbc225 being activated only at high CO 2 concentration, besides leading to the loss of circadian rhythm. The results from this study on promoter features and regulation will help expand the repertoire of tools for pathway engineering in cyanobacteria.

The present study was aimed at understanding the effects of heat stress on selected physiological and biochemical parameters of a model cyanobacterium, Anabaena PCC 7120 in addition to amelioration strategy using exogenous Ca 2+ . A comparison of the cells exposed to heat stress (0-24 h) in the presence or absence of Ca 2+ clearly showed reduction in colony-forming ability and increase in reactive oxygen species (ROS) leading to loss in the viability of cells of Ca 2+ -deficient cultures. There was higher level of saturation in membrane lipids of the cells supplemented with Ca 2+ along with higher accumulation of proline. Similarly, higher quantum yield (7.8-fold) in Ca 2+ -supplemented cultures indicated role of Ca 2+ in regulation of photosynthesis. Relative electron transport rate (rETR) decreased in both the sets with the difference in the rate of decrease (slow) in Ca 2+ -supplemented cultures. The Ca 2+ -supplemented sets also maintained high levels of open reaction centers of PS II in comparison to Ca 2+ -deprived cells. Increase in transcripts of both subunits ((rbcL and rbcS) of RubisCO from Ca 2+ -supplemented Anabaena cultures pointed out the role of Ca 2+ in sustenance of photosynthesis of cells via CO 2 fixation, thus, playing an important role in maintaining metabolic status of the heat-stressed cyanobacterium.

The unique medicinal and nutritional properties of honey are determined by its chemical composition. To evaluate the quality of honey, it is essential to study the surrounding vegetation where honeybees forage. In this study we used conventional melissopalynological and DNA barcoding techniques to determine the floral source of honey samples collected from different districts of the state of Mizoram, India. Pollen grains were isolated and genomic DNA was extracted from the honey samples. PCR amplification was carried out using universal barcode candidates ITS2 and rbcL to identify the plant species. Furthermore, TA cloning was carried out to screen the PCR amplicon libraries to identify the presence of multiple plant species. Results from both the melissopalynological and DNA barcoding analyses identified almost exactly the same 22 species, suggesting that both methods are suitable for analysis. However, DNA barcoding is easier and widely practiced. Hence, it can be concluded that DNA barcoding is a useful tool in determining the medicinal and commercial value of honey.

Brown algae play a dominant role in the primary productivity of coastal ecosystems and may have an efficient carbon fixation. In this work, 56 genes involved in inorganic carbon fixation were identified from the Saccharina japonica genome. Sequence structure analysis of these genes showed the existence of corresponding function domains and active amino acid sites highly conserved with other stramenopile species. The predicted subcellular localizations showed that Calvin cycle-related enzymes predominantly reside in the plastid and that putative C 4 -related enzymes are mainly distributed in the mitochondrion. We determined the transcriptional profiles and enzymatic activities of these C 4 -related enzymes in response to the KHCO 3 concentrations and light intensities. Pyruvate orthophosphate dikinase (PPDK) presented the greatest response to low HCO 3 - concentrations and high light intensity. Phosphoenolpyruvate carboxykinase (PEPCK) was up-regulated at low HCO 3 - concentrations to compensate for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and might be the crucial decarboxylase in this kelp. We propose that S. japonica might possess a PPDK- and PEPCK-dependent C 4 -like pathway that enables its rapid growth in natural coastal environments., (Copyright © 2019. Published by Elsevier Masson SAS.)

In the carboxylation reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco), which is fundamental to photosynthesis, scission of a C-C bond in the six-carbon gemdiolate intermediate forms a carbanion that must be protonated stereospecifically to form product. It is thought that a conserved lysine side chain (LYS175 in spinach Rubisco), in the immediate vicinity of the carbanion, provides the necessary proton. Here, we endeavor to determine from the electronic-structure calculations whether protonation via this route is energetically possible. The two-dimensional energy surface was mapped to determine the minimum energy path (MEP) using density functional theory (B3LYP) and incorporating basis set superposition and classical (London) dispersion corrections. The potential of mean force (free energy) was then calculated from ab initio molecular dynamics simulations with umbrella sampling in the vicinity of the MEP on the scission-protonation reaction coordinate. MEP calculations were also carried out to evaluate the possibility of an active-site water near the phosphate (P1) of RuBP, with an excess proton positioned at P1, as an alternative facilitator of stereospecific protonation via a classical Grotthuss mechanism. In both cases, the C-C bond scission in the six-carbon intermediate and proton transfer from the donor was found to be concerted and highly asynchronous, without a stable carbanion intermediate. However, the free energy change was unfavorable for direct protonation by the LYS175 side chain. In contrast, the Grotthuss mechanism yielded stable products and an activation energy in good agreement with experiment. It also provides a plausible mechanism for alternative product formed in enzyme mutations at the LYS175 position and is consistent with the observed deuterium isotope effects.

Improving the performance of the key photosynthetic enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by protein engineering is a critical strategy for increasing crop yields. The extensive chaperone requirement of plant Rubisco for folding and assembly has long been an impediment to this goal. Production of plant Rubisco in Escherichia coli requires the coexpression of the chloroplast chaperonin and four assembly factors. Here, we demonstrate that simultaneous expression of Rubisco and chaperones from a T7 promotor produces high levels of functional enzyme. Expressing the small subunit of Rubisco with a C-terminal hexahistidine-tag further improved assembly, resulting in a ~ 12-fold higher yield than the previously published procedure. The expression system described here provides a platform for the efficient production and engineering of plant Rubisco., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is considered to be the most abundant enzyme on Earth. Despite this, its full diversity and distribution across the domains of life remain to be determined. Here, we leverage a large set of bacterial, archaeal, and viral genomes recovered from the environment to expand our understanding of existing RuBisCO diversity and the evolutionary processes responsible for its distribution. Specifically, we report a new type of RuBisCO present in Candidate Phyla Radiation (CPR) bacteria that is related to the archaeal Form III enzyme and contains the amino acid residues necessary for carboxylase activity. Genome-level metabolic analyses supported the inference that these RuBisCO function in a CO2-incorporating pathway that consumes nucleotides. Importantly, some Gottesmanbacteria (CPR) also encode a phosphoribulokinase that may augment carbon metabolism through a partial Calvin-Benson-Bassham cycle. Based on the scattered distribution of RuBisCO and its discordant evolutionary history, we conclude that this enzyme has been extensively laterally transferred across the CPR bacteria and DPANN archaea. We also report RuBisCO-like proteins in phage genomes from diverse environments. These sequences cluster with proteins in the Beckwithbacteria (CPR), implicating phage as a possible mechanism of RuBisCO transfer. Finally, we synthesize our metabolic and evolutionary analyses to suggest that lateral gene transfer of RuBisCO may have facilitated major shifts in carbon metabolism in several important bacterial and archaeal lineages., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Carboxysomes are compartments in bacterial cells that promote efficient carbon fixation by sequestering RubisCO and carbonic anhydrase within a protein shell that impedes CO 2 escape. The key to assembling this protein complex is CcmM, a multidomain protein whose C-terminal region is required for RubisCO recruitment. This CcmM region is built as a series of copies (generally 3-5) of a small domain, CcmM S , joined by unstructured linkers. CcmM S domains have weak, but significant, sequence identity to RubisCO's small subunit, RbcS, suggesting that CcmM binds RubisCO by displacing RbcS. We report here the 1.35-Å structure of the first Thermosynechococcus elongatus CcmM S domain, revealing that it adopts a compact, well-defined structure that resembles that of RbcS. CcmM S , however, lacked key RbcS RubisCO-binding determinants, most notably an extended N-terminal loop. Nevertheless, individual CcmM S domains are able to bind RubisCO in vitro with 1.16 μm affinity. Two or four linked CcmM S domains did not exhibit dramatic increases in this affinity, implying that short, disordered linkers may frustrate successive CcmM S domains attempting to simultaneously bind a single RubisCO oligomer. Size-exclusion chromatography-coupled right-angled light scattering (SEC-RALS) and native MS experiments indicated that multiple CcmM S domains can bind a single RubisCO holoenzyme and, moreover, that RbcS is not released from these complexes. CcmM S bound equally tightly to a RubisCO variant in which the α/β domain of RbcS was deleted, suggesting that CcmM S binds RubisCO independently of its RbcS subunit. We propose that, instead, the electropositive CcmM S may bind to an extended electronegative pocket between RbcL dimers., (© 2019 Ryan et al.)

Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light-dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C 4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1 , a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C 4 enzyme genes and RUBISCO SMALL SUBUNIT2 Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C 4 photosynthesis., Competing Interests: The authors declare no conflict of interest.

The aim of this study is to assess the effect of methyl jasmonate (MeJA) and temperature on the valuable pharmaceuticals expression in a virus-mediated transient expression system, and so the Zuchini Yellow Mosaic Virus (ZYMV) based vector was used for transferring the GFP reporter gene and recombinant tissue plasminogen activator (rtPA) gene (K2S) to cucurbit (Cucurbita pepo L.). MeJA, temperature and time (days after inoculation), were evaluated as a factorial experiment in a completely randomized design (CRD). At first, the effect of all treatment combinations on GFP expression was assessed. At this step, the ELISA test was used to select the optimum treatment combination. ELISA method revealed the significant difference between applied treatments. The optimized treatment significantly increased the expression of rtPA compared to the control. The Real-Time PCR reaction for both GFP and rtPA genes showed no significant differences between optimum and control treatments, however, transcripts of the small subunit of RuBisCO were extremely down-regulated in optimum treatment condition. Reduction in RuBisCO expression at protein level was tangible under treatment condition based on the ELISA test. Therefore, it can be inferred that suppressing the expression of RuBisCO, probably resulted in higher access of expression system to free amino acids inside the cell. In this study, MeJA has been shown to be a positive factor, but the low temperature (17 °C), unlike previous studies, suppressed the expression of recombinant protein unexpectedly, probably due to the incompatibility of the viral construct with low temperature. In conclusion, the use of a suitable gene construct, which is not sensitive to temperature, is likely to result in a more favorable outcome.

Ribosyl 1,5-bisphosphate (PRibP) was discovered 65 years ago and was believed to be an important intermediate in ribonucleotide metabolism, a role immediately taken over by its "big brother" phosphoribosyldiphosphate. Only recently has PRibP come back into focus as an important player in the metabolism of ribonucleotides with the discovery of the pentose bisphosphate pathway that comprises, among others, the intermediates PRibP and ribulose 1,5-bisphosphate (cf. ribose 5-phosphate and ribulose 5-phosphate of the pentose phosphate pathway). Enzymes of several pathways produce and utilize PRibP not only in ribonucleotide metabolism but also in the catabolism of phosphonates, i.e., compounds containing a carbon-phosphorus bond. Pathways for PRibP metabolism are found in all three domains of life, most prominently among organisms of the archaeal domain, where they have been identified either experimentally or by bioinformatic analysis within all of the four main taxonomic groups, Euryarchaeota , TACK, DPANN, and Asgard. Advances in molecular genetics of archaea have greatly improved the understanding of the physiology of PRibP metabolism, and reconciliation of molecular enzymology and three-dimensional structure analysis of enzymes producing or utilizing PRibP emphasize the versatility of the compound. Finally, PRibP is also an effector of several metabolic activities in many organisms, including higher organisms such as mammals. In the present review, we describe all aspects of PRibP metabolism, with emphasis on the biochemical, genetic, and physiological aspects of the enzymes that produce or utilize PRibP. The inclusion of high-resolution structures of relevant enzymes that bind PRibP provides evidence for the flexibility and importance of the compound in metabolism., (Copyright © 2018 American Society for Microbiology.)

In C3 plants, photorespiration is an energy-expensive process, including the oxygenation of ribulose-1,5-bisphosphate (RuBP) by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the ensuing multi-organellar photorespiratory pathway required to recycle the toxic byproducts and recapture a portion of the fixed carbon. Photorespiration significantly impacts crop productivity through reducing yields in C3 crops by as much as 50% under severe conditions. Thus, reducing the flux through, or improving the efficiency of photorespiration has the potential of large improvements in C3 crop productivity. Here, we review an array of approaches intended to engineer photorespiration in a range of plant systems with the goal of increasing crop productivity. Approaches include optimizing flux through the native photorespiratory pathway, installing non-native alternative photorespiratory pathways, and lowering or even eliminating Rubisco-catalyzed oxygenation of RuBP to reduce substrate entrance into the photorespiratory cycle. Some proposed designs have been successful at the proof of concept level. A plant systems-engineering approach, based on new opportunities available from synthetic biology to implement in silico designs, holds promise for further progress toward delivering more productive crops to farmer's fields., (© 2018 Institute of Botany, Chinese Academy of Sciences.)

Soil is often collected from a suspect's tire, vehicle, or shoes during a criminal investigation and subsequently submitted to a forensic laboratory for analysis. Plant and insect material recovered in such samples is rarely analyzed, as morphological identification is difficult. In this study, DNA barcoding was used for taxonomic identifications by targeting the gene regions known to permit discrimination in plants [maturase K (matK) and ribulose 1,5-biphosphate carboxylase (rbcL)] and insects [cytochrome oxidase subunit I (COI)]. A DNA barcode protocol suitable for processing forensic-type biological fragments was developed and its utility broadly tested with forensic-type fragments (e.g., seeds, leaves, bark, head, legs; n, 213) isolated from soils collected within Virginia, USA (n, 11). Difficulties with PCR inhibitors in plant extracts and obtaining clean Sanger sequence data from insect amplicons were encountered during protocol development; however, the final protocol produced sequences specific to the expected locus and taxa. The overall quantity and quality of DNA extracted from the 213 forensic-type biological fragments was low (< 15 ng/μL). For plant fragments, only the rbcL sequence data was deemed reliable; thus, taxonomic identifications were limited to the family level. The majority of insect sequences matched COI in both GenBank and Barcode of Life DataSystems; however, they were identified as an undescribed environmental contaminant. Although limited taxonomic information was gleaned from the forensic-type fragments processed in this study, the new protocol shows promise for obtaining reliable and specific identifications through DNA barcoding, which could ultimately enhance the information gleaned from soil examinations.

Alcaligenes hydrogenophilus was used to verify the role of the electron donor and acceptor in apparent CO 2 fixation of chemoautotrophic bacteria. The response mechanisms underlying the apparent CO 2 fixation characteristics with different concentrations of electron donor and acceptor were elucidated by analyzing the transcription characteristics of the cbbL gene, cytoskeleton synthesis efficiency and extracellular free organic carbon concentration. The results showed that the apparent CO 2 fixation efficiency of A. hydrogenophilus was significantly influenced by the electron donor (H 2 ), and the degree of electron donor oxidization was responsible for the variation in apparent CO 2 fixation efficiency. Furthermore, transcription efficiency of the cbbL gene at low electron donor concentration was lower than that at high electron donor concentration, but excessive electron donor concentrations did not further increase cbbL gene transcription efficiency significantly. High oxygen concentration was not advantageous to cbbL gene transcription efficiency in A. hydrogenophilus, but could improve cell growth rate (protein synthesis rate) and apparent CO 2 fixation efficiency, implying that cytoskeleton synthesis efficiency is another important factor determining apparent CO 2 fixation efficiency and its contribution maybe greater than that of cbbL transcription. The results also indicated that high apparent CO 2 fixation efficiency required the matching of electron donor and acceptor., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The traditional green algal genus Chloromonas accommodates mesophilic, cold-tolerant and cold-adapted microorganisms. In this paper, we studied a new strain isolated from a wet hummock meadow in the High Arctic. We used morphological, ultrastructural and molecular data to assess the taxonomic position and phylogenetic relationships of the new isolate. The observed morphological features generally corresponded to the cold-tolerant Chloromonas characteristics. However, ellipsoidal or wide ellipsoidal vegetative cells, a massive parietal cup-shaped chloroplast with a number of continuously connected lobes, a thick cell wall, a prominent hemispherical papilla and the anterior position of an oblong or round eyespot distinguished the alga from all previously described Chloromonas species. Analyses of rbcL and 18S rRNA genes showed that the new strain formed an independent lineage within a clade containing mesophilic and psychrotolerant Chloromonas species. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker supported a separate species identity of the new isolate. Considering the morphological and molecular differences from its relatives, a new psychrotolerant species, Chloromonas svalbardensis, is proposed. Further, our results demonstrated the paraphyletic origin of Chloromonas within Chloromonadinia with genetically, morphologically and ecologically well-defined clades. We discuss a scenario of a possible Chloromonas split and revision., (© 2018 International Society of Protistologists.)

A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO 2 -concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO 2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO 2 around the enzyme Rubisco in specialized protein micro-compartments called carboxysomes. To date, chloroplastic expression of carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified carboxysomes, isometric with those of the source organism Cyanobium, within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-carboxysome structural proteins. This minimal gene set produces carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO 2 . This result demonstrates the formation of α-carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-carboxysomes in chloroplasts.

The relationship between ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase (Rca) levels was studied using transgenic rice overexpressing maize Rca (OX-mRca) and knockdown transgenic rice expressing antisense Rca (KD-Rca). The ratio of Rubisco to total soluble protein was lower in OX-mRca, whereas it was higher in KD-Rca than in WT, indicating that Rca expression was negatively correlated with Rubisco content. The expressions of other Calvin-Benson-Bassham cycle enzymes such as sedoheptulose-1,7-bisphosphatase and phosphoribulokinase analyzed by immunoblotting did not show such a negative correlation with Rca, suggesting that the effect of Rca on protein expression may be specific for Rubisco. Although Rubisco content was decreased in OX-mRca, the transcript levels of the Rubisco large subunit (OsRbcL) and the Rubisco small subunit mostly increased in OX-mRca as well as in KD-Rca. Additionally, polysome loading of OsRbcL was slightly higher in OX-mRca than it was in WT, suggesting that the OsRbcL translation activity was likely stimulated by overexpression of Rca. 35 S-methionine labeling experiments demonstrated that there was no significant difference in the stability of newly synthesized Rubisco among genotypes. However, 35 S-methionine-labeled Rubisco was marginally decreased in OX-mRca and increased in KD-Rca compared to the WT. These results suggest that Rca negatively affects the Rubisco content, possibly in the synthesis step.

The catalytic performance of the major CO 2 -assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO 2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from arctic waters were collected, cultivated, and analyzed for their CO 2 -fixing capability. We characterized the kinetic properties of five and determined the crystal structures of four Rubiscos selected for their high CO 2 -fixing efficiency. The DNA sequences of the rbc L and rbc S genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The V max and K m for the oxygenase and carboxylase activities at 25 °C and the specificity factors ( S c/o ) at 15, 25, and 35 °C were determined. The S c/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO 2 relative to O 2 Structurally, diatom Rubiscos belong to form I C/D, containing small subunits characterized by a short βA-βB loop and a C-terminal extension that forms a β-hairpin structure (βE-βF loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxyproline, β-hydroxyleucine, hydroxylated and nitrosylated cysteine, mono- and dihydroxylated lysine, and trimethylated lysine. Our studies suggest adaptation toward achieving efficient CO 2 fixation in arctic diatom Rubiscos., (© 2018 Valegård et al.)

Increasing agricultural productivity is one of the major challenges our society faces. While multiple strategies to enhance plant carbon fixation have been suggested, and partially implemented, most of them are restricted to relatively simple modifications of endogenous metabolism, i.e., "low hanging fruit". Here, I portray the next generation of metabolic solutions to increase carbon fixation rate and yield. These strategies involve major rewiring of central metabolism, including dividing Rubisco's catalysis between several enzymes, replacing Rubisco with a different carboxylation reaction, substituting the Calvin Cycle with alternative carbon fixation pathways, and engineering photorespiration bypass routes that do not release carbon. While the barriers for implementing these elaborated metabolic architectures are quite significant, if we truly want to revolutionize carbon fixation, only daring engineering efforts will lead the way., (Copyright © 2017 The Author. Published by Elsevier B.V. All rights reserved.)

Oxidation of the cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) leads to inactivation and promotes structural changes that increase the proteolytic sensitivity and membrane association propensity related to its catabolism. To uncover the individual role of the different cysteines, the sequential order of modification under increasing oxidative conditions was determined using chemical labeling and mass spectrometry. Besides, site-directed RubisCO mutants were obtained in Chlamydomonas reinhardtii replacing single conserved cysteines (Cys84, Cys172, Cys192, Cys247, Cys284, Cys427, Cys459 from the large and sCys41, sCys83 from the small subunit) and the redox properties of the mutant enzymes were determined. All mutants retained significant carboxylase activity and grew photoautotrophically, indicating that these conserved cysteines are not essential for catalysis. Cys84 played a noticeable structural role, its replacement producing a structurally altered enzyme. While Cys247, Cys284, and sCys83 were not affected by the redox environment, all other residues were oxidized using a disulfide/thiol ratio of around two, except for Cys172 whose oxidation was distinctly delayed. Remarkably, Cys192 and Cys427 were apparently protective, their absence leading to a premature oxidation of critical residues (Cys172 and Cys459). These cysteines integrate a regulatory network that modulates RubisCO activity and conformation in response to oxidative conditions.

The analysis of RubisCO genes is a highly useful instrument to explore the diversity of chemoautotrophic bacteria using the Calvin-Benson-Bassham cycle for CO 2 fixation. However, because of the wide taxonomic distribution of phylogenetically related RubisCO forms, environmental studies targeting chemoautotrophs are hampered in habitats dominated by phototrophs. Here, we report the development of a gene marker that specifically detects form IA RubisCO genes in bacteria, excluding photoautotrophic representatives. The high specificity of the PCR assay was confirmed by sequence analysis of DNA obtained from the photic zone of six lakes, were chemoautotrophs are outnumbered by Cyanobacteria also using form IA RubisCO for CO 2 assimilation., (© 2018 The Authors. Journal of Basic Microbiology Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The CO 2 -concentrating mechanism (CCM) has evolved to improve the efficiency of photosynthesis in autotrophic cyanobacteria. CmpR, a LysR-type transcriptional regulator (LTTR) from Synechococcus elongatus PCC 7942, was found to regulate CCM-related genes under low-CO 2 conditions. Here, the dimeric structure of the effector-binding domain of CmpR (CmpR-EBD) in complex with the co-activator ribulose 1,5-bisphosphate (RuBP) is reported at 2.15 Å resolution. One RuBP molecule binds to the inter-domain cleft between the two subunits of the CmpR-EBD dimer. Structural comparison combined with sequence analyses demonstrated that CmpR-EBD has an overall structure similar to those of LTTRs of known structure, but possesses a distinctly different effector-binding pattern.

Among green freshwater microalgae, Chlamydomonas reinhardtii has the most comprehensive and developed molecular toolkit, however, advanced genetic and metabolic engineering driven from the nuclear genome is generally hindered by inherently low transgene expression levels. Progressive strain development and synthetic promoters have improved the capacity of transgene expression; however, the responsible regulatory mechanisms are still not fully understood. Here, we elucidate the sequence specific dynamics of native regulatory element insertion into nuclear transgenes. Systematic insertions of the first intron of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 (rbcS2i1) throughout codon-optimized coding sequences (CDS) generates optimized algal transgenes which express reliably in C. reinhardtii. The optimal rbcS2i1 insertion site for efficient splicing was systematically determined and improved gene expression rates were shown using a codon-optimized sesquiterpene synthase CDS. Sequential insertions of rbcS2i1 were found to have a step-wise additive effect on all levels of transgene expression, which is likely correlated to a synergy of transcriptional machinery recruitment and mimicking the short average exon lengths natively found in the C. reinhardtii genome. We further demonstrate the value of this optimization with five representative transgene examples and provide guidelines for the design of any desired sequence with this strategy.

Two new genera (Streptosarcina and Streptofilum) and three new species (Streptosarcina arenaria, S. costaricana and Streptofilum capillatum) of streptophyte algae were detected in cultures isolated from terrestrial habitats of Europe and Central America and described using an integrative approach. Additionally, a strain isolated from soil in North America was identified as Hormidiella parvula and proposed as an epitype of this species. The molecular phylogeny based on 18S rRNA and rbcL genes, secondary structure of ITS-2, as well as the morphology of vegetative and reproductive stages, cell ultrastructure, ecology and distribution of the investigated strains were assessed. The new genus Streptosarcina forms a sister lineage to the genus Hormidiella (Klebsormidiophyceae). Streptosarcina is characterized by packet-like (sarcinoid) and filamentous thalli with true branching and a cell organization typical for Klebsormidiophyceae. Streptofilum forms a separate lineage within Streptophyta. This genus represents an easily disintegrating filamentous alga which exhibits a cell coverage of unique structure: layers of submicroscopic scales of piliform shape covering the plasmalemma and exfoliate inside the mucilage envelope surrounding cells. The implications of the discovery of the new taxa for understanding evolutionary tendencies in the Streptophyta, a group of great evolutionary interest, are discussed., (Copyright © 2018 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The protein coding genes (rbcL/cbbL/cbbM) for RuBisCO large subunit, the most abundant protein on earth that drives biological CO2 fixation, were considered as useful marker genes in characterizing CO2-assimilating plankton. However, their community specificity has hindered comprehensive screening of genetic diversity. In this study, six different rbcL/cbbL/cbbM primers were employed to screen clone libraries to identify CO2-assimilating plankton in Jiaozhou Bay. The following community compositions were observed: the community components in Form I A/B rbcL/cbbL clone library mainly comprised Chlorophyta and Proteobacteria, Form ID2 and ID3 libraries consisted of Bacillariophyta, Form II cbbM library consisted of Proteobacteria and Alveolata, and both Form I green and red libraries included Proteobacteria, respectively. At the genus taxonomic level, no overlaps among these clone libraries were observed, except for ID2 and ID3. Overall, the phytoplankton in Jiaozhou Bay mainly consists of Bacillariophyta, Chlorophyta, Cryptophyta, Haptophyceae and Alveolata. The CO2-assimilating prokaryotes mainly consist of Proteobacteria. Considering the high-sequence specificities of these marker genes, we propose that the joint use of multiple primers may be utilized in unveiling the diversity of CO2-assimilating organisms. In addition, designing novel RuBisCO gene primers that generate longer amplicons and have broader phylogenetic coverage may be necessary in the future.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a ∼530 kDa complex of 8 large (RbcL) and 8 small subunits (RbcS), mediates the fixation of atmospheric CO 2 into usable sugars during photosynthesis. Despite its fundamental role, Rubisco is a remarkably inefficient enzyme and thus is produced by plants in huge amounts. It has long been a key target for bioengineering with the goal to increase crop yields. However, such efforts have been hampered by the complex requirement of Rubisco biogenesis for molecular chaperones. Recent studies have identified an array of auxiliary factors needed for the folding and assembly of the Rubisco subunits. The folding of plant RbcL subunits is mediated by the cylindrical chloroplast chaperonin, Cpn60, and its cofactor Cpn20. Folded RbcL requires a number of additional Rubisco specific assembly chaperones, including RbcX, Rubisco accumulation factors 1 (Raf1) and 2 (Raf2), and the Bundle sheath defective-2 (BSD2), to mediate the assembly of the RbcL 8 intermediate complex. Incorporation of the RbcS and displacement of the assembly factors generates the active holoenzyme. An Escherichia coli strain expressing the chloroplast chaperonin and auxiliary factors now allows the expression of functional plant Rubisco, paving the way for Rubisco engineering by large scale mutagenesis. Here, we review our current understanding on how these chaperones cooperate to produce one of the most important enzymes in nature.

Chrysanthemum carinatum Schousb and Kalimeris indica are widely distributed edible vegetables and the sources of the Chinese medicine Asteraceae . The complete chloroplast (cp) genome of Asteraceae usually occurs in the inversions of two regions. Hence, the cp genome sequences and structures of Asteraceae species are crucial for the cp genome genetic diversity and evolutionary studies. Hence, in this paper, we have sequenced and analyzed for the first time the cp genome size of C. carinatum Schousb and K. indica , which are 149,752 bp and 152,885 bp, with a pair of inverted repeats (IRs) (24,523 bp and 25,003) separated by a large single copy (LSC) region (82,290 bp and 84,610) and a small single copy (SSC) region (18,416 bp and 18,269), respectively. In total, 79 protein-coding genes, 30 distinct transfer RNA (tRNA) genes, four distinct rRNA genes and two pseudogenes were found not only in C. carinatum Schousb but also in the K. indica cp genome. Fifty-two (52) and fifty-nine (59) repeats, and seventy (70) and ninety (90) simple sequence repeats (SSRs) were found in the C. carinatum Schousb and K. indica cp genomes, respectively. Codon usage analysis showed that leucine, isoleucine, and serine are the most frequent amino acids and that the UAA stop codon was the significantly favorite stop codon in both cp genomes. The two inversions, the LSC region ranging from trnC-GCA to trnG-UCC and the whole SSC region were found in both of them. The complete cp genome comparison with other Asteraceae species showed that the coding area is more conservative than the non-coding area. The phylogenetic analysis revealed that the rbcL gene is a good barcoding marker for identifying different vegetables. These results give an insight into the identification, the barcoding, and the understanding of the evolutionary model of the Asteraceae cp genome.

Rubisco-based engineered Escherichia coli MZLFB (E. coli BL21(DE3) Δzwf, Δldh, Δfrd) containing heterologous phosphoribulokinase (Prk) and Ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) was constructed for the mixotrophic growth. However, in situ CO 2 recycling was hindered by clogs of pyruvate during glucose metabolism, which consequently resulted in an insufficient regeneration of NAD + through the pflB-mediated ethanol production. Recombinant plasmid pLOI295 (encodes pyruvate decarboxylase and alcohol dehydrogenase II, referred to as the Pdc-based carbon tap valve (CTV) for convenience) was introduced into E. coli MZLFB + CTV to bypass the pflB-mediated ethanol production. Results show that while the C-2/C-1 ratio (i.e., the molar ratio of ethanol and acetate to formate and total CO 2 ) for parental strain MZLFB was 1.0 ± 0.1, the C-2/C-1 for MZLFB + CTV increased to 1.6 ± 0.1. This indicates that the Pdc-based CTV enhanced the performance of in situ CO 2 recycling. By simultaneously utilizing glucose and CO 2 , the fermentation product yield of MZLFB + CTV exceeded the normal theoretical yield and reached 2.2 ± 0.0 (mol/mol). In silico analysis shows that 61% of the glucose consumption went through the Rubisco-based engineered pathway when the CTV was equipped. Also shown are the average CO 2 consumption rate of 55.3 mg L -1 ·h -1 and an average ethanol production rate of 144.8 mg L -1 ·h -1 . The conversion of CO 2 to ethanol through the Rubisco-based engineered pathway and the Pdc-based carbon tap valve is important for mixotrophic growth, since these two modules serve as the energy sink to achieve intracellular energy balance. Also, during mixotrophic growth, ATP production from a certain percentage (39% in this study) of the EMP pathway activity is needed for mixotrophic growth., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)