Plant-based Expression Systems Research Articles

Enhanced efficacy of glycoengineered rice cell-produced trastuzumab.

For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 μg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

Green Biologics: Harnessing the Power of Plants to Produce Pharmaceuticals.

Plants are increasingly used for the production of high-quality biological molecules for use as pharmaceuticals and biomaterials in industry. Plants have proved that they can produce life-saving therapeutic proteins (Elelyso™-Gaucher's disease treatment, ZMapp™-anti-Ebola monoclonal antibodies, seasonal flu vaccine, Covifenz™-SARS-CoV-2 virus-like particle vaccine); however, some of these therapeutic proteins are difficult to bring to market, which leads to serious difficulties for the manufacturing companies. The closure of one of the leading companies in the sector (the Canadian biotech company Medicago Inc., producer of Covifenz) as a result of the withdrawal of investments from the parent company has led to the serious question: What is hindering the exploitation of plant-made biologics to improve health outcomes? Exploring the vast potential of plants as biological factories, this review provides an updated perspective on plant-derived biologics (PDB). A key focus is placed on the advancements in plant-based expression systems and highlighting cutting-edge technologies that streamline the production of complex protein-based biologics. The versatility of plant-derived biologics across diverse fields, such as human and animal health, industry, and agriculture, is emphasized. This review also meticulously examines regulatory considerations specific to plant-derived biologics, shedding light on the disparities faced compared to biologics produced in other systems.

Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs.

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant.

Antimicrobial Peptides: The Production of Novel Peptide-Based Therapeutics in Plant Systems.

The increased prevalence of antibiotic resistance is alarming and has a significant impact on the economies of emerging and underdeveloped nations. The redundancy of antibiotic discovery platforms (ADPs) and injudicious use of conventional antibiotics has severely impacted millions, across the globe. Potent antimicrobials from biological sources have been extensively explored as a ray of hope to counter the growing menace of antibiotic resistance in the population. Antimicrobial peptides (AMPs) are gaining momentum as powerful antimicrobial therapies to combat drug-resistant bacterial strains. The tremendous therapeutic potential of natural and synthesized AMPs as novel and potent antimicrobials is highlighted by their unique mode of action, as exemplified by multiple research initiatives. Recent advances and developments in antimicrobial discovery and research have increased our understanding of the structure, characteristics, and function of AMPs; nevertheless, knowledge gaps still need to be addressed before these therapeutic options can be fully exploited. This thematic article provides a comprehensive insight into the potential of AMPs as potent arsenals to counter drug-resistant pathogens, a historical overview and recent advances, and their efficient production in plants, defining novel upcoming trends in drug discovery and research. The advances in synthetic biology and plant-based expression systems for AMP production have defined new paradigms in the efficient production of potent antimicrobials in plant systems, a prospective approach to countering drug-resistant pathogens.

Characterisation of two snake toxin-targeting human monoclonal immunoglobulin G antibodies expressed in tobacco plants

Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro antigen binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies.

Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40–60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients’ sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

Current Advances of Plant-Based Vaccines for Neurodegenerative Diseases

Neurodegenerative diseases (NDDs) are characterized by the progressive degeneration and/or loss of neurons belonging to the central nervous system, and represent one of the major global health issues. Therefore, a number of immunotherapeutic approaches targeting the non-functional or toxic proteins that induce neurodegeneration in NDDs have been designed in the last decades. In this context, due to unprecedented advances in genetic engineering techniques and molecular farming technology, pioneering plant-based immunogenic antigen expression systems have been developed aiming to offer reliable alternatives to deal with important NDDs, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Diverse reports have evidenced that plant-made vaccines trigger significant immune responses in model animals, supported by the production of antibodies against the aberrant proteins expressed in the aforementioned NDDs. Moreover, these immunogenic tools have various advantages that make them a viable alternative for preventing and treating NDDs, such as high scalability, no risk of contamination with human pathogens, cold chain free production, and lower production costs. Hence, this article presents an overview of the current progress on plant-manufactured vaccines for NDDs and discusses its future prospects.

Facilitating viral vector movement enhances heterologous protein production in an established plant system.

Molecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system-wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant-based expression system that utilizes viral-based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%.

Comparative analysis of plant transient expression vectors for targeted N-glycosylation.

While plant-based transient expression systems have demonstrated their potency to rapidly express economically feasible quantities of complex human proteins, less is known about their compatibility with posttranslational modification control. Here we investigated three commonly used transient expression vectors, pEAQ, magnICON and pTra for their capability to express a multi-component protein with controlled and modified N-glycosylation. Cetuximab (Cx), a therapeutic IgG1 monoclonal antibody, which carries next to the conserved Fc an additional N-glycosylation site (GS) in the Fab-domain, was used as model. While pEAQ and pTra produce fully assembled Cx at similar levels in N. benthamiana, the yield of magnICON-Cx was twice as high. When expressed in wild type plants, both Cx-GSs exhibited typical plant N-glycans decorated with plant-specific xylose and fucose. Likewise, Cx generated in the glycoengineered ΔXTFT line carried mainly complex N-glycans lacking plant specific residues. Exposure to different engineering settings (encompassing stable lines and transient approaches) towards human galactosylation and sialylation resulted in Cx carrying targeted N-glycans at similar quantities using all three expression vectors. Collectively, our results exhibit the universal application of plant-based glycoengineering, thereby increasing the attractivity of the ambitious expression platform.

Plant-based expression platforms to produce high-value metabolites and proteins.

Plant-based heterologous expression systems can be leveraged to produce high-value therapeutics, industrially important proteins, metabolites, and bioproducts. The production can be scaled up, free from pathogen contamination, and offer post-translational modifications to synthesize complex proteins. With advancements in molecular techniques, transgenics, CRISPR/Cas9 system, plant cell, tissue, and organ culture, significant progress has been made to increase the expression of recombinant proteins and important metabolites in plants. Methods are also available to stabilize RNA transcripts, optimize protein translation, engineer proteins for their stability, and target proteins to subcellular locations best suited for their accumulation. This mini-review focuses on recent advancements to enhance the production of high-value metabolites and proteins necessary for therapeutic applications using plants as bio-factories.

An Extensive Review on the Development of Plant-Based Vaccines against SARS-Cov-2 and Respiratory Disorders Using Plant Biotechnology Platform

The COVID-19 pandemic is the most recent of numerous pathogenic viral pandemics that have posed risks to global health. Its exponentially expanding global transmission is placing pressure on the global health system. There are currently a large number of COVID-19 candidate vaccines on the market, and there is fierce worldwide competition to obtain the most vaccinations for each nation. The main objective among several nations during the exceptional COVID-19 epidemic has been to build herd immunity through the planning of large-scale vaccination efforts. However, the availability of vaccines has been a challenge for developing countries. However, there are strains in the world's vaccine manufacturing as a result of the high demand. This international effort aims to develop plant-based heterologous expression systems, virus-like particles (VLPs)-vaccines, antiviral drugs, a rapid supply of antigen-antibodies for detecting kits, and plant-origin bioactive compounds that boost immunity and provide tolerance to fight against virus infection, also includes the use of a plant biotechnology-based expression system for vaccine production. Due to possible benefits including low cost, high manufacturing volume, and physicochemical characteristics, the idea of a plant-based vaccination has gained more and more momentum in recent years. By utilising local commodities, we suggest plant-based vaccines as an appealing alternative to widespread and inexpensive vaccination methods against COVID-19 in this review. Additionally, we discussed the processes of action, necessary standards, and prospects for applying unique biotechnology tools in near future.

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead, and an orally active analgesic is demonstrated.

Synthesis of single-chain antibody fragment fused to the elastin-like polypeptide in Nicotiana benthamiana and its application in affinity precipitation of difficult to produce proteins.

Plants are economical and sustainable factories for the production of recombinant proteins. Currently, numerous proteins produced using different plant-based systems with applications as cosmetic and tissue culture ingredients, research and diagnostic reagents, and industrial enzymes are marketed worldwide. In this study, we aimed to demonstrate the usefulness of a plant-based system to synthesize a single-chain antibody (scFv)-elastin-like polypeptide (ELP) fusion to be applied as an affinity precipitation reagent of the difficult to produce recombinant proteins. We used the human tissue transglutaminase (TG2), the main celiac disease autoantigen, as a proof of concept. We cloned a TG2-specific scFv and fused it to a short hydrophobic ELP tag. The anti-TG2-scFv-ELP was produced in Nicotiana benthamiana and was efficiently recovered by an inverse transition cycling procedure improved by coaggregation with bacteria-made free ELP. Finally, the scFv-ELP was used to purify both plant-synthesized human TG2 and also Caco-2-TG2. In conclusion, this study showed for the first time the usefulness of a plant-based expression system to produce an antibody-ELP fusion designed for the purification of low-yield proteins.

Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6

Cytochrome P450 monooxygenases (CYPs) are important tools for regio- and stereoselective oxidation of target molecules or engineering of metabolic pathways. Functional heterologous expression of eukaryotic CYPs is often problematic due to their dependency on the specific redox partner and the necessity of correct association with the membranes for displaying enzymatic activity. Plant hosts offer advantages of accessibility of reducing partners and a choice of membranes to insert heterologous CYPs. For the evaluation of plant systems for efficient CYP expression, we established transplastomic plants and hairy root cultures of Nicotiana tabacum carrying the gene encoding human CYP2D6 with broad substrate specificity. The levels of CYP2D6 transcript accumulation and enzymatic activity were estimated and compared with the data of CYP2D6 transient expression in N. benthamiana. The relative level of CYP2D6 transcripts in transplastomic plants was 2–3 orders of magnitude higher of that observed after constitutive or transient expression from the nucleus. CYP2D6 expressed in chloroplasts converted exogenous synthetic substrate loratadine without the need for co-expression of the cognate CYP reductase. The loratadine conversion rate in transplastomic plants was comparable to that in N. benthamiana plants transiently expressing a chloroplast targeted CYP2D6 from the nucleus, but was lower than the value reported for transiently expressed CYP2D6 with the native endoplasmic reticulum signal-anchor sequence. Hairy roots showed the lowest substrate conversion rate, but demonstrated the ability to release the product into the culture medium. The obtained results illustrate the potential of plant-based expression systems for exploiting the enzymatic activities of eukaryotic CYPs with broad substrate specificities.

Process Engineering of Biopharmaceutical Production in Moss Bioreactors via Model-Based Description and Evaluation of Phytohormone Impact.

The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 µM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-kynurenine (100 µM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 h after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first 2 hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

Safety and immunogenicity of a plant-derived recombinant protective antigen (rPA)-based vaccine against Bacillus anthracis: A Phase 1 dose-escalation study in healthy adults

BackgroundThe potential use of Bacillus anthracis as a bioterrorism weapon requires a safe and effective vaccine that can be immediately distributed for mass vaccination. Protective antigen (PA), a principal component of virulence factors edema toxin and lethal toxin of B. anthracis, has been the topic of extensive research. Previously, full-length PA (PA83) was manufactured using a transient plant-based expression system. Immunization with this PA83 antigen formulated with Alhydrogel® adjuvant elicited strong neutralizing immune responses in mice and rabbits and protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. This Phase 1 study evaluates this vaccine’s safety and immunogenicity in healthy human volunteers. MethodsThis first-in-human, single-blind, Phase 1 study was performed at a single center to investigate the safety, reactogenicity, and immunogenicity of the plant-derived PA83-FhCMB vaccine at four escalating dose levels (12.5, 25, 50 or 100 µg) with Alhydrogel® in healthy adults 18–49 years of age (inclusive). Recipients received three doses of vaccine intramuscularly at 28-day intervals. Safety was evaluated on days 3, 7, and 14 following vaccination. Immunogenicity was assessed using an enzyme-linked immunosorbent assay (ELISA) and a toxin neutralizing antibody (TNA) assay on days 0, 14, 28, 56, 84, and 180. ResultsAll four-dose ranges were safe and immunogenic, with no related serious adverse events observed. Peak ELISA Geometric Mean Concentration (GMC) and TNA ED50 Geometric Mean Titer (GMT) were noted at Day 84, 1 month after the final dose, with the most robust response detected in the highest dose group. Antibody responses decreased by Day 180 across all dose groups. Long-term immunogenicity data beyond six months was not collected. ConclusionsThis is the first study demonstrating a plant-derived subunit anthrax vaccine’s safety and immunogenicity in healthy adults. The results support further clinical investigation of the PA83-FhCMB vaccine.ClinicalTrials.gov identifier.NCT02239172.

Plant proteins as natural, biodegradable, low cost larvicides against mosquitoes

Vectors spread infectious and deadly diseases among human beings and animals. The best-known vector of many infectious diseases is mosquito. Some other vectors are flies, ticks, fleas, aquatic snails and triatomine bugs. Mosquitoes are the major group of arthropods which causes millions of deaths every year. Mosquitoes breed in the water and the larval stage is attractive target for botanical insecticides to deal with them in their habitat. Plant contains many phytochemicals which act as mosquito repellents, larvicidal agents, insect growth regulators and transmission blocking sources. There are many reports available showing larvicidal activities of plant crude extracts and some compounds isolated from crude extracts. But there is very little information available about plant proteins as larvicides. The present study shade light on mosquito larvicidal role of crude proteins extracts, some toxic proteins (lectins, RIPs, proteinases inhibitors) derived from different plants and plant-based expression system for transmission blocking vaccine. This review may straighten out the complete investigations of the potency of anti-larvicidal properties of plant derived proteins and these proteins could be targeted for drug formation, natural bio-insecticide and transmission blocking agents for the control of vector borne diseases.

Safety and immunogenicity studies in animal models support clinical development of a bivalent norovirus-like particle vaccine produced in plants

Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide. A safe and effective vaccine that prevents NoV infection or minimizes NoV disease burden is needed, especially for children and the elderly who are particularly susceptible to NoV disease. A plant-based expression system (magnICON®) was used to manufacture two different virus-like particle (VLP) immunogens derived from human NoV genogroups I and II, genotype 4 (GI.4 and GII.4), which were subsequently blended 1:1 (w/w) into a bivalent vaccine composition (rNV-2v). Here, we report on the safety and immunogenicity of rNV-2v from one pilot and two GLP-compliant toxicity studies in New Zealand White rabbits administered the vaccine subcutaneously (SC) or intramuscularly (IM). Strong genogroup-specific immune responses were induced by vaccination without adjuvant at various doses (200 to 400 μg VLP/administration) and administration schedules (Days 1 and 7; or Days 1, 15 and 29). The results showed sporadic local irritation at the injection site, which resolved over time, and was non-adverse and consistent with expected reactogenicity. There were no signs of systemic toxicity related to vaccine administration relative to vehicle-treated controls with respect to clinical chemistry, haematology, organ weights, macroscopic examinations, or histopathology. In a 3-administration regimen (n + 1 the clinical regimen), the NOAEL for rNV-2v via the SC or IM route was initially determined to be 200 μg. An improved GI.4 VLP variant mixed 1:1 (w/w) with the wild-type GII.4 VLP was subsequently evaluated via the IM route at a higher dose in the same 3-administration model, and the NOAEL was raised to 300 µg. Serology performed in samples of both toxicity studies showed significant and substantial anti-VLP-specific antibody titers for rNV-2v vaccines administered via the IM or SC route, as well as relevant NoV blocking antibody responses. These results support initiation of clinical development of the plant-made NoV vaccine.

Effects of plant growth regulators on transient expression of foreign gene in Nicotiana benthamiana L. leaves

BackgroundIn the last decades, replicating expression vectors based on plant geminivirus have been widely used for enhancing the efficiency of plant transient expression. By using the replicating expression vector derived from bean yellow dwarf virus and green fluorescent protein as a reporter, we investigated the effects of α-naphthalene acetic acid, gibberellins3, and 6-benzyladenine, as three common plant growth regulators, on the plant biomass and efficiency of transient expression during the process of transient expression in Nicotiana benthamiana L. leaves.ResultsWith the increase of the concentration of α-naphthalene acetic acid, gibberellins3, and 6-benzyladenine (from 0.1 to 1.6 mg/L), the fresh weight, dry weight, and leaf area of the seedlings increased first and then returned to the levels similar to the controls (without chemical treatment). The treatment with α-naphthalene acetic acid at 0.2 and 0.4 mg/L can enhance the level of transient expression of green fluorescent protein, which peaked at 0.4 mg/L α-naphthalene acetic acid and was increased about by 19%, compared to the controls. Gibberellins3 at 0.1–0.4 mg/L can enhance the level of transient expression of green fluorescent protein, which peaked at 0.2 mg/L gibberellins3 and was increased by 25%. However, the application of 6-benzyladenine led to decrease in the level of transient expression of green fluorescent protein.ConclusionsThe appropriate plant growth regulators at moderate concentration could be beneficial to the expression of foreign genes from the Agrobacterium-mediated transient expression system in plants. Thus, appropriate plant growth regulators could be considered as exogenous components that are applied for the production of recombinant protein by plant-based transient expression systems.

Robust Agrobacterium-Mediated Transient Expression in Two Duckweed Species (Lemnaceae) Directed by Non-replicating, Replicating, and Cell-to-Cell Spreading Vectors.

Plant-based transient expression systems have recognized potential for use as rapid and cost-effective alternatives to expression systems based on bacteria, yeast, insect, or mammalian cells. The free-floating aquatic plants of the Lemnaceae family (duckweed) have compact architecture and can be vegetatively propagated on low-cost nutrient solutions in aseptic conditions. These features provide an economically feasible opportunity for duckweed-based production of high-value products via transient expression of recombinant products in fully contained, controlled, aseptic and bio-safe conditions in accordance with the requirements for pharmaceutical manufacturing and environmental biosafety. Here, we demonstrated Agrobacterium-mediated high-yield transient expression of a reporter green fluorescent protein using deconstructed vectors based on potato virus X and sweet potato leaf curl virus, as well as conventional binary vectors, in two representatives of the Lemnaceae (Spirodela polyrhiza and Landoltia punctata). Aseptically cultivated duckweed populations yielded reporter protein accumulation of >1 mg/g fresh biomass, when the protein was expressed from a deconstructed potato virus X-based vector, which is capable of replication and cell-to-cell movement of the replicons in duckweed. The expression efficiency demonstrated here places duckweed among the most efficient host organisms for plant-based transient expression systems, with the additional benefits of easy scale-up and full containment.