Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine–Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5′-untranslated region (5′-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5′-UTR and DB region promotes accumulation of NPTII up to ~16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield (~23% of TSP) was obtained from a construct in which the T7g10 5′-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.     

High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions

The Thermobifida fusca cel6A gene encoding an endoglucanase was fused to three different downstream box (DB) regions to generate cel6A genes with 14 amino acid fusions. The DB-Cel6A fusions were inserted into the tobacco (Nicotiana tabacum cv. Samsun) chloroplast genome for protein expression. Accumulation of Cel6A protein in transformed tobacco leaves varied over approximately two orders of magnitude, dependent on the identity of the DB region fused to the cel6A open reading frame (ORF). Additionally, the DB region fused to the cel6A ORF affected the accumulation of Cel6A protein in aging leaves, with the most effective DB regions allowing for high level accumulation of Cel6A protein in young, mature, and old leaves, while Cel6A protein accumulation decreased with leaf age when less effective DB regions were fused to the cel6A ORF. In the most highly expressed DB-Cel6A construct, enzymatically active Cel6A protein accumulated at up to 10.7% of total soluble leaf protein (%TSP). The strategy used for high-level endoglucanase expression may be useful for expression of other cellulolytic enzymes in chloroplasts, ultimately leading to cost-effective heterologous enzyme production for cellulosic ethanol using transplastomic plants.     

Three amino acid changes contribute markedly to the thermostability of β-glucosidase BglC from Thermobifida fusca

Thermostability of β-glucosidase was enhanced by family shuffling, site saturation mutagenesis, and site-directed mutagenesis. Family shuffling was carried out based on β-glucosidase BglC from Thermobifida fusca and β-glucosidase BglB from Paebibacillus polymxyxa with the help of synthetic primers. High-throughput screening revealed mutants with higher thermostability than both parental enzymes. The most thermostable mutant VM2 containing three key amino acid changes in L444Y/G447S/A433V had a 144-fold increase in half-life of inactivation as compared to the parental enzyme BglC. The mutant VM2 showed 28% and 94% increase in k(cat) towards p-nitrophenyl-β-D-glucopyranoside (pNPG) and cellobiose, respectively. The mutant with enhanced stability would facilitate the recycle of β-glucosidase in the bioconversion of cellulosic biomass.     

Identification of an Extracellular Endoglucanase That Is Required for Full Virulence in Xanthomonas citri subsp. citri

Xanthomonas citri subsp. citri causes citrus canker disease, which is characterized by the formation of water-soaked lesions, white or yellow spongy pustules and brown corky canker. In this work, we report the contribution of extracellular endoglucanase to canker development during infection. The ectopic expression of nine putative cellulases in Escherichia coli indicated that two endoglucanases, BglC3 and EngXCA, show carboxymethyl cellulase activity. Both bglC3 and engXCA genes were transcribed in X. citri subsp. citri, however, only BglC3 protein was detected outside the cell in western blot analysis. The deletion of bglC3 gene resulted in complete loss of extracellular carboxymethyl cellulase activity and delayed the onset of canker symptoms in both infiltration- and wound-inoculation assays. When growing in plant tissue, the cell density of bglC3 mutant was lower than that of the wild type. Our data demonstrated that BglC3 is an extracellular endoglucanase required for the full virulence of X. citri subsp. citri.     

Overexpression of a multifunctional β-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter

The β-glucosidase, which hydrolyzes the β(1–4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of β-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including β-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2–4. We overexpressed the β-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with β-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of β-glucosidase in plants could further increase. The plant-expressed β-glucosidase had optimal activity at 80 °C and pH 5–6. In addition, the plant-expressed β-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with β-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial β-glucosidase gene as a reporter in plants based on alternative β-galactosidase activity.

     

High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants

Chloroplast transformation has an extraordinary potential for antigen production in plants because of the capacity to accumulate high levels of recombinant proteins and increased biosafety due to maternal plastid inheritance in most crops. In this article, we evaluate tobacco chloroplasts transformation for the production of a highly immunogenic epitope containing amino acid residues 135–160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). To increase the accumulation levels, the peptide was expressed as a fusion protein with the β-glucuronidase reporter gene (uidA). The recombinant protein represented the 51% of the total soluble proteins in mature leaves, a level higher than those of the Rubisco large subunit, the most abundant protein in the leaf of a wild-type plant. Despite this high accumulation of heterologous protein, the transplastomic plants and wild-type tobacco were phenotypically indistinguishable. The FMDV epitope expressed in transplastomic plants was immunogenic in mice. These results show that transplastomic tobacco express efficiently the recombinant protein, and we conclude that this technology allows the production of large quantities of immunogenic proteins.     

Cell surface display of a β-glucosidase employing the type V secretion system on ethanologenic Escherichia coli for the fermentation of cellobiose to ethanol

We used the autodisplay system AIDA-I, which belongs to the type V secretion system (TVSS), to display the β-glucosidase BglC from Thermobifida fusca on the outer membrane of the ethanologenic Escherichia coli strain MS04 (MG1655 ?pflB, ?adhE, ?frdA, ?xylFGH, ?ldhA, PpflB::pdc (Zm)-adhB (Zm)). MS04 that was transformed with the plasmid pAIDABglCRHis showed cellobiase activity (171 U/g(CDW)) and fermented 40 g/l cellobiose in mineral medium in 60 h with an ethanol yield of 81 % of the theoretical maximum. Whole-cell protease treatment, SDS-PAGE, and Western-blot analysis demonstrated that BglC was attached to the external surface of the outer membrane of MS04. When attached to the cells, BglC showed 93.3 % relative activity in the presence of 40 g/l ethanol and retained 100 % of its activity following 2 days of incubation at 37 °C with the same ethanol concentration. This study shows the potential of the TVSS (AIDA-I) and BglC as tools for the production of lignocellulosic bio-commodities.     

Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.     

Accumulation of recombinant SARS-CoV spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines

Plants are promising candidates as bioreactors for the production of oral recombinant proteins in the biopharmaceutical industry. As an initial step toward provision of an oral vaccine against the severe acute respiratory syndrome coronavirus (SARS-CoV), we have expressed a partial spike (S) protein of SARS-CoV in the cytosol of nuclear-transformed plants and in the chloroplasts of plastid-transformed plants. In the construction of both nuclear and plastid transformation vectors, a 2-kilobase nucleotide sequence encoding amino acids 1-658 of the SARS-CoV spike protein (S1) was modified with nucleotide changes, but not amino acid changes, to optimize codon usage for expression in plants. To investigate the subcellular localization of S1 during transient expression in tobacco leaves, a translational fusion consisting of S1 and the green fluorescent protein (GFP) was generated. Following agroinfiltration of tobacco leaves, analysis by laser confocal scanning microscopy revealed that the S1:GFP fusion protein was localized to the cytosol. In stable transgenic tobacco plants and lettuce plants generated by Agrobacterium-mediated transformation, tobacco and lettuce leaves were observed to express the S1 at high levels from the Cauliflower Mosaic Virus 35S promoter with Northern blot analysis. When the S1 was expressed in transplastomic tobacco, S1 messenger RNA and its corresponding protein were detected on Northern and Western blot analyses, respectively. Our results demonstrate the feasibility of producing S1 in nuclear- and chloroplast-transformed plants, indicating its potential in subsequent development of a plant-derived and safe oral recombinant subunit vaccine against the SARS-CoV in edible plants.     

The effects of inhibitors of cell wall synthesis on tobacco protoplast development

The effect of 2,6-dichlorobenzonitrile (DB) and coumarin on tobacco protoplast development has been studied using a combination of flow microfluorimetry and quantitative fluorescence microscopy. The results indicated that, over the initial period of culture, DB largely inhibited cellulose production at the cell surface, but did not inhibit DNA synthesis or protein accumulation by the protoplasts. Continuous culture of protoplasts in DB resulted in the accumulation of an increased capacity for cellulose synthesis as expressed following the removal of inhibitor. The possible sites of the specific inhibitory action of DB are discussed.     

Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco

Fragments from camelid single-chain antibodies known as VHHs or nanobodies represent a valuable tool in diagnostics, investigation and passive immunity therapy. Here, we explored different strategies to improve the accumulation of a neutralizing VHH antibody against rotavirus in tobacco transplastomic plants. First, we attempted to express the VHH in the chloroplast stroma and then two alternative strategies were carried out to improve the expression levels: expression as a translational fusion to the β-glucuronidase enzyme (GUS-E-VHH), and redirection of the VHH into the thylakoid lumen (pep-VHH). Every attempt to produce transplastomic plants expressing the VHH in the stroma was futile. The transgene turned out to be unstable and the presence of the VHH protein was almost undetectable. Although pep-VHH plants also presented some of the aforementioned problems, higher accumulation of the nanobody was observed (2-3% of the total soluble proteins). The use of β-glucuronidase as a partner protein turned out to be a successful strategy and expression levels reached 3% of the total soluble proteins. The functionality of the VHHs produced by pep-VHH and GUS-E-VHH plants was studied and compared with that of the antibody produced in Escherichia coli. This work contributes to optimizing the expression of VHH in transplastomic plants. Recombinant proteins could be obtained either by accumulation in the thylakoid lumen or as a fusion protein with β-glucuronidase, and both strategies allow for further optimization.     

Synergistic growth in bacteria depends on substrate complexity

Both positive and negative interactions among bacteria take place in the environment. We hypothesize that the complexity of the substrate affects the way bacteria interact with greater cooperation in the presence of recalcitrant substrate. We isolated lignocellulolytic bacteria from salt marsh detritus and compared the growth, metabolic activity and enzyme production of pure cultures to those of three-species mixed cultures in lignocellulose and glucose media. Synergistic growth was common in lignocellulose medium containing carboxyl methyl cellulose, xylan and lignin but absent in glucose medium. Bacterial synergism promoted metabolic activity in synergistic mixed cultures but not the maximal growth rate (μ). Bacterial synergism also promoted the production of β-1,4-glucosidase but not the production of cellobiohydrolase or β-1,4-xylosidase. Our results suggest that the chemical complexity of the substrate affects the way bacteria interact. While a complex substrate such as lignocellulose promotes positive interactions and synergistic growth, a labile substrate such as glucose promotes negative interactions and competition. Synergistic interactions among indigenous bacteria are suggested to be important in promoting lignocellulose degradation in the environment.     

Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA.

The plastid psbA mRNA is present in all tissues, while the encoded 32 kDa D1 protein of photosystem II accumulates tissue-specifically and in response to light. To study the regulation of D1 accumulation, a chimeric uidA gene encoding beta-glucuronidase (GUS) under control of the psbA 5'- and 3'-regulatory regions (224 and 393 bp, respectively), was integrated into the tobacco plastid genome. A high level of GUS accumulation in leaves and the lack of GUS in roots, with uidA mRNA present in both tissues, indicated tissue-specific accumulation of the chimeric gene product. Light-regulated accumulation of GUS in seedlings was shown. (i) Light-induced accumulation (100-fold) of GUS in etiolated cotyledons was accompanied by only a modest increase in mRNA levels. (ii) Inhibition of GUS synthesis was observed in cotyledons when light-grown seedlings were transferred to the dark, with no reduction in mRNA levels. Tissue-specific and light-regulated accumulation of GUS indicates that D1 accumulation is controlled via cis-acting regulatory elements in the untranslated region of the psbA mRNA. We propose that in tobacco, control of translation initiation is the primary mechanism regulating D1 protein accumulation.     

Gene activation in plastids by the CRE site-specific recombinase

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.     

Cytoplasm and chloroplasts are not suitable subcellular locations for beta-zein accumulation in transgenic plants

Zeins, the main storage proteins of maize that accumulate in the endoplasmic reticulum of the endosperm cells, are particularly interesting because they are rich in the essential sulphur amino acids. Overexpression of certain zein genes in plants such as alfalfa would be expected to improve the nutritional characteristics of this crop. Recently, significant accumulation values have been reached, but still far from those considered useful for nutritional purposes. This study investigates whether targeting to compartments other than the endoplasmic reticulum (cytosol and chloroplasts) could result in increasing beta-zein accumulation in transgenic plants. To address beta-zein to the cytosol, the fragment which codes for the signal peptide has been removed. beta-zein has also been targeted to alfalfa and tobacco chloroplasts by a transit peptide signal. Both tobacco, as a model plant species, and alfalfa have been transformed with the assembled constructs. An alternative route to accumulate beta-zein in the chloroplasts is to synthesize beta-zein directly in the plastid lumen. Thus, the beta-zein gene has also been inserted into tobacco plastid DNA. The beta-zein gene in each different type of transformed plant was properly transcribed, as determined by northern blot analysis, but no accumulation of beta-zein was detected, either in the cytoplasm or in the chloroplasts of alfalfa and tobacco transformed plants. Therefore, it is concluded that chloroplasts and the cytosol are not favourable subcellular locations for zein protein accumulation.     

Complementation of the nuclear antisense rbcS-induced photosynthesis deficiency by introducing an rbcS gene into the tobacco plastid genome

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line alpha5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.