Plant-based Production Systems Research Articles

How to find alternative crops for climate-resilient regional food production

CONTEXTAgricultural food production is both affected by and contributing to climate change. At the global scale, agri-food systems are responsible for one-third of total greenhouse gas emissions. With progressing climate change, the risks of crop failure increase. Thus, an urgent need is to reduce emissions from food systems while increasing their resilience to climate change. Enormous untapped potentials to achieve these dual goals lie in transforming agri-food systems towards more diverse, plant-based, and regional food production systems. OBJECTIVEIn this paper, we present an innovative approach for identifying climate-adapted alternative food crops that could (1) help to diversify existing cropping systems and thus increase their climate resilience and can be (2) nutritious elements of plant-based regional diets with reduced emissions. METHODSThe approach builds on the model ecocrop to select food crops that could benefit from regionally projected changes in climate. The model-based analysis is complemented with a literature review to examine the ecocrop results for their plausibility and provide a broader assessment of potentials for cultivation, utilization, and nutritional values of model-selected crops. RESULTS AND CONCLUSIONSThe approach is applied to Switzerland, where we identify eight alternative crops with the potential to increase climate resilience while contributing to healthy human diets of regional consumers with benefits for climate mitigation (almond, pecan, sesame, durum wheat, quinoa, lentil, lupine, and borage). The literature review indicated that the increasing demand for many of these crops suggests great potential for regional marketing of crop products. The results produced in this study provide an initial guide for researchers and innovative farmers interested in experimenting with alternative crops in Switzerland, thus promoting climate-smart food system transformation from the production side. SIGNIFICANCEUsing our unbiased bottom-up screening approach, we identified climate-adapted alternative crops that can provide essential nutrients, cover nutritional gaps in Switzerland, diversify existing production systems, and improve sustainability.

Substrate and fertigation management modulate microgreens production, quality and resource efficiency

Long-term space missions will require a self-sustaining food production system to meet the crew’s nutritional and health needs. For this purpose, plant-based food production systems with elevated resource efficiency are required, based on advanced agricultural technologies that produce phytonutrient-rich crops. In addition to the resource requirements for crop production on Earth, volume and time efficiency become essential factors to consider for food production in space. Microgreens represent a promising candidate for space farming as they have a high harvest index, short cultivation cycle, and high nutritional potential. However, the development of specific technical protocols for growing microgreens in space is essential since different agronomic inputs, such as substrates and fertigation, can modulate productivity, quality and resource efficiency of microgreens cultivation. The current work examines the effects of different substrates (coconut fiber and cellulose sponge) and nutrient solution (NS) management strategies (quarter strength Hoagland and half strength Hoagland/osmotic water) on the production of two species of microgreens [Raphanus sativus cv. Saxa 2 (Radish); Brassica oleracea var. capitata f. sabauda cv. Vertus (Savoy cabbage)]. The appraisal focused on (i) biomass production and quality, and (ii) sizing of space facilities devoted to the production of phytonutrients required for the astronauts’ wellbeing. In our study, the interaction among species, substrate and NS significantly affected the accumulation of fructose, sucrose, total soluble non-structural carbohydrates and nitrate as well as the daily production of total ascorbic acid and, in turn, the required microgreens serving to supply its adequate daily intake. Species-substrate interaction effects on fresh yield, dry yield, dry matter, anthocyanins, TPC, β-carotene and sulfate content as well as the cultivation surface required to produce the adequate daily intake of ascorbic acid (AscA) were assessed. Substrate-NS interaction modulated the anthocyanins, violaxanthin and sulfate contents independently of species. On the other hand, single factor effects were identified with respect to the accumulation of lutein, chlorophylls, glucose, and starch. Therefore, the management of microgreens cultivation in terms of NS and substrate is an effective tool to meet the phytochemical requirements of the crew.

Seed-Specific Expression of Apolipoprotein A-IMilano Dimer in Engineered Rice Lines

Apolipoprotein A-IMilano (ApoA-IM) has been shown to significantly reduce coronary atherosclerotic plaques. However, the preparation of cost-effective pharmaceutical formulations of ApoA-IM can be limited by the high cost and difficulty of purifying the protein and producing the highly effective dimeric form. The aim of this study was to create an expression cassette that specifically drives the expression of dimeric ApoA-IM in the protein bodies of rice seeds. The ApoA-IM protein under control of the 13 kDa prolamin promoter is expressed exclusively in its dimeric form within the seeds, and immunocytochemical and immunogold analyses confirmed its expression in different caryopsis tissue such as seed coat, aleurone cell and endosperm, particularly in amyloplast and storage vacuoles. A plant-based ApoA-IM production system offered numerous advantages over current production systems, including the direct production of the most therapeutically effective dimeric ApoA-IM forms, long-term protein storage in seeds, and ease of protein production by simply growing plants. Therefore, seeds had the potential to serve as a cost-effective source of therapeutic ApoA-IM.

Genomic and transcriptomic analysis of camptothecin producing novel fungal endophyte: Alternaria burnsii NCIM 1409

Camptothecin is an important anticancer alkaloid produced by particular plant species. No suitable synthetic route has been established for camptothecin production yet, imposing a stress on plant-based production systems. Endophytes associated with these camptothecin-producing plants have been reported to also produce camptothecin and other high-value phytochemicals. A previous study identified a fungal endophyte Alternaria burnsii NCIM 1409, isolated from Nothapodytes nimmoniana, to be a sustainable producer of camptothecin. Our study provides key insights on camptothecin biosynthesis in this recently discovered endophyte. The whole genome sequence of A. burnsii NCIM 1409 was assembled and screened for biosynthetic gene clusters. Comparative studies with related fungi supported the identification of candidate genes involved in camptothecin synthesis and also helped to understand some aspects of the endophyte’s defense against the toxic effects of camptothecin. No evidence for horizontal gene transfer of the camptothecin biosynthetic genes from the host plant to the endophyte was detected suggesting an independent evolution of the camptothecin biosynthesis in this fungus.

Plantibodies: a plant based production system

Plantibodies: a plant based production system

Improvement of recombinant miraculin production in transgenic tomato by crossbreeding-based genetic background modification.

An important optimization step in plant-based recombinant protein production systems is the selection of an appropriate cultivar after a potential host has been determined. Previously, we have shown that transgenic tomatoes of the variety 'Micro-Tom' accumulate incredibly high levels of miraculin (MIR) due to the introduction of MIR gene controlled by a CaMV35S promoter and a heat-shock protein terminator. However, 'Micro-Tom' is unsuitable for commercial production of MIR as it is a dwarf cultivar characterized by small-sized fruit and poor yield. Here, we used the crossbreeding approach to transfer the high MIR accumulation trait of transgenic 'Micro-Tom' tomatoes to 'Natsunokoma' and 'Aichi First', two commercial cultivars producing medium and large fruit sizes, respectively. Fruits of the resultant crossbred lines were larger (~ 95 times), but their miraculin accumulation levels (~ 1,062μg/g fresh mass) were comparable to the donor cultivar, indicating that the high miraculin accumulation trait was preserved regardless of fruit size or cultivar. Further, the transferred trait resulted in a 3-4fold increase in overall miraculin production than that of the previously reported line 5B. These findings demonstrate the effectiveness of crossbreeding in improving MIR production in tomatoes and could pave the way for a more efficient production of recombinant proteins in other plants.

Reducing water uptake into BY-2 cells by systematically optimizing the cultivation parameters increases product yields achieved by transient expression in plant cell packs.

Plant-based production systems are inexpensive and easy to handle, allowing them to complement existing platforms for the production of protein-based vaccines, therapeutics and diagnostic reagents. However, screening product candidates in whole plants requires a large facility footprint and is challenging due to natural variations in recombinant protein accumulation. In contrast, plant cell packs (PCPs) allow more than 1000 samples to be screened per day in microtiter plates. PCPs enable rapid development cycles based on transient expression in as little as 3 days, and yield milligram quantities of product for initial quality assessment and functional testing. However, this requires high-level expression in BY-2 cells and consistent cell quality across batches. We therefore used a statistical design of experiments (DoE) approach to systematically assess factors that contribute to consistent high yields of recombinant proteins in PCPs. Specifically, we tested the osmolality, pH, carbon source, light source, and additives during cell cultivation, as well as cell and PCP harvest times. The careful adjustment of these factors increased overall productivity by approximately fourfold. Remarkably all cultivation conditions leading to high productivities during transient expression in PCPs were associated with a reduced water uptake into the central vacuole. The universal presence of a vacuole in plant cells indicates that our results should be transferrable to other cells lines. Our findings therefore support the broad application of PCPs for screening and product analysis during the development of protein-based pharmaceuticals and reagents in plants.

Plants as Sources of Natural and Recombinant Antimalaria Agents.

Malaria is one of the severe infectious diseases that has victimized about half a civilization billion people each year worldwide. The application of long-lasting insecticides is the main strategy to control malaria; however, a surge in antimalarial drug development is also taking a leading role to break off the infections. Although, recurring drug resistance can compromise the efficiency of both conventional and novel antimalarial medicines. The eradication of malaria is significantly contingent on discovering novel potent agents that are low cost and easy to administer. In this context, plant metabolites inhibit malaria infection progression and might potentially be utilized as an alternative treatment for malaria, such as artemisinin. Advances in genetic engineering technology, especially the advent of molecular farming, have made plants more versatile in producing protein drugs (PDs) to treat infectious diseases, including malaria. These recent developments in genetic modifications have enabled the production of native pharmaceutically active compounds and the accumulation of diverse heterologous proteins such as human antibodies, booster vaccines, and many PDs to treat infectious diseases and genetic disorders. This review will discuss the pivotal role of a plant-based production system that expresses natural antimalarial agents or host protein drugs to cure malaria infections. The potential of these natural and induced compounds will support modern healthcare systems in treating malaria infections, especially in developing countries to mitigate human fatalities.

Proteomic Profiling of Interplay Between Agrobacterium tumefaciens and Nicotiana benthamiana for Improved Molecular Pharming Outcomes.

Transient expression of recombinant proteins in plants is being used as a platform for production of therapeutic proteins. Benefits of this system include a reduced cost of drug development, rapid delivery of new products to the market, and an ability to provide safe and efficacious medicines for diseases. Although plant-based production systems offer excellent potential for therapeutic protein production, barriers, such as plant host defense response, exist which negatively impact the yield of product. Here we provide a protocol using tandem mass tags and mass spectrometry-based proteomics to quickly and robustly quantify the change in abundance of host defense proteins produced during the production process. These proteins can then become candidates for genetic manipulation to create host plants with reduced plant defenses capable of producing higher therapeutic protein yields.

Integrated Pest Management Protocols for Space-Based Bioregenerative Life Support Systems

Human missions to the Moon and Mars will necessarily increase in both duration and complexity over the coming decades. In the past, short-term missions to low-Earth orbit (LEO) or the Moon (e.g., Apollo) utilized physiochemical life support systems for the crews. However, as the spatial and temporal durations of crewed missions to other planetary bodies increase, physiochemical life support systems become burdened with the requirement of frequent resupply missions. Bioregenerative life support systems (BLSS) have been proposed to replace much of the resupply required of physiochemical systems with modules that can regenerate water, oxygen, and food stocks with plant-based biological production systems. In order to protect the stability and productivity of BLSS modules (i.e., small scale units) or habitats (i.e., large scale systems), an integrated pest management (IPM) program is required to prevent, mitigate, and eliminate both insect pests and disease outbreaks in space-based plant-growing systems. A first-order BLSS IPM program is outlined herein that summarizes a collection of protocols that are similar to those used in field, greenhouse, and vertical-farming agricultural systems. However, the space environment offers numerous unusual stresses to plants, and thus, unique space-based IPM protocols will have to be developed. In general, successful operation of space-based BLSS units will be guided by IPM protocols that (1) should be established early in the mission design phase to be effective, (2) will be dynamic in nature changing both spatially and temporally depending on the successional processes afoot within the crewed spacecraft, plant-growing systems, and through time; and (3) can prevent insect/phytopathology outbreaks at very high levels that can approach 100% if properly implemented.

Use of the DELTA Model to Understand the Food System and Global Nutrition

BackgroundIncreasing attention is being directed at the environmental, social, and economic sustainability of the global food system. However, a key aspect of a sustainable food system should be its ability to deliver nutrition to the global population. Quantifying nutrient adequacy with current tools is challenging. ObjectiveTo produce a computational model illustrating the nutrient adequacy of current and proposed global food systems. MethodsThe DELTA Model was constructed using global food commodity balance sheet data, alongside demographic and nutrient requirement data from UN and European Food Safety Authority sources. It also includes nutrient bioavailability considerations for protein, the indispensable amino acids, iron, and zinc, sourced from scientific literature. ResultsThe DELTA Model calculates global per capita nutrient availability under conditions of equal distribution and identifies areas of nutrient deficiency for various food system scenarios. Modeling the 2018 global food system showed that it supplied insufficient calcium (64% of demographically weighted target intake) and vitamin E (69%), despite supplying sufficient macronutrients. Several future scenarios were modeled, including variations in waste; scaling up current food production for the 2030 global population; plant-based food production systems; and removing sugar crops from the global food system. Each of these scenarios fell short of meeting requirements for multiple nutrients. These results emphasize the need for a balanced approach in the design of future food systems. ConclusionsNutrient adequacy must be at the forefront of the sustainable food system debate. The DELTA Model was designed for both experts and nonexperts to inform this debate as to what may be possible, practical, and optimal for our food system. The model results strongly suggest that both plant and animal foods are necessary to achieve global nutrition. The model is freely available for public use so that anyone can explore current and simulated global food systems.

Production of a Bacteria-like Particle Vaccine Targeting Rock Bream (Oplegnathus fasciatus) Iridovirus Using Nicotiana benthamiana.

Viral diseases are extremely widespread infections that change constantly through mutations. To produce vaccines against viral diseases, transient expression systems are employed, and Nicotiana benthamiana (tobacco) plants are a rapidly expanding platform. In this study, we developed a recombinant protein vaccine targeting the major capsid protein (MCP) of iridovirus fused with the lysine motif (LysM) and coiled-coil domain of coronin 1 (ccCor1) for surface display using Lactococcus lactis. The protein was abundantly produced in N. benthamiana in its N-glycosylated form. Total soluble proteins isolated from infiltrated N. benthamiana leaves were treated sequentially with increasing ammonium sulfate solution, and recombinant MCP mainly precipitated at 40–60%. Additionally, affinity chromatography using Ni–NTA resin was applied for further purification. Native structure analysis using size exclusion chromatography showed that recombinant MCP existed in a large oligomeric form. A minimum OD600 value of 0.4 trichloroacetic acid (TCA)-treated L. lactis was required for efficient recombinant MCP display. Immunogenicity of recombinant MCP was assessed in a mouse model through enzyme-linked immunosorbent assay (ELISA) with serum-injected recombinant MCP-displaying L. lactis. In summary, we developed a plant-based recombinant vaccine production system combined with surface display on L. lactis.

Plant-Based Vaccines: The Way Ahead?

Severe virus outbreaks are occurring more often and spreading faster and further than ever. Preparedness plans based on lessons learned from past epidemics can guide behavioral and pharmacological interventions to contain and treat emergent diseases. Although conventional biologics production systems can meet the pharmaceutical needs of a community at homeostasis, the COVID-19 pandemic has created an abrupt rise in demand for vaccines and therapeutics that highlight the gaps in this supply chain’s ability to quickly develop and produce biologics in emergency situations given a short lead time. Considering the projected requirements for COVID-19 vaccines and the necessity for expedited large scale manufacture the capabilities of current biologics production systems should be surveyed to determine their applicability to pandemic preparedness. Plant-based biologics production systems have progressed to a state of commercial viability in the past 30 years with the capacity for production of complex, glycosylated, “mammalian compatible” molecules in a system with comparatively low production costs, high scalability, and production flexibility. Continued research drives the expansion of plant virus-based tools for harnessing the full production capacity from the plant biomass in transient systems. Here, we present an overview of vaccine production systems with a focus on plant-based production systems and their potential role as “first responders” in emergency pandemic situations.

Plant production and functional characterization of catfish interleukin-22 as a natural immune stimulant for aquaculture fish

As the world population increases and wild caught fisheries decline, aquaculture offers a sustainable solution addressing this global challenge. However, disease management remains difficult. With limited options, there is a need for innovative solutions. The cytokine interleukin-22 (IL-22) has emerged as a possible therapeutic target for fish and has been correlated with protection under pathogen challenge. Plant-based production systems have the potential to effectively manufacture and bring unique efficacy-enhancing features to the aquaculture industry; namely, the advantages of low cost for this commodity market, ready scalability, and reduced environmental impact. Catfish IL-22 produced at significant yield and purity highlights the use of plants as a promising production platform for therapeutic proteins with utility to the aquaculture industry. Purified cfIL-22 shows similar in vitro bioactivity to its mammalian homolog that include increased proliferation of catfish cells highlighting the tissue preservation capabilities associated with this protein. Recombinant cfIL-22 also upregulated expression of genes encoding a tissue repair protein, fibronectin, an antimicrobial peptide, Natural killer lysin-1, and a common innate immune protein, interferon. These findings support plant-made recombinant catfish interleukin-22 as a potential therapeutic for the aquaculture industry and further analysis of this protein for promoting animal health.

Increased brazzein expression by abiotic stress and bioreactor culture system for the production of sweet protein, brazzein

Plant-based recombinant protein production systems are powerful tools that can be used instead of microbial and animal cell culture systems. The aim of this study was to establish a stable brazzein production system from carrot cell suspension using bioreactor. Using Agrobacterium-mediated transformation (stress-inducible SWPA2 promoter and brazzein gene), three transgenic lines (TC1, 11, 12) were generated. During the TC12 culture period, cell proliferation increased rapidly up to 15 days, and the maximum cell division rate (log phase) was reached after 6 days of culture. Fresh weight peaked at day 18, 3.8-fold (7.6 g 100 mL−1) higher than on day 1, and gene expression was tenfold higher on day 27 than on day 0. The brazzein-encoding gene was also analyzed after treatment with various stress factors for increase of brazzein gene expression, and abscisic acid (ABA) and hydrogen peroxide (H2O2) proved most effective. In particular, the gene expression was enhanced 2.5-fold with 220 μM H2O2 and 2.8-fold with 50 μM ABA, compared with controls. Transgenic cells were then transferred to various types of air-lift bioreactor, and the column bioreactor achieved a higher biomass (238.9 g L−1) than cone and balloon bioreactors. These results demonstrate an efficient protein production system for brazzein using an air-lift bioreactor that may be suitable for the use in the food industry.

Biotechnological Production of the Cell Penetrating Antifungal PAF102 Peptide in Pichia pastoris.

Antimicrobial peptides (AMPs) have potent and durable antimicrobial activity to a wide range of fungi and bacteria. The growing problem of drug-resistant pathogenic microorganisms, together with the lack of new effective compounds, has stimulated interest in developing AMPs as anti-infective molecules. PAF102 is an AMP that was rationally designed for improved antifungal properties. This cell penetrating peptide has potent and specific activity against major fungal pathogens. Cecropin A is a natural AMP with strong and fast lytic activity against bacterial and fungal pathogens, including multidrug resistant pathogens. Both peptides, PAF102 and Cecropin A, are alternative antibiotic compounds. However, their exploitation requires fast, cost-efficient production systems. Here, we developed an innovative system to produce AMPs in Pichia pastoris using the oleosin fusion technology. Oleosins are plant-specific proteins with a structural role in lipid droplet formation and stabilization, which are used as carriers for recombinant proteins to lipid droplets in plant-based production systems. This study reports the efficient production of PAF102 in P. pastoris when fused to the rice plant Oleosin 18, whereas no accumulation of Cecropin A was detected. The Ole18-PAF102 fusion protein targets the lipid droplets of the heterologous system where it accumulates to high levels. Interestingly, the production of this fusion protein induces the formation of lipid droplets in yeast cells, which can be additionally enhanced by the coexpression of a diacylglycerol transferase gene that allows a three-fold increase in the production of the fusion protein. Using this high producer strain, PAF102 reaches commercially relevant yields of up to 180 mg/l of yeast culture. Moreover, the accumulation of PAF102 in the yeast lipid droplets facilitates its downstream extraction and recovery by flotation on density gradients, with the recovered PAF102 being biologically active against pathogenic fungi. Our results demonstrate that plant oleosin fusion technology can be transferred to the well-established P. pastoris cell factory to produce the PAF102 antifungal peptide, and potentially other AMPs, for multiple applications in crop protection, food preservation and animal and human therapies.

Current state-of-the-art in plant-based antibody production systems

Monoclonal antibodies represent the major class of biopharmaceutical products (for therapeutics and diagnostics) with an increasing demand that reaches several tons per year worldwide. Traditional large-scale manufacturing processes are based on stirred tank bioreactors for the growth of Chinese Hamster Ovary cells (CHO) which requires high initial investments and production costs. Therefore, there is an urgent need for alternative production platforms that can at least act as a complement to the over-exploited mammalian fermentation systems. In this perspective, the use of plants for the large-scale production of biopharmaceuticals ('Molecular farming') represents an interesting and mature technology that has already proved its benefits in terms of safety, scalability, rapidity and reduced manufacturing costs. Here we discuss the recent advances in the production of monoclonal antibodies (mAbs) in plant-based platforms such as transgenic plants, tissue and cell cultures and transient expression systems.

Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing.

Plants have unique advantages over other systems such as mammalian cells for the production of valuable small molecules and proteins. The benefits cited most often include safety due to the absence of replicating human pathogens, simplicity because sterility is not required during production, scalability due to the potential for open-field cultivation with transgenic plants, and the speed of transient expression potentially providing gram quantities of product in less than 4 weeks. Initially there were also significant drawbacks, such as the need to clarify feed streams with a high particle burden and the large quantities of host cell proteins, but efficient clarification is now readily achieved. Several additional advantages have also emerged reflecting the fact that plants are essentially biodegradable, single-use bioreactors. This article will focus on the exploitation of this concept for the production of biopharmaceutical proteins, thus improving overall process economics. Specifically, we will discuss the single-use properties of plants, the sustainability of the production platform, and the commercial potential of different biomass side streams. We find that incorporating these side streams through rational process integration has the potential to more than double the revenue that can currently be achieved using plant-based production systems.

Lignocellulosic Bioethanol Production: Perspectives and Challenges

Biofuels produced from different lignocellulosic materials have the potential to be a valuable substitute for gasoline. Many physicochemical structural and compositional factors hinder the hydrolysis of cellulose present in biomass to sugars and other organic compounds that can later be converted to fuels. Different pretreatment techniques can change the physical and chemical structure of the lignocellulosic biomass and improve their hydrolysis rates. Many methods have been shown to result in high sugar yields, above 90% of the theoretical yield. Advances in pretreatment and biological-processing steps clearly provide the greatest opportunity to reduce bioethanol costs, and much more emphasis is needed in these areas. For pretreatment, improved process configurations are needed that reduce chemical costs for hemicellulose hydrolysis and subsequent conditioning for biological processing. In addition, energy requirements for biomass milling and heating must be reduced, and less corrosive environments are desired to reduce the cost of vessels. Future biotechnology-based developments in processing technology will likely include: Improved cellulase and hemicellulase production economics via microbe or plant-based production systems; improved fermentation strains that efficiently utilize both hemicellulose and cellulosic sugars; consolidated bioprocessing microbes which combine the ability to break down cellulosic materials with the ability to efficiently ferment sugars to ethanol.

Towards development of a universal dengue vaccine - How close are we?

Dengue has been ranked as one of the top emerging diseases in Asia and Latin America. Current epidemiological data may not even reflect the true burden of disease due to under-reported figures. Vector control programmes have failed to contain the disease and worst of all, no specific treatment is available at the moment. Thereby, this pushes the demand for a dengue vaccine as a long-term protective approach. Despite there are numerous vaccine candidates ahead, they could be held back by different aspects in promoting vaccine implementation. Particularly for developing nations, logistics and cost are the major hurdles that need to be addressed in order to provide a quick yet affordable medical relief. As an alternative, plant-based vaccine production system is able to offer an attractive prospect given to its advantages of biocontainment warranty, low operation cost, rapid scalability and logistics flexibility. Researches that have embarked on this scope are laid out and reviewed in terms of the feasibility of plant system to serve as a biofactory for dengue vaccine.