Tobacco Chloroplasts Research Articles

High-level production of single chain monellin mutants with enhanced sweetness and stability in tobacco chloroplasts

Plastid-based MNEI protein mutants retain the structure, stability and sweetness of their bacterial counterparts, confirming the attractiveness of the plastid transformation technology for high-yield production of recombinant proteins. The prevalence of obesity and diabetes has dramatically increased the industrial demand for the development and use of alternatives to sugar and traditional sweeteners. Sweet proteins, such as MNEI, a single chain derivative of monellin, are the most promising candidates for industrial applications. In this work, we describe the use of tobacco chloroplasts as a stable plant expression platform to produce three MNEI protein mutants with improved taste profile and stability. All plant-based proteins were correctly expressed in tobacco chloroplasts, purified and subjected to in-depth chemical and sensory analyses. Recombinant MNEI mutants showed a protein yield ranging from 5% to more than 50% of total soluble proteins, which, to date, represents the highest accumulation level of MNEI mutants in plants. Comparative analyses demonstrated the high similarity, in terms of structure, stability and function, of the proteins produced in plant chloroplasts and bacteria. The high yield and the extreme sweetness perceived for the plant-derived proteins prove that plastid transformation technology is a safe, stable and cost-effective production platform for low-calorie sweeteners, with an estimated production of up to 25-30mg of pure protein/plant.

Red algal Rubisco fails to accumulate in transplastomic tobacco expressing Griffithsiamonilis RbcL and RbcS genes.

In C3 plants, the carbon fixation step catalyzed by ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) represents a major rate‐limiting step due to the competing oxygenation reaction, which leads to the energy‐intensive photorespiration and lowers the overall photosynthetic efficiency. Hence, there is great biotechnological interest in replacing the Rubisco in C3 crops with a more efficient enzyme. The Rubisco enzymes from red algae are among the most attractive choices due to their remarkable preference for carboxylation over oxygenation reaction. However, the biogenesis of Rubisco is extremely complex. The Rubisco enzymes in plants, algae, and cyanobacteria are made up of eight large and eight small subunits. The folding of the large subunits and the assembly of the large subunits with the small subunits to form a functional holoenzyme require specific chaperonin complexes and assembly factors. As a result, previous success in expressing foreign Rubisco in plants has been limited to Rubisco large subunits from closely related plant species and simpler bacterial enzymes. In our previous work, we successfully replaced the Rubisco in tobacco with a cyanobacterial enzyme, which was able to support the phototrophic growth of the transgenic plants. In this work, we used the same approach to express the Rubisco subunits from the red alga Griffithsia monilis in tobacco chloroplasts in the absence of the tobacco Rubisco large subunit. Although the red algal Rubisco genes are being transcribed in tobacco chloroplasts, the transgenic plants lack functional Rubisco and can only grow in a medium containing sucrose. Our results suggest that co‐expression of compatible chaperones will be necessary for successful assembly of red algal Rubisco in plants.

Sustained Photosynthesis in Tobacco Leaves by Fast CO<sub>2</sub>-Fixing Enzymes Encapsulated in Micro-Compartments

Improvement of yield potential in global food crops has hit a breeding road-block which could be solved through genetic engineering. One promising strategy is the introduction of cyanobacterial CO2-concentrating-mechanisms (CCMs) into plant chloroplasts to enhance photosynthesis and yield. CCMs actively accumulate CO2 within specialized microcompartments called carboxysomes, where the enzyme Rubisco rapidly fixes CO2. Here we replaced the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco and key structural proteins of α- carboxysomes in tobacco chloroplasts. Transformed plants produced purifiable carboxysomes, isometric with those of the source organism Cyanobium marinum. Carboxysomes encapsulated the introduced Rubisco and enabled autotrophic growth at elevated CO2. Our results demonstrate the formation of α-carboxysomes from a minimal gene set, and that cyanobacterial Form-1A Rubisco supports C3 photosynthesis at high CO2. This major advance informs the step-wise construction of fully-functional α-carboxysomes in chloroplasts which, along with other CCM components, are expected to improve crop plant performance.

Blue Native/SDS‐PAGE and iTRAQ‐Based Chloroplasts Proteomics Analysis of Nicotiana tabacum Leaves Infected with M Strain of Cucumber Mosaic Virus Reveals Several Proteins Involved in Chlorosis Symptoms

Virus infection in plants involves necrosis, chlorosis, and mosaic. The M strain of cucumber mosaic virus (M-CMV) has six distinct symptoms: vein clearing, mosaic, chlorosis, partial green recovery, complete green recovery, and secondary mosaic. Chlorosis indicates the loss of chlorophyll which is highly abundant in plant leaves and plays essential roles in photosynthesis. Blue native/SDS-PAGE combined with mass spectrum was performed to detect the location of virus, and proteomic analysis of chloroplast isolated from virus-infected plants was performed to quantify the changes of individual proteins in order to gain a global view of the total chloroplast protein dynamics during the virus infection. Among the 438 proteins quantified, 33 showed a more than twofold change in abundance, of which 22 are involved in the light-dependent reactions and five in the Calvin cycle. The dynamic change of these proteins indicates that light-dependent reactions are down-accumulated, and the Calvin cycle was up-accumulated during virus infection. In addition to the proteins involved in photosynthesis, tubulin was up-accumulated in virus-infected plant, which might contribute to the autophagic process during plant infection. In conclusion, this extensive proteomic investigation on intact chloroplasts of virus-infected tobacco leaves provided some important novel information on chlorosis mechanisms induced by virus infection.

Experimental reconstruction of double-stranded break repair-mediated plastid DNA insertion into the tobacco nucleus.

The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes. DNA from the endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a precursor of the nucleated eukaryotic host. An experimental confirmation regarding the molecular mechanisms responsible for organelle DNA incorporation into nuclei has not been performed until the present analysis. Here we introduced double-stranded DNA breaks into the nuclear genome of tobacco through inducible expression of I-SceI, and showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through double-stranded DNA break repair. Microhomology-mediated linking of disparate segments of chloroplast DNA occurs frequently during healing of induced nuclear double-stranded breaks (DSB) but the resulting nuclear integrants are often immediately unstable. Non-Mendelian inheritance of a selectable marker (neo), used to identify plastid DNA transfer, was observed in the progeny of about 50% of lines emerging from the screen. The instability of these de novo nuclear insertions of plastid DNA (nupts) was shown to be associated with deletion not only of the nupt itself but also of flanking nuclear DNA within one generation of transfer. This deletion of pre-existing nuclear DNA suggests that the genetic impact of organellar DNA transfer to the nucleus is potentially far greater than previously thought.

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice.

SummaryInhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder haemophilia A and occurs in 20%–30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4+ T‐cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐ to 42‐fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII‐HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titres ~10‐fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4+ CD25+FoxP3+ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.

Relative association of Rubisco with manganese and magnesium as a regulatory mechanism in plants

Rubisco, the enzyme that constitutes as much as half of the protein in a leaf, initiates either the photorespiratory pathway that supplies reductant for the assimilation of nitrate into amino acids or the C3 carbon fixation pathway that generates carbohydrates. The relative rates of these two pathways depend both on the relative extent to which O2 and CO2 occupies the active site of Rubisco and on whether manganese or magnesium is bound to the enzyme. This study quantified the activities of manganese and magnesium in isolated tobacco chloroplasts and the thermodynamics of binding of these metals to Rubisco purified from tobacco or a bacterium. In tobacco chloroplasts, manganese was less active than magnesium, but Rubisco purified from tobacco had a higher affinity for manganese. The activity of each metal in the chloroplast was similar in magnitude to the affinity of tobacco Rubisco for each. This indicates that, in tobacco chloroplasts, Rubisco associates almost equally with both metals and rapidly exchanges one metal for the other. Binding of magnesium was similar in Rubisco from tobacco and a bacterium, whereas binding of manganese differed greatly between the Rubisco from these species. Moreover, the ratio of leaf manganese to magnesium in C3 plants increased as atmospheric CO2 increased. These results suggest that Rubisco has evolved to improve the energy transfers between photorespiration and nitrate assimilation and that plants regulate manganese and magnesium activities in the chloroplast to mitigate detrimental changes in their nitrogen/carbon balance as atmospheric CO2 varies.

Faster photosynthetic induction in tobacco by expressing cyanobacterial flavodiiron proteins in chloroplasts.

Plants grown in the field experience sharp changes in irradiation due to shading effects caused by clouds, other leaves, etc. The excess of absorbed light energy is dissipated by a number of mechanisms including cyclic electron transport, photorespiration, and Mehler-type reactions. This protection is essential for survival but decreases photosynthetic efficiency. All phototrophs except angiosperms harbor flavodiiron proteins (Flvs) which relieve the excess of excitation energy on the photosynthetic electron transport chain by reducing oxygen directly to water. Introduction of cyanobacterial Flv1/Flv3 in tobacco chloroplasts resulted in transgenic plants that showed similar photosynthetic performance under steady-state illumination, but displayed faster recovery of various photosynthetic parameters, including electron transport and non-photochemical quenching during dark-light transitions. They also kept the electron transport chain in a more oxidized state and enhanced the proton motive force of dark-adapted leaves. The results indicate that, by acting as electron sinks during light transitions, Flvs contribute to increase photosynthesis protection and efficiency under changing environmental conditions as those found by plants in the field.

Over-expression of the Arabidopsis formate dehydrogenase in chloroplasts enhances formaldehyde uptake and metabolism in transgenic tobacco leaves.

Over-expression of AtFDH controlled by the promoter of Rubisco small subunit in chloroplasts increases formaldehyde uptake and metabolism in tobacco leaves. Our previous study showed that formaldehyde (HCHO) uptake and resistance in tobacco are weaker than in Arabidopsis. Formate dehydrogenase in Arabidopsis (AtFDH) is a key enzyme in HCHO metabolism by oxidation of HCOOH to CO2, which enters the Calvin cycle to be assimilated into glucose. HCHO metabolic mechanism in tobacco differs from that in Arabidopsis. In this study, AtFDH was over-expressed in the chloroplasts of transgenic tobacco using a light inducible promoter. 13C-NMR analysis showed that the carbon flux from H13CHO metabolism was not introduced into the Calvin cycle to produce glucose in transgenic tobacco leaves. However, the over-expression of AtFDH significantly enhanced the HCHO metabolism in transgenic leaves. Consequently, the productions of [4-13C]Asn, [3-13C]Gln, [U-13C]oxalate, and H13COOH were notably greater in transgenic leaves than in non-transformed leaves after treatment with H13CHO. The increased stomatal conductance and aperture in transgenic leaves might be ascribed to the increased yield of oxalate in the guard cells with over-expressed AtFDH in chloroplasts. Accordingly, the transgenic plants exhibited a stronger capacity to absorb gaseous HCHO. Furthermore, the higher proline content in transgenic leaves compared with non-transformed leaves under HCHO stress might be attributable to the excess formate accumulation and Gln production. Consequently, the HCHO-induced oxidative stress was reduced in transgenic leaves.

LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/.

Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes.

Halogenation of natural compounds in planta is rare. Herein, a successful engineering of tryptophan 6-halogenation into the plant context by heterologous expression of the Streptomyces toxytricini Stth gene and localization of its enzymatic product in various tobacco cell compartments is described. When co-expressed with the flavin reductase rebF from Lechevalieria aerocolonigenes, Stth efficiently produced chlorinated tryptophan in the cytosol. Further, supplementation of KBr yielded the brominated metabolite. More strikingly, targeting of the protein to the chloroplasts enabled effective halogenation of tryptophan even in absence of the partner reductase, providing crucial evidence for sufficient, organelle-specific supply of the FADH2 cofactor to drive halogen integration. Incorporation of an alternative enzyme, the 7-halogenase RebH from L. aerocolonigenes, into the metabolic set-up resulted in the formation of 6,7-dichlorotryptophan. Finally, expression of tryptophan decarboxylase (tdc) in concert with stth led to the generation of 6-chlorotryptamine, a new-to-nature precursor of monoterpenoid indole alkaloids. In sum, the report highlights the tremendous application potential of plants as a unique chassis for the engineering of rare and valuable halogenated natural products, with chloroplasts as the cache of reduction equivalents driving metabolic reactions.

Comparison of effectiveness of 5'-regulatory sequences in transplastomic tobacco chloroplasts.

The development of tools which ensure the desired level of transgene expression in plastids is a prerequisite for the effective utilization of these plant organelles for the deployment of bioactive proteins. High-level accumulation of target proteins is considered as a positive feature of transplastomic plants, but excessive accumulation of foreign proteins may have deleterious effects on host plants. On the other hand, expression at low levels can result in ineffective phenotypes. We compared the effectiveness of different 5'-regulatory sequences in driving the expression of a reporter gene, β-glucuronidase (uidA), in tobacco chloroplasts. To achieve varying expression levels, we have chosen heterologous 5'-regulatory sequences which either differ significantly from their homologous counterparts or depend on specific nuclear encoded factors. The Medicago truncatula psbA promoter/5'-UTR supported the highest levels of protein accumulation, surpassing the other tested sequences by two to three orders of magnitude. The heterologous regulatory sequence of Phaseolus vulgaris rbcL gene was as efficient in tobacco chloroplasts as the corresponding homologous promoter/5'-UTR. The Arabidopsis thaliana ndhF promoter/5'-UTR supported as high reporter activity levels as the rbcL 5'-sequences, whereas the effectiveness of A. thaliana psbN promoter/5'-UTR was three fold lower. The characterized regulatory sequences can be utilized to establish transplastomic lines with desirable levels of target protein accumulation. The ability to control transgene expression should be useful for achieving appropriate levels of protein accumulation and thereby avoid their negative impacts on host plant physiology.

Transferring a bioactive

Plant Science![Figure][1] Once only found in small hairs on sweet wormwood plants, artemisinin can now be produced by tobacco plants. PHOTO: MANFRED RUCKSZIO / ALAMY STOCK PHOTO Artemisinin, which is critical in defense against malaria, was originally found in tiny hairs on the surface of leaves of the plant Artemisia annua. But this source plant does not support a sufficiently stable supply of artemisinin for worldwide use. Fuentes et al. have developed a synthetic biology approach termed COSTREL (combinatorial supertransformation of transplastomic recipient lines) that produces the immediate precursor of artemisinin, artemisinic acid, in widely cultivated tobacco ( Nicotiana tabacum cv. Petit Havana). The first stage of COSTREL involves transformation of tobacco chloroplasts with genes encoding the core enzymes of the artemisinin biosynthetic pathway. With that pathway established, the second stage involves combinatorial nuclear transformation to superimpose genes that regulate flux through the biosynthetic pathway. A transformation mix supports selection of the most optimal gene combination. The resulting tobacco plants produce artemisinic acid at up to ~4.8 kg per acre. eLife 10.7554/eLife.13664 (2016). [1]: pending:yes

Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants

Plants are low cost platforms for the production of recombinant proteins, but their complexity renders the purification of plant recombinant proteins more difficult than proteins expressed in yeast or bacteria. Plastid transformation enables high-level expression of foreign genes and the accumulation of recombinant proteins in plastid organelles. Histidine (His) tags are widely used for affinity purification of recombinant proteins in a nickel column. The human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of the body. The truncated form of the tissue plasminogen activator (K2S) has a longer plasma half-life, better diffusion into the clot, and higher fibrinolytic activity. In a construct designed to insert the K2S gene in the tobacco chloroplast, the sequence of six histidines and a factor Xa protease site was fused to the C-terminus of the K2S protein. The presence and amount of tPA recombinant protein in transplastomic tobacco plants was estimated by ELISA analysis using a specific antibody. The protein was purified from total soluble protein, insoluble protein aggregates and the protein was extracted from the isolated chloroplast using nickel resin and a chromatography column. After digestion of the purified protein with factor Xa, the presence of the purified tPA protein was confirmed by western blot analysis.

Testing the Role of the N-Terminal Tail of D1 in the Maintenance of Photosystem II in Tobacco Chloroplasts.

A key step in the repair of photoinactivated oxygen-evolving photosystem II (PSII) complexes is the selective recognition and degradation of the damaged PSII subunit, usually the D1 reaction center subunit. FtsH proteases play a major role in D1 degradation in both cyanobacteria and chloroplasts. In the case of the cyanobacterium Synechocystis sp. PCC 6803, analysis of an N-terminal truncation mutant of D1 lacking 20 amino-acid residues has provided evidence that FtsH complexes can remove damaged D1 in a processive reaction initiated at the exposed N-terminal tail. To test the importance of the N-terminal D1 tail in higher plants, we have constructed the equivalent truncation mutant in tobacco using chloroplast transformation techniques. The resulting mutant grew poorly and only accumulated about 25% of wild-type levels of PSII in young leaves which declined as the leaves grew so that there was little PSII activity in mature leaves. Truncating D1 led to the loss of PSII supercomplexes and dimeric complexes in the membrane. Extensive and rapid non-photochemical quenching (NPQ) was still induced in the mutant, supporting the conclusion that PSII complexes are not required for NPQ. Analysis of leaves exposed to high light indicated that PSII repair in the truncation mutant was impaired at the level of synthesis and/or assembly of PSII but that D1 could still be degraded. These data support the idea that tobacco plants possess a number of back-up and compensatory pathways for removal of damaged D1 upon severe light stress.

Changes in chloroplast ultrastructure of tobacco plants in the course of protection from oxidative stress under hypothermia

Changes of ultrastructural organization of tobacco (Nicotiana tabacum L. cv. Samsun) chloroplasts associated with plant protection from oxidative stress during hypothermia were studied. It was found that the chilling hardening (6 days at 8°C) was accompanied by the significant reduction in the number of grana in a chloroplast simultaneously with area reduction of a granum that led to 30% decrease in the total area of grana in tobacco chloroplasts. In the course of tobacco plant hardening, approximately twofold decrease in generation rate of superoxide anion radical and 30% decrease in content of hydrogen peroxide occurred, which indicates retardation of oxidative processes in plant cells during the cold exposure. It is suggested that the ultrastructural changes in chloroplast organization that were found may prevent an overreduction of an electron-transport chain under hypothermia, when the ability of the Calvin cycle to utilize ATP and NADP·H is significantly reduced. The balanced work of components of light and dark photosynthetic phases may prevent the excessive generation of reactive oxygen species and render formation of tobacco plant tolerance to hypothermia.

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6% of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression.

Cooperation between the chloroplast psbA 5'-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region.

Chloroplast mRNA translation is regulated by the 5'-untranslated region (5'-UTR). Chloroplast 5'-UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5'-UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5'-UTR with the E. coli phage T7 gene 10 5'-UTR, a highly active 5'-UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5'-UTR with a cognate 5'-coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5'-UTR and its coding region is important for translational initiation.

Production and characterization of fungal β-glucosidase and bacterial cellulases by tobacco chloroplast transformation

The high capacity of the chloroplast genome to integrate and express transgenes at high levels makes transplastomic technology a good option for overexpressing proteins of interest. This report presents the stable expression of β-glucosidase (bgl1 gene) from Aspergillus niger and two cellulases (celA and celB genes) from Thermotoga neapolitana into the chloroplast genome of tobacco. The pES6, pHM4, pHM5 and pHM6 vectors were derived from the pES4 plasmid containing bgl1, celA-celB, celA and celB synthetic genes, respectively. All of the genes were flanked by a synthetic rrn16 promoter and the 3′UTR from rbcL gene. The integration of the genes into intergenic regions rrn16 and 3′rps12 of the inverted repeats was confirmed by Southern blot analysis. Stable expression and processing of monocistronic mRNA were confirmed by Northern blot analysis, and protein functionality was analysed via enzymatic activity assay. The recombinant enzymes exhibited high enzymatic activity at pH 5 (β-glucosidase: 30.45 U mg−1 of TSP, celA-celB 58 U mg−1 of TSP, celA 49.10 U mg−1 of TSP and celB 48.72 U mg−1 of TSP). In addition, β-glucosidase exhibited high activity at 40 °C, whereas cellulases type A (celA) and type B (celB) showed high activity at 65 °C. NtpES6, NtpHM5 and NtpHM6 plants showed a similar phenotype compared with the wild type plants; however, NtpHM4 plants presented an abnormal phenotype with variegated leaves. This study, demonstrated that hydrolytic genes such as bgl1, celA and celB could be integrated and expressed correctly in the chloroplast genome. This work provides new information on methods and strategies for the expression of hydrolytic enzymes that are potentially useful for biotechnological applications using transplastomic plants.

Expression of the Multimeric and Highly Immunogenic Brucella spp. Lumazine Synthase Fused to Bovine Rotavirus VP8d as a Scaffold for Antigen Production in Tobacco Chloroplasts

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein which can accommodate foreign polypeptides or protein domains fused to its N-termini, markedly increasing their immunogenicity. The inner core domain (VP8d) of VP8 spike protein from bovine rotavirus is responsible for viral adhesion to sialic acid residues and infection. It also displays neutralizing epitopes, making it a good candidate for vaccination. In this work, the BLS scaffold was assessed for the first time in plants for recombinant vaccine development by N-terminally fusing BLS to VP8d and expressing the resulting fusion (BLSVP8d) in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern, northern and western blot. BLSVP8d was highly expressed, representing 40% of total soluble protein (4.85 mg/g fresh tissue). BLSVP8d remained soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature. Soluble protein extracts from fresh and lyophilized leaves were able to induce specific neutralizing IgY antibodies in a laying hen model. This work presents BLS as an interesting platform for highly immunogenic injectable, or even oral, subunit vaccines. Lyophilization of transplastomic leaves expressing stable antigenic fusions to BLS would further reduce costs and simplify downstream processing, purification and storage, allowing for more practical vaccines.