Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension.

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

Performance of plant-produced RBDs as SARS-CoV-2 diagnostic reagents: a tale of two plant platforms.

The COVID-19 pandemic has underscored the need for rapid and cost-effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development.

Improving yield of a recombinant biologic in a Brassica hairy root manufacturing process.

Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha- l-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4- d + 20.5 g/l KNO3 ) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.

Tailoring sensory properties of plant cell cultures for food use.

The nutritional value of Rowan (Sorbus aucuparia L.) and Arctic bramble (Rubus arcticus L.) plant cell cultures in terms of protein and dietary fibre contents is very good, ∼ 18-22% and âˆ¼ 28-29% on dry matter basis, respectively. The aim of this study was to evaluate various processing methods and formulation to modulate sensory profiles of these plant cell cultures for food purposes. For fresh unprocessed plant cell cultures, treatment with sugar or sugar in combination with citric acid significantly improved the mouthfeel and flavour. The sugar and sugar + citric acid treated plant cell culture samples were perceived more moist, softer, less sandy and they had a less coarse mouthfeel when compared to untreated plant cell cultures. Freeze-drying produced sweet, intense, berry-like flavour and resulted in most promising sensory attributes for the studied plant cell cultures. When freeze-dried Rowan plant cell culture was further processed, the most balanced sweetness/sourness ratio was reached by using 9.5 % (w/w) sucrose and 0.1 % (w/w) citric acid or 4.8 % w/w fructose and 0.1 % w/w citric acid. We conclude that formulation and processing can greatly improve the performance of plant cell cultures for food use.

The role of single cell protein in cellular agriculture.

More food needs to be produced for the growing human population, but the possibilities of expanding the area of arable land are limited. Cellular Agriculture is an emerging field of biotechnology, aimed at finding alternatives to agricultural production of various commodities. As a part of Cellular Agriculture, the use of microbes and microalgae as food and feed with high protein content, so-called single cell protein (SCP), is gaining renewed scientific and commercial interest. In this review, we give an introduction to SCP production by heterotrophic microbial species, phototrophs, methanotrophs and autotrophic hydrogen oxidizers, as well as highlight some challenges and the latest developments in the growing SCP industry.

Life cycle assessment of plant cell cultures.

A novel food such as plant cell culture (PCC) is an important complementary asset for traditional agriculture to tackle global food insecurity. To evaluate environmental impacts of PCC, a life cycle assessment was applied to tobacco bright yellow-2 and cloudberry PCCs. Global warming potential (GWP), freshwater eutrophication potential (FEUP), marine eutrophication potential, terrestrial acidification potential (TAP), stratospheric ozone depletion, water consumption and land use were assessed. The results showed particularly high contributions (82-93%) of electricity consumption to GWP, FEUP and TAP. Sensitivity analysis indicated that using wind energy instead of the average Finnish electricity mix reduced the environmental impacts by 34-81%. Enhancement in the energy efficiency of bioreactor mixing processes and reduction in cultivation time also effectively improved the environmental performance (4-47% reduction of impacts). In comparison with other novel foods, the environmental impacts of the PCC products studied were mostly comparable to those of microalgae products but higher than those of microbial protein products produced by autotrophic hydrogen-oxidizing bacteria. Assayed fresh PCC products were similar or close to GWP of conventionally grown food products and, with technological advancements, can be highly competitive.

Plant cell cultures as food-aspects of sustainability and safety.

KEY MESSAGE: Sustainability and safety aspects of plant cell cultures as food are presented. Applicability of dairy side streams as carbon source and use of natural growth enhancers in cultivation are shown. Biotechnologically produced cellular products are currently emerging to replace and add into the portfolio of agriculturally derived commodities. Plant cell cultures used for food could supplement current food production. However, still many aspects need to be resolved before this new food concept can enter the market. Issues related to sustainability and safety for human consumption are relevant for both consumers and regulators. In this study, two plant cell cultures, deriving from arctic bramble (Rubus arcticus) and birch (Betula pendula), were cultivated using lactose-rich dairy side streams as alternative carbon sources to replace sucrose. Biomasses were comparable to those of original plant cell culture media when up to 83% and 75% of the original sucrose was replaced by these side streams for arctic bramble and birch cell cultures, respectively. Furthermore, nutritional composition or sensory properties were not compromised. Synthetic plant growth regulators were replaced by natural components, such as coconut water and IAA for several subculture cycles. Finally, it was shown that only trace amounts of free growth regulators are present in the cells at the harvesting point and assessment by freshwater crustaceans assay indicated that toxicity of the cells was not exceeding that of traditionally consumed bilberry fruit.

Hairy Root Cultures-A Versatile Tool With Multiple Applications.

Hairy roots derived from the infection of a plant by Rhizobium rhizogenes (previously referred to as Agrobacterium rhizogenes) bacteria, can be obtained from a wide variety of plants and allow the production of highly diverse molecules. Hairy roots are able to produce and secrete complex active glycoproteins from a large spectrum of organisms. They are also adequate to express plant natural biosynthesis pathways required to produce specialized metabolites and can benefit from the new genetic tools available to facilitate an optimized production of tailor-made molecules. This adaptability has positioned hairy root platforms as major biotechnological tools. Researchers and industries have contributed to their advancement, which represents new alternatives from classical systems to produce complex molecules. Now these expression systems are ready to be used by different industries like pharmaceutical, cosmetics, and food sectors due to the development of fully controlled large-scale bioreactors. This review aims to describe the evolution of hairy root generation and culture methods and to highlight the possibilities offered by hairy roots in terms of feasibility and perspectives.

Plant-Made Nervous Necrosis Virus-Like Particles Protect Fish Against Disease.

Virus-like particles (VLPs) of the fish virus, Atlantic Cod Nervous necrosis virus (ACNNV), were successfully produced by transient expression of the coat protein in Nicotiana benthamiana plants. VLPs could also be produced in transgenic tobacco BY-2 cells. The protein extracted from plants self-assembled into T = 3 particles, that appeared to be morphologically similar to previously analyzed NNV VLPs when analyzed by high resolution cryo-electron microscopy. Administration of the plant-produced VLPs to sea bass (Dicentrarchus labrax) showed that they could protect the fish against subsequent virus challenge, indicating that plant-produced vaccines may have a substantial future role in aquaculture.

Impact of Cereal Seed Sprouting on Its Nutritional and Technological Properties: A Critical Review.

Sprouting induces activation and de novo synthesis of hydrolytic enzymes that make nutrients available for plant growth and development. Consumption of sprouted grains is suggested to be beneficial for human health. Positive consumer perceptions about sprouted cereals have resulted in new food and beverage product launches. However, because there is no generally accepted definition of "sprouting," it is unclear when grains are to be called sprouted. Moreover, guidelines about how much sprouted grain material food products should contain to exert health benefits are currently lacking. Accordingly, there is no regulatory base to develop appropriate food labeling for "sprouted foods." This review describes the nutritional and technological properties of sprouted grains in relation to processing conditions and provides guidelines to optimize sprouting practices in order to maximize nutritive value. Relatively long sprouting times (3 to 5 days) and/or high processing temperatures (25 to 35 °C) are needed to maximize the de novo synthesis and/or release of plant bioactive compounds. Nutrient compositional changes resulting from sprouting are often associated with health benefits. However, supportive data from clinical studies are very scarce, and at present it is impossible to draw any conclusion on health benefits of sprouted cereals. Finally, grains sprouted under the above-mentioned conditions are generally unfit for use in traditional food processing and it is challenging to use sprouted grains as ingredients without compromising their nutrient content. The present review provides a basis for better defining what "sprouting" is, and to help further research and development efforts in this field as well as future food regulations development.

Tobacco BY-2 Media Component Optimization for a Cost-Efficient Recombinant Protein Production.

Plant cells constitute an attractive platform for production of recombinant proteins as more and more animal-free products and processes are desired. One of the challenges in using plant cells as production hosts has been the costs deriving from expensive culture medium components. In this work, the aim was to optimize the levels of most expensive components in the nutrient medium without compromising the accumulation of biomass and recombinant protein yields. Wild-type BY-2 culture and transgenic tobacco BY-2 expressing green fluorescent protein-Hydrophobin I (GFP-HFBI) fusion protein were used to determine the most inexpensive medium composition. One particularly high-accumulating BY-2 clone, named 'Hulk,' produced 1.1 ± 0.2 g/l GFP-HFBI in suspension and kept its high performance during prolonged subculturing. In addition, both cultures were successfully cryopreserved enabling truly industrial application of this plant cell host. With the optimized culture medium, 43-55% cost reduction with regard to biomass and up to 69% reduction with regard to recombinant protein production was achieved.

Scandinavian perspectives on plant gene technology: applications, policies and progress.

Plant research and breeding has a long and successful history in the Scandinavian countries, Denmark, Finland, Norway and Sweden. Researchers in the region have been early in adopting plant gene technologies as they developed. This review gives a background, as well as discuss the current and future progress of plant gene technology in these four countries. Country-specific details of the regulation of genetically modified plants are described, as well as similarities and differences in the approach to regulation of novel genome-editing techniques. Also, the development of a sustainable bioeconomy may encompass the application of plant gene technology and we discuss whether or not this is reflected in current associated national strategies. In addition, country-specific information about the opinion of the public and other stakeholders on plant gene technology is presented, together with a country-wise political comparison and a discussion of the potential reciprocal influence between public opinion and the political process of policy development. The Scandinavian region is unique in several aspects, such as climate and certain agriculturally related regulations, and at the same time the region is vulnerable to changes in plant breeding investments due to the relatively small market sizes. It is therefore important to discuss the role and regulation of innovative solutions in Scandinavian plant research and breeding.

In-solution antibody harvesting with a plant-produced hydrophobin-Protein A fusion.

Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low-cost and scalable antibody capturing in solutions. Immunoglobulin-binding Protein A is widely used in affinity-based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two-phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35-fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB-induced protein body formation. We also demonstrated production of HFBI-Protein A fusion protein in tobacco BY-2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody-binding capacity of the Protein A block was similar to the nonfused Protein A. The best-performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two-phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.

Single Cell Protein-State-of-the-Art, Industrial Landscape and Patents 2001-2016.

By 2050, the world would need to produce 1,250 million tonnes of meat and dairy per year to meet global demand for animal-derived protein at current consumption levels. However, growing demand for protein will not be met sustainably by increasing meat and dairy production because of the low efficiency of converting feed to meat and dairy products. New solutions are needed. Single cell protein (SCP), i.e., protein produced in microbial and algal cells, is an option with potential. Much of the recent interest in SCP has focused on the valorisation of side streams by using microorganisms to improve their protein content, which can then be used in animal feed. There is also increased use of mixed populations, rather than pure strains in the production of SCP. In addition, the use of methane as a carbon source for SCP is reaching commercial scales and more protein-rich products are being derived from algae for both food and feed. The following review addresses the latest developments in SCP production from various organisms, giving an overview of commercial exploitation, a review of recent advances in the patent landscape (2001-2016) and a list of industrial players in the SCP field.

Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells.

The encapsulation of drugs to nanoparticles may offer a solution for targeted delivery. Here, we set out to engineer a self-assembling targeting ligand by combining the functional properties of human transferrin and fungal hydrophobins in a single fusion protein. We showed that human transferrin can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of-concept for the functionalization of hydrophobin coatings with transferrin as a targeting ligand.

Novel Hydrophobin Fusion Tags for Plant-Produced Fusion Proteins.

Hydrophobin fusion technology has been applied in the expression of several recombinant proteins in plants. Until now, the technology has relied exclusively on the Trichoderma reesei hydrophobin HFBI. We screened eight novel hydrophobin tags, T. reesei HFBII, HFBIII, HFBIV, HFBV, HFBVI and Fusarium verticillioides derived HYD3, HYD4 and HYD5, for production of fusion proteins in plants and purification by two-phase separation. To study the properties of the hydrophobins, we used N-terminal and C-terminal GFP as a fusion partner. Transient expression of the hydrophobin fusions in Nicotiana benthamiana revealed large variability in accumulation levels, which was also reflected in formation of protein bodies. In two-phase separations, only HFBII and HFBIV were able to concentrate GFP into the surfactant phase from a plant extract. The separation efficiency of both tags was comparable to HFBI. When the accumulation was tested side by side, HFBII-GFP gave a better yield than HFBI-GFP, while the yield of HFBIV-GFP remained lower. Thus we present here two alternatives for HFBI as functional fusion tags for plant-based protein production and first step purification.