Improved recombinant cellulase expression in chloroplast of tobacco through promoter engineering and 5′ amplification promoting sequence

Economical production of bioethanol from lignocellulosic biomass still faces many technical limitations. Cost-effective production of fermentable sugars is still not practical for large-scale production of bioethanol due to high costs of lignocellulolytic enzymes. Therefore, plant molecular farming, where plants are used as bioreactors, was developed for the mass production of cell wall degrading enzymes that will help reduce costs. Subcellular targeting is also potentially more suitable for the accumulation of recombinant cellulases. Herein, we generated transgenic tobacco plants (Nicotiana tabacum cv. SR1) that accumulated Thermotoga maritima BglB cellulase, which was driven by the alfalfa RbcsK-1A promoter and contained a small subunit of the rubisco complex transit peptide. The generated transformants possessed high specific BglB activity and did not show any abnormal phenotypes. Furthermore, we genetically engineered the RbcsK-1A promoter (MRbcsK-1A) and fused the amplification promoting sequence (aps) to MRbcsK-1A promoter to obtain high expression of BglB in transgenic plants. AMRsB plant lines with aps-MRbcsK-1A promoter showed the highest specific activity of BglB, and the accumulated BglB protein represented up to 9.3 % of total soluble protein. When BglB was expressed in Arabidopsis and tobacco plants, the maximal production capacity of recombinant BglB was 0.59 and 1.42 mg/g wet weight, respectively. These results suggests that suitable recombinant expression of cellulases in subcellular compartments such as chloroplasts will contribute to the cost-effective production of enzymes, and will serve as the solid foundation for the future commercialization of bioethanol production via plant molecular farming.     

Overexpression of the anaphase-promoting complex (APC) genes in Nicotiana tabacum promotes increasing biomass accumulation

The anaphase-promoting complex (APC) plays pivotal roles in cell cycle pathways related to plant development. In this study, we present evidence that overproduction of APC10 from Arabidopsis thaliana in tobacco (Nicotiana tabacum) plants promotes significant increases in biomass. Analyzes of plant’s fresh and dried weight, root length, number of days to flower and number of seeds of plants overexpressing AtAPC10 verified an improved agronomic performance of the transgenic plants. Detailed analyzes of the leaf growth at the cellular level, and measurements of leaf cell number, showed that AtAPC10 also produce more cells, showing an enhancement of proliferation in these plants. In addition, crossing of plants overexpressing AtAPC10 and AtCDC27a resulted in a synergistic accumulation of biomass and these transgenic plants exhibited superior characteristics compared to the parental lines. The results of the present study suggest that transgenic plants expressing AtAPC10 and AtAPC10/AtCDC27a concomitantly are promising leads to develop plants with higher biomass.     

Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants

Monoclonal antibody production from transgenic tobacco plants offers many advantages over other heterologous production systems, creating the prospect of production at a scale that will allow new prophylactic and therapeutic applications in global human and animal health. However, information on the major processing factors to consider for large-scale purification of antibodies from transgenic plants is currently limited, and is in urgent need of attention. The purpose of this project was to investigate methods for the initial extraction of recombinant immunoglobulin G (IgG) antibodies from transgenic tobacco leaf tissue. Three different transgenic plant lines were studied in order to establish the parameters for optimal extraction of monoclonal antibodies that accumulate in the apoplasm, at the plasma membrane or within the endoplasmic reticulum. For each transgenic line, seven techniques for physical extraction were compared. The factors that determine the optimal extraction of antibodies from plants have a direct influence on the initial choice of expression strategy, and so must be considered at an early stage. The use of small-scale techniques that are applicable to large-scale purification was a particularly important consideration. The optimal extraction technique varied with the target location of IgG in the plant cell, and the dependence of antibody yield on the physical extraction methodology employed, the pH of the extraction buffer and the extraction temperature was demonstrated in each case. The addition of detergent to the extraction buffer may improve the yield, but this was found to be dependent on the site of accumulation of IgG within the plant cell.     

Recombinant monoclonal antibody yield in transgenic tobacco plants is affected by the wounding response via an ethylene dependent mechanism

Variability in recombinant IgG yield in transgenic tobacco plants has previously been observed in relation to leaf position, and is interpreted as a function of ageing and the senescence process, leading to increasing protein degradation. Here, similar findings are demonstrated in plants of different ages, expressing IgG but not IgG-HDEL, an antibody form that accumulates within the endoplasmic reticulum. Antibody yields declined following wounding in young transgenic plants expressing IgG but not in those expressing IgG-HDEL. However, in mature IgG plants, the opposite was demonstrated, with significant boosts in yield, while mature IgG-HDEL plants could not be boosted. The lack of response in IgG-HDEL plants suggests that the changes induced by wounding occur post-translationally, and the findings might be explained by wounding responses that differ in plants according to their developmental stages. Plant mechanisms involved in senescence and wounding overlap to a significant degree and compounds such as ethylene, jasmonic acid and salicylic acid are important for mediating downstream effects. Treatment of transgenic plants with ethylene also resulted in a decrease in recombinant IgG yield, which was consistent with the finding that wounded plants could induce lower IgG yields in neighbouring non-wounded plants. Treatment with 1-MCP, an ethylene antagonist, abrogated the IgG yield drop that resulted from wounding, but had no effect on the more gradual IgG yield loss associated with increasing plant age.     

Heterologous expression of chloroplast‐localized geranylgeranyl pyrophosphate synthase confers fast plant growth,early flowering and increased seed yield

Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast‐targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS‐transgenic lines (control) or wild‐type plants. The gibberellin levels in HaGGPS‐transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS‐transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS‐expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.     

Transgenically Enhanced Sorbitol Synthesis Facilitates Phloem Boron Transport and Increases Tolerance of Tobacco to Boron Deficiency

The mobility of elements within plants contributes to a plant species' tolerance of nutrient deficiencies in the soil. The genetic manipulation of within-plant nutrient movement may therefore provide a means to enhance plant growth under conditions of variable soil nutrient availability. In these experiments tobacco (Nicotiana tabacum) was engineered to synthesize sorbitol, and the resultant effect on phloem mobility of boron (B) was determined. In contrast to wild-type tobacco, transgenic tobacco plants containing sorbitol exhibit a marked increase in within-plant B mobility and a resultant increase in plant growth and yield when grown with limited or interrupted soil B supply. Growth of transgenic tobacco could be maintained by reutilization of B present in mature tissues or from B supplied as a foliar application to mature leaves. In contrast, B present in mature leaves of control tobacco lines could not be used to provide the B requirements for new plant growth. ¹⁰B-labeling experiments verified that B is phloem mobile in transgenic tobacco but is immobile in control lines. These results demonstrate that the transgenic enhancement of within-plant nutrient mobility is a viable approach to improve plant tolerance of nutrient stress.     

Enhanced expression of the human CD14 protein in tobacco using a 22-kDa alpha-zein signal peptide

The human CD14, a high affinity receptor for lipopolysaccharides (LPS), is involved in the innate immunity system and the inflammatory response. There is increasing interest in using recombinant approaches to produce purified CD14 protein for therapeutic uses. Plants provide ideal expression systems for the production of recombinant proteins, but the levels of expression of recombinant proteins produced in planta are still not high. To improve expression levels of CD14 the 22-kDa alpha-zein signal peptide (ZSP) from maize was fused to the human CD14 cDNA so that recombinant CD14 could stably accumulate in plant cells. The human CD14 gene and the modified human CD14 cDNA with the 22-kDa ZSP were respectively transformed into tobacco to produce transgenic plants. Western blot analysis confirmed human CD14 accumulation in the transgenic tobacco. The concentration of the recombinant protein in the tobacco leaves was measured by ELISA, and the results suggested that fusion with the 22-kDa alpha-ZSP effectively increased the accumulation of the recombinant protein (rCD14). The concentration of rCD14 in some of the transgenic lines was 19.54???g?g^?1 tobacco leaf (fw), which was about 0.6?% of the total soluble protein. The rCD14 protein showed natural LPS-binding bioactivity by using U937 cells mensuration. Our results suggested that the maize 22-kDa alpha-zein signal peptide could be used to increase the accumulation of recombinant protein in tobacco leaves so that proteins can be produced in abundant biomass.     

Characterization of Transgenic Tobacco Plants Containing Bacterial bphc Gene and Study of Their Phytoremediation Ability

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene – enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified after purification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected.Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with non-transgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.     

High-Level Transient Expression of ER-Targeted Human Interleukin 6 in Nicotiana benthamiana

Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T₀ transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T₂ plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein.     

Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis

Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable α-amylase) was evaluated in numerous species and cultivars of Nicotiana. Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for recombinant protein production.     

Monocotyledonous C4 NADP+ -malate dehydrogenase is efficiently synthesized,targeted to chloroplasts and processed to an active form in transgenic plants of the C3 dicotyledon tobacco

Chloroplastic NADP⁺-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbonfixation pathway of some C₄ plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C₃ plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C₃-C₄ photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic proteinimport machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of l-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.     

Enhancement of Reproductive Heat Tolerance in Plants

Comparison of average crop yields with reported record yields has shown that major crops exhibit annual average yields three- to seven-fold lower than record yields because of unfavorable environments. The current study investigated the enhancement of pollen heat tolerance through expressing an Arabidopsis thaliana heat shock protein 101 (AtHSP101) that is not normally expressed in pollen but reported to play a crucial role in vegetative thermotolerance. The AtHSP101 construct under the control of the constitutive ocs/mas ‘superpromoter’ was transformed into cotton Coker 312 and tobacco SRI lines via Agrobacterium mediated transformation. Thermotolerance of pollen was evaluated by in vitro pollen germination studies. Comparing with those of wild type and transgenic null lines, pollen from AtHSP101 transgenic tobacco and cotton lines exhibited significantly higher germination rate and much greater pollen tube elongation under elevated temperatures or after a heat exposure. In addition, significant increases in boll set and seed numbers were also observed in transgenic cotton lines exposed to elevated day and night temperatures in both greenhouse and field studies. The results of this study suggest that enhancing heat tolerance of reproductive tissues in plant holds promise in the development of crops with improved yield production and yield sustainability in unfavorable environments.     

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy’s 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 μg g⁻¹ leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.     

Overexpression of the Gossypium barbadense Actin-Depolymerizing Factor 1 Gene Mediates Biological Changes in Transgenic Tobacco

The function of a member of the actin-depolymerizing factor family from Gossypium barbadense, GbADF1, was investigated. Tobacco (Nicotiana tabacum) lines expressing GbADF1 were produced by Agrobacterium-mediated transformation. Southern and northern blot analyses showed that GbADF1 was successfully incorporated as a single copy into the tobacco genome and stably expressed in three lines of T₁ transgenic tobacco plants. Biological changes were detected in these transgenic lines, wherein GbADF1 transgenic seedlings exhibited shorter hypocotyls along with fewer root hairs than those of control plants. Moreover, guard cells of leaves of the transgenic plants were induced to close stomata, while flowering was delayed 5 days in T₁ lines compared to those of empty vector transgenic control plants. Segregation of GbADF1 in the T₂ generation fits the expected 3:1 ratio corresponding to a single dominant gene. Subsequently, GbADF1 was fused to the green fluorescent protein gene to generate a fusion expression vector. Transient expression analysis indicated that this fusion protein was localized in the nucleus and cytoskeleton of epidermal cells of onion. These results suggest that actin-depolymerizing factor 1 gene from G. barbadense plays an important role in the process of plant cell morphogenesis.     

Improving biomass production and saccharification in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> through overexpression of a sucrose-phosphate synthase from sugarcane

The substitution of fossil by renewable energy sources is a major strategy in reducing CO₂ emission and mitigating climate change. In the transport sector, which is still mainly dependent on liquid fuels, the production of second generation ethanol from lignocellulosic feedstock is a promising strategy to substitute fossil fuels. The main prerequisites on designated crops for increased biomass production are high biomass yield and optimized saccharification for subsequent use in fermentation processes. We tried to address these traits by the overexpression of a sucrose-phosphate synthase gene (SoSPS) from sugarcane (Saccharum officinarum) in the model grass Brachypodium distachyon. The resulting transgenic B. distachyon lines not only revealed increased plant height at early growth stages but also higher biomass yield from fully senesced plants, which was increased up to 52 % compared to wild-type. Additionally, we determined higher sucrose content in senesced leaf biomass from the transgenic lines, which correlated with improved biomass saccharification after conventional thermo-chemical pretreatment and enzymatic hydrolysis. Combining increased biomass production and saccharification efficiency in the generated B. distachyon SoSPS overexpression lines, we obtained a maximum of 74 % increase in glucose release per plant compared to wild-type. Therefore, we consider SoSPS overexpression as a promising approach in molecular breeding of energy crops for optimizing yields of biomass and its utilization in second generation biofuel production.