Transplastomic Lines Research Articles

Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

High Quality and Purified DNA Extraction Protocol from Transplastomic Potato Lines by Cetyltrimethylammonium Bromide (CTAB)-Based Method

For the advanced molecular techniques high quality and pure DNA is important requirement where a quick and cost-effective extraction method is a crucial factor. Commonly, prolong incubation period, multiple precipitations, and highly expensive commercial kits are remaining basic barrier to introduce swift and easiest method of DNA extraction from transgenic plants. Non-cellulose components present in the leaf and elevated level of starch content as well as protein content also makes this extraction procedure challenging and difficult. Present study was conducted to establish a unique DNA isolation method with some modification of existing CTAB method to ensure high-quality and purified genomic DNA from the potato plants. Through the implementation of solution based and selective DNA precipitation method, 100 mg of potato leaves from every transplastomic potato lines was applied to extract genomic DNA. Ethanol (70%) was used as elution buffer where maximum DNA yield was 3415.500 ng/µl and the ranges of DNA purity (A260/A280 nm) was recorded as 1.954-2.048, respectively. Compared to existing methods, this modified CTAB mediated procedure is swift (at least one hour can be saved ) and proficient enough for high quality DNA yield, free from the selective precipitation, regents and laboratory equipment’s are cost-effective and non-toxic to human health.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Successful production of human epidermal growth factor in tobacco chloroplasts in a biologically active conformation

Human epidermal growth factor (hEGF) is an important therapeutic compound with multiple applications particularly in pharmaceutical industry. Human EGF has already been expressed in different expression systems, however, the production of hEGF with bioactivity in chloroplasts has not been successful so far. In this study, we expressed a 6 × His-tagged hEGF in tobacco chloroplasts in its native conformation for the potential of large-scale production of hEGF for industrial applications. Several transplastomic plant lines were obtained, which were screened by PCR (polymerase chain reaction) using primers specific to selectable gene aadA, hEGF- and GFP-coding sequences that were included in the chloroplast expression vector. The selected lines were confirmed to be homoplasmic by PCR verification and Southern blot analysis. Immunoblotting assays of homoplasmic lines using antibodies raised against hEGF confirmed the accumulation of hEGF in transplastomic plants and the ELISA results demonstrated the expression levels of hEGF were between 0.124% and 0.165% of the total soluble proteins (TSP), namely, 23.16–25.77 ng/g of the fresh weight. In terms of activity, the data from cell proliferation and elongation assays showed that the tobacco-derived recombinant hEGF was as bioactive as its commercial counterpart. To our knowledge, this is the first report of recombinant production of hEGF with native bioactivity form in the chloroplast stroma. Overall, our results demonstrate the potential of higher plant chloroplasts for the production of a human therapeutic, hEGF, in an active conformation.

Producing fluorescent plants to lure and trap insect pests.

Producing fluorescent plants to lure and trap insect pests.

High-level expression of anti-interleukin-6 single chain variable fragment through plastid transformation technology

Interleukin-6 (IL-6) is a pleiotropic cytokine which participates in the pathogenesis of a variety of clinical disorders, including many kinds of cancers. Anti-IL-6 antibody was proved to be useful for the immunotherapy of various inflammatory diseases. Plants are low-cost platforms for producing specific proteins of therapeutic interest. Two dependent transplastomic tobacco lines expressing murine anti-IL-6 single chain variable fragment (scFv) were generated after bombardment and regeneration, homoplasmy was then verified by Southern blotting analysis. The anti-IL-6 scFv gene was successfully expressed at both transcriptional and translational levels in transplastomic tobacco plants. Functional anti-IL-6 scFv accumulated to 1% of total soluble proteins, namely 41 mg/kg fresh weight. There was no obvious phenotypic difference between the wild-type and the transplastomic tobacco plants, including the growth rate, the height of mature plants and the number of siliques. The high-level expression of anti-IL-6 scFv indicates the potential for cost-effective production of scFV using transplastomic plants.

Construction and Validation of a Chloroplast Expression Vector for the Production of Recombinant Proteins in Chlorella vulgaris

Microalgae constitute a diverse group of photosynthetic unicellular microorganisms that have gained immense attention for biotechnological applications. They are promising platforms for the production of high-value metabolites and biopharmaceuticals for commercial and therapeutic applications because of their physiological properties and capability to grow easily in natural and artificial environments. Although the proof-of-concept for some applications have been achieved for model species, such as Chlamydomonas reinhardtii, the genetic engineering methods for microalgae are still in their infancy. Thus, an expansion of this field is required. Chlorella vulgaris is an important algal species with a high protein content and requires focus for the development of an efficient nuclear and chloroplast transformation process. This research aimed to establish a chloroplast transformation method for the freshwater green-algae species C. vulgaris based on a specific expression vector (pCMCC, which was named after Chula Mexico Chlorella chloroplast) constructed with endogenous recombination regions, namely, 16S–trn I (left) and trn A–23S (right), and the Prrn promoter. Human basic fibroblast growth factor (bFGF) was adopted as a target biopharmaceutical to establish the chloroplast expression approach. The plasmid pCMCC:bFGF was transformed into C. vulgaris via electroporation using simple carbohydrate-based buffers, which aided in the transfer of the transgene into the chloroplast genome. Cells transformed with the pCMCC:bFGF vector were selected using kanamycin, and resistant colonies were analyzed by polymerase chain reaction and Western blotting to confirm the presence of the transgene and the recombinant bFGF, respectively. The bFGF that accumulated in the transplastomic C. vulgaris clones ranged from 0.26 to 1.42 ng/g fresh weight of biomass, and it was quantified by enzyme-linked immunosorbent assay. Therefore, the designed expression vector, in combination with an optimized electroporation protocol, constitutes a viable approach to successfully develop transplastomic lines of C. vulgaris for the potential low-cost production of biopharmaceuticals using this algal species. This study paves the way for the establishment of chloroplast biotechnology in microalgae other than the model organism C. reinhardtii.

Plastidial Expression of 3β-Hydroxysteroid Dehydrogenase and Progesterone 5β-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco.

The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3β-hydroxysteroid dehydrogenase (3β-HSD) and progesterone 5-β-reductases (P5βR1, P5βR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3β-HSD, P5βR1 and P5βR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3β-HSD and P5βR1 are cytoplasmic, and P5βR2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3β-HSD, P5βR1 and P5βR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering.

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism.

Sustainable and economically viable support for an ever-increasing global population requires a paradigm shift in agricultural productivity, including the application of biotechnology to generate future crop plants. Current genetic engineering approaches aimed at enhancing the photosynthetic efficiency or composition of the harvested tissues involve relatively simple manipulations of endogenous metabolism. However, radical rewiring of central metabolism using new-to-nature pathways, so-called “synthetic metabolism”, may be needed to really bring about significant step changes. In many cases, this will require re-programming the metabolism of the chloroplast, or other plastids in non-green tissues, through a combination of chloroplast and nuclear engineering. However, current technologies for sophisticated chloroplast engineering (“transplastomics”) of plants are limited to just a handful of species. Moreover, the testing of metabolic rewiring in the chloroplast of plant models is often impractical given their obligate phototrophy, the extended time needed to create stable non-chimeric transplastomic lines, and the technical challenges associated with regeneration of whole plants. In contrast, the unicellular green alga, Chlamydomonas reinhardtii is a facultative heterotroph that allows for extensive modification of chloroplast function, including non-photosynthetic designs. Moreover, chloroplast engineering in C. reinhardtii is facile, with the ability to generate novel lines in a matter of weeks, and a well-defined molecular toolbox allows for rapid iterations of the “Design-Build-Test-Learn” (DBTL) cycle of modern synthetic biology approaches. The recent development of combinatorial DNA assembly pipelines for designing and building transgene clusters, simple methods for marker-free delivery of these clusters into the chloroplast genome, and the pre-existing wealth of knowledge regarding chloroplast gene expression and regulation in C. reinhardtii further adds to the versatility of transplastomics using this organism. Herein, we review the inherent advantages of the algal chloroplast as a simple and tractable testbed for metabolic engineering designs, which could then be implemented in higher plants.

Affordable oral health care: dental biofilm disruption using chloroplast made enzymes with chewing gum delivery.

SummaryCurrent approaches for oral health care rely on procedures that are unaffordable to impoverished populations, whereas aerosolized droplets in the dental clinic and poor oral hygiene may contribute to spread of several infectious diseases including COVID‐19, requiring new solutions for dental biofilm/plaque treatment at home. Plant cells have been used to produce monoclonal antibodies or antimicrobial peptides for topical applications to decrease colonization of pathogenic microbes on dental surface. Therefore, we investigated an affordable method for dental biofilm disruption by expressing lipase, dextranase or mutanase in plant cells via the chloroplast genome. Antibiotic resistance gene used to engineer foreign genes into the chloroplast genome were subsequently removed using direct repeats flanking the aadA gene and enzymes were successfully expressed in marker‐free lettuce transplastomic lines. Equivalent enzyme units of plant‐derived lipase performed better than purified commercial enzymes against biofilms, specifically targeting fungal hyphae formation. Combination of lipase with dextranase and mutanase suppressed biofilm development by degrading the biofilm matrix, with concomitant reduction of bacterial and fungal accumulation. In chewing gum tablets formulated with freeze‐dried plant cells, expressed protein was stable up to 3 years at ambient temperature and was efficiently released in a time‐dependent manner using a mechanical chewing simulator device. Development of edible plant cells expressing enzymes eliminates the need for purification and cold‐chain transportation, providing a potential translatable therapeutic approach. Biofilm disruption through plant enzymes and chewing gum‐based delivery offers an effective and affordable dental biofilm control at home particularly for populations with minimal oral care access.

Preclinical development of plant-based oral immune modulatory therapy for haemophilia B.

SummaryAnti‐drug antibody (ADA) formation is a major complication in treatment of the X‐linked bleeding disorder haemophilia B (deficiency in coagulation factor IX, FIX). Current clinical immune tolerance protocols are often not effective due to complications such as anaphylactic reactions against FIX. Plant‐based oral tolerance induction may address this problem, as illustrated by the recent first regulatory approval of orally delivered plant cells to treat peanut allergy. Our previous studies showed that oral delivery of plant cells expressing FIX fused to the transmucosal carrier CTB (cholera toxin subunit B) in chloroplasts suppressed ADA in animals with haemophilia B. We report here creation of the first lettuce transplastomic lines expressing a coagulation factor, in the absence of antibiotic resistance gene. Stable integration of the CTB‐FIX gene and homoplasmy (transformation of ˜10 000 copies in each cell) were maintained in both T1 and T2 generation marker‐free plants. CTB‐FIX expression in lyophilized leaves of T1 and T2 marker‐free plants was 1.0–1.5 mg/g dry weight, confirming that the marker excision did not affect antigen levels. Oral administration of CTB‐FIX to Sprague Dawley rats at 0.25, 1 or 2.5 mg/kg did not produce overt adverse effects or toxicity. The no‐observed‐adverse‐effect level (NOAEL) is at least 2.5 mg/kg for a single oral administration in rats. Oral administration of CTB‐FIX at 0.3 or 1.47 mg/kg either mixed in food or as an oral suspension to Beagle dogs did not produce any observable toxicity. These toxicology studies should facilitate filing of regulatory approval documents and evaluation in haemophilia B patients.

Chloroplast Transformation in Arabidopsis.

Plastids (chloroplasts) are the defining organelles of plants and eukaryotic algae. In addition to performing photosynthesis, plastids harbor numerous other metabolic pathways and therefore are often referred to as the biosynthetic center of the plant cell. The chloroplasts of seed plants possess dozens of copies of a circular genome of ∼150 kb that contains a conserved set of 120 to 130 genes. The engineering of this genome by genetic transformation is technically challenging and currently only possible in a small number of species. In this article, we describe the methods involved in generating stable chloroplast-transformed (transplastomic) plants in the model species Arabidopsis (Arabidopsis thaliana). The protocols presented here can be applied to (1) target genes in the Arabidopsis chloroplast genome by reverse genetics and (2) express reporter genes or other foreign genes of interest in plastids of Arabidopsis plants. © 2021 The Authors. Basic Protocol 1: Generation of root-derived microcallus material for biolistic transformation Basic Protocol 2: Chloroplast transformation by biolistic bombardment of root-derived microcalli Basic Protocol 3: Regeneration of transplastomic lines and seed production.

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco.

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.

Knockdown of the plastid-encoded acetyl-CoA carboxylase gene uncovers functions in metabolism and development.

De novo fatty acid biosynthesis in plants relies on a prokaryotic-type acetyl-CoA carboxylase (ACCase) that resides in the plastid compartment. The enzyme is composed of four subunits, one of which is encoded in the plastid genome, whereas the other three subunits are encoded by nuclear genes. The plastid gene (accD) encodes the β-carboxyltransferase subunit of ACCase and is essential for cell viability. To facilitate the functional analysis of accD, we pursued a transplastomic knockdown strategy in tobacco (Nicotiana tabacum). By introducing point mutations into the translational start codon of accD, we obtained stable transplastomic lines with altered ACCase activity. Replacement of the standard initiator codon AUG with UUG strongly reduced AccD expression, whereas replacement with GUG had no detectable effects. AccD knockdown mutants displayed reduced ACCase activity, which resulted in changes in the levels of many but not all species of cellular lipids. Limiting fatty acid availability caused a wide range of macroscopic, microscopic, and biochemical phenotypes, including impaired chloroplast division, reduced seed set, and altered storage metabolism. Finally, while the mutants displayed reduced growth under photoautotrophic conditions, they showed exaggerated growth under heterotrophic conditions, thus uncovering an unexpected antagonistic role of AccD activity in autotrophic and heterotrophic growth.

Plastid Transformation in Physcomitrium (Physcomitrella) patens: An Update.

The moss Physcomitrium (Physcomitrella) patens performs efficient homologous recombination in both the nucleus and plastid enabling the study of individual gene function by generating precise inactivation or modification of genes. Polyethylene glycol (PEG)-mediated transformation of protoplasts is routinely used to study the nuclear gene function of P. patens. PEG-mediated protoplast transformation is also applied for plastid transformation of this moss. The efficiency of plastid transformation is quite reliable and one or two homoplasmic transplastomic lines are obtained in a plastid transformation experiment (5×105 protoplasts) by selection for spectinomycin resistance.

Plastid Transformation in Poplar: A Model for Perennial Trees.

Poplar (Populus) is an important forest tree and considered model for perennial trees. Here we describe a method for poplar plastid transformation, which involves preparation of explants, vector construction, biolistic bombardment, regeneration and selection of transplastomic poplar plants. The young leaves of 4-week-old poplar plants are used for biolistic bombardment and aadA gene as selectable marker. Homoplasmic transplastomic lines are obtained after regeneration and several rounds of selection with spectinomycin over 10months. Homoplasmy is further confirmed by Southern blot. The establishment of a plastid transformation system for Populus is likely to make a significant contribution to tree genetic improvement.

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology

Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

An AMA1/MSP119 Adjuvanted Malaria Transplastomic Plant-Based Vaccine Induces Immune Responses in Test Animals

Malaria is a tropical human disease, caused by protozoan parasites, wherein a significant number of the world's population is at risk. Annually, more than 219 million new cases are reported. Although there are prevention treatments, there are no highly and widely effective licensed anti-malarial vaccines available for use. Opportunities for utilization of plant-based vaccines as novel platforms for developing safe, reliable, and affordable treatments offer promise for developing such a vaccine against malaria. In this study, a Malchloroplast candidate vaccine was designed, composed of segments of AMA1 and MSP1 proteins, two epitopes of Plasmodium falciparum, along with a GK1 peptide from Taenia solium as adjuvant, and this was expressed in tobacco chloroplasts. Transplastomic tobacco lines were generated using biolistic transformation, and these were confirmed to carry the synthetic gene construct. Expression of the synthetic GK1 peptide was confirmed using RT-PCR and Western blots. Furthermore, the GK1 peptide was detected by HPLC at levels of up to 6µgg-1 dry weight of tobacco leaf tissue. The plant-derived Malchloroplast candidate vaccine was subsequently tested in BALB/c female mice following subcutaneous administration, and was found to elicit specific humoral responses. Furthermore, components of this candidate vaccine were recognized by antibodies in Plasmodium falciparum malaria patients and were immunogenic in test mice. Thus, this study provided a 'proof of concept' for a promising plant-based candidate subunit vaccine against malaria.

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii.

Heterologous expression of the NAD+-dependent phosphite dehydrogenase (PTXD) bacterial enzyme from Pseudomonas stutzerii enables selective growth of transgenic organisms by using phosphite as sole phosphorous source. Combining phosphite fertilization with nuclear expression of the ptxD transgene was shown to be an alternative to herbicides in controlling weeds and contamination of algal cultures. Chloroplast expression of ptxD in Chlamydomonas reinhardtii was proposed as an environmentally friendly alternative to antibiotic resistance genes for plastid transformation. However, PTXD activity in the chloroplast is low, possibly due to the low NAD+/NADP+ ratio, limiting the efficiency of phosphite assimilation. We addressed the intrinsic constraints of the PTXD activity in the chloroplast and improved its catalytic efficiency in vivo via rational mutagenesis of key residues involved in cofactor binding. Transplastomic lines carrying a mutagenized PTXD version promiscuously used NADP+ and NAD+ for converting phosphite into phosphate and grew faster compared to those expressing the wild type protein. The modified PTXD enzyme also enabled faster and reproducible selection of transplastomic colonies by directly plating on phosphite-containing medium. These results allow using phosphite as selective agent for chloroplast transformation and for controlling biological contaminants when expressing heterologous proteins in algal chloroplasts, without compromising on culture performance.