Expression of tetanus toxin Fragment C in tobacco chloroplasts

Fragment C (TetC) is a non-toxic 47 kDa polypeptide fragment of tetanus toxin that can be used as a subunit vaccine against tetanus. Expression of TetC in Escherichia coli and yeast was dependent on the availability of synthetic genes that were required to improve translation efficiency and stabilize the mRNA. To explore the feasibility of producing TetC in tobacco leaves, we attempted expression of both the bacterial high-AT (72.3% AT) and the synthetic higher-GC genes (52.5% AT) in tobacco chloroplasts. We report here that the bacterial high-AT mRNA is stable in tobacco chloroplasts. Significant TetC accumulation was obtained from both genes, 25 and 10% of total soluble cellular protein, respectively, proving the versatility of plastids for expression of unmodified high-AT and high-GC genes. Mucosal immunization of mice with the plastid- produced TetC induced protective levels of TetC antibodies. Thus, expression of TetC in chloroplasts provides a potential route towards the development of a safe, plant-based tetanus vaccine for nasal and oral applications.     

Expression of a thermostable bacterial cellulase in transgenic tobacco plants

The bacterial gene of the thermostable endo-beta-1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased business and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.     

Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts

Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.     

Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.     

Production and characterization of thermostable cellulases from Streptomyces transformant T3-1

A total of 26 thermophilic isolates, selected from a compost of agricultural waste, which was mostly composed of vegetable, corncob and rice straw, were cultivated at 50 °C for further studies of thermostable cellulase production. The thermostable cellulase gene from the chromosomal DNA of actinomycetes isolate no. 10 was shotgun-cloned and transformed into Streptomyces sp. IAF 10-164. A transformant, T3-1, was found to be a good strain for the production of thermostable cellulases. Cultivation of T3-1 in modified Mandels–Reese broth containing 1% carboxymethylcellulose (CMC)-sodium salt and the optimal condition for microbial growth were studied. Batch cultivation in a flask revealed that CMCase and Avicelase production reached the maximum between the third to fifth day, whereas maximum -glucosidase production occurred on the ninth day. Microbial biomass increased from the first day to the fifth day and then decreased. The crude enzyme had the highest activity at 50 °C and at pH 6.5. The enzyme was shown to be a thermostable cellulase whose activities were stable at 50 °C for more than 7 days.     

Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts

The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.     

Expression of a bacterial lysine decarboxylase gene and transport of the protein into chloroplasts of transgenic tobacco

A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. Theldcgene fromHafnia alvei was therefore (a) placed under the control of the 1 promoter of the bidirectional Tr promoter fromAgrobacterium tumefaciens Ti- plasmid, and (b) cloned behind therbcS promoter from potato fused to the coding region of therbcS transit peptide. Bothldc constructs, introduced intoNicotiana tabacum with the aid ofA. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of therbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3–1% of dry mass.     

Herbicide resistance of tobacco chloroplasts expressing the bar gene

The chloroplast transformation system has the potential advantages of maternal inheritance and high-level expression of heterologous genes. We studied the expression of the bar gene in tobacco chloroplasts to test these ideas. The bar gene conferring tolerance to the herbicide phosphinothricin (PPT) encodes phosphinothricin acetyltransferase (PAT). It was introduced into the chloroplast genome at a targeted site by homologous recombination. Transplastomic plantlets were selected in medium supplemented with PPT (up to 50 mg l(-1)). The polymerase chain reaction (PCR) and Southern blot analysis confirmed that bar had been inserted at the specified site in the chloroplast genome. The transplastomic plants transferred to a greenhouse proved to be resistant to 2% PPT. Reciprocal crosses between wild type and transplastomic plants confirmed maternal inheritance of the PPT resistance and high levels of PAT activity in the transplastomic plants were confirmed by assays of PAT and of ammonium evolution. The technology demonstrated here could perhaps be usefully transferred to other crop species.     

Three-dimensional reconstruction of anomalous chloroplasts in transgenic ipt tobacco

Anomalies in the ultrastructure of chloroplasts, from transgenic ipt tobacco, overproducing endogenous cytokinins (CKs) were studied. Detailed analyses of CKs and their metabolites showed that Pssu-ipt tobacco contained enhanced contents of CKs both in leaves and in isolated chloroplasts. The role of CKs in the formation of anomalous structures is suggested. Pssu-ipt chloroplasts frequently formed the distinct peripheral reticulum with a system of caverns that often involved mitochondria and/or peroxisomes. Large crystalloids, which were found in chloroplasts of Pssu-ipt, occupied up to 16% of chloroplast volume. We suggested that the crystalloids were formed by LHC II aggregates. This was supported by analysis of the fluorescence emission spectra at 77°K, chlorophyll a/b ratio, immunogold staining of the structures, and crystallographic unit size analysis.     

Heterogeneity of 5S RNA species in tobacco chloroplasts

Summary Tobacco chloroplasts were found to contain three species of 5S RNA with different electrophoretic mobility. The nucleotide sequences of two species of the 5S RNA have been determined. The large 5S RNA species (5S RNA_L) is composed of 121 nucleotides and the small 5S RNA species (5S RNA_s) of 119 nucleotides. The 5S RNA_L contains and extra uridine residue at both 5 and 3ends of the 5S RNA_s.     

Pathways of silicomolybdate photoreduction and the associated photophosphorylation in tobacco chloroplasts

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (> 5 mg/ml)) is associated with Photosystem I, since it supports phosphorylation with a P/e₂ value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e₂ value routinely observed is about 0.2. Bovine serum albumin (1–2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even in the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis

Bacterial lipoproteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N-terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten-fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N-terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration-dependent interference with essential functions of chloroplastic membranes required for primary metabolism.     

耐高温α—淀粉酶基因在马铃薯中的表达

The thermostable alpha-amylase gene cloned from Bacillus lichemiformis was reconstructed into an expression vector pAMY721M under the CaMV35S promoter. The vector was transferred into A. tumerfaciens ABI. The thermostable alpha-amylase gene was transferred into Solanum tuberosum L. via Agrobacterium mediation according to the revised method of ZHAO Shu Juan et al (1997) and YANG Mei Zhu et al (1992). Shoots were induced and regenerated on MS medium with 2 mg/L ZT, 0.1 mg/L IAA and 100 mg/L kanamycin. Putative transformants were selected with kanamycin and roots induced on MS medium with 0.15 mg/L IAA. PCR analysis and thermostable alpha-amylase activity assay were done to identify the transgenic plantlets. Among them, 102,001, 102,607, and 110,402 were showed to have a higher alpha-amylase activity than untransformed control.     

Stable expression of the sweet protein monellin variant MNEI in tobacco chloroplasts

Monellin is a naturally sweet protein that consists of two polypeptide chains and has potential uses as a highly potent non-carbohydrate sweetener. We aimed to make this protein more usable by increasing its stability and expressing it in a high-yielding system. MNEI is a modified version of the protein that consists of the two natural chains of monellin joined via a dipeptide linkage. In the thermostability analysis of MNEI variants, four mutated MNEIs, MNEI-E24L, MNEI-E24F, MNEI-E24W, and MNEI-E24A, had higher melting temperatures than wild-type MNEI and retained their sweet flavor even at temperatures above 70?°C. Our findings indicate that the increased stability of monellin allows it to retain its strong sweetness even under extreme conditions. We successfully overexpressed the thermostable MNEI mutants in tobacco chloroplasts. Here, we report that the MNEI mutants showed enhanced thermostability, and the stable forms of MNEI can be produced through plastid transformation in tobacco.     

High-yield production of a human therapeutic protein in tobacco chloroplasts

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.