Production Of Proteins In Plants Research Articles

Sustainable Valorization of Coffee Silverskin: Extraction of Phenolic Compounds and Proteins for Enzymatic Production of Bioactive Peptides.

Coffee silverskin (CS), a by-product of the coffee roasting process, has high protein content (16.2-19.0%, w/w), making it a potential source for plant protein and bioactive peptide production. This study aims to develop innovative extraction methods for phenolic compounds and proteins from CS. The conditions for hydrothermal (HT) extraction of phenolic compounds from CS were optimized by varying CS loading (2.5-10%, w/v), temperature (110-130 °C), and time (5-30 min) using a one-factor-at-a-time (OFAT) approach. The highest TPC of 55.59 ± 0.12 µmole GAE/g CS was achieved at 5.0% (w/v) CS loading and autoclaving at 125 °C for 25 min. Following hydrothermal extraction, CS protein was extracted from HT-extracted solid fraction by microwave-assisted alkaline extraction (MAE) using 0.2 M NaOH at 90 W for 2 min, resulting in a protein recovery of 12.19 ± 0.39 mg/g CS. The CS protein was then subjected to enzymatic hydrolysis using protease from Bacillus halodurans SE5 (protease_SE5). Protease_SE5-derived CS protein hydrolysate had a peptide concentration of 0.73 ± 0.09 mg/mL, with ABTS, DPPH, and FRAP values of 15.71 ± 0.10, 16.63 ± 0.061, and 6.48 ± 0.01 µmole TE/mL, respectively. Peptide identification by LC-MS/MS revealed several promising biological activities without toxicity or allergenicity concerns. This study's integrated approach offers a sustainable and efficient method for extracting valuable compounds from CS, with potential applications in the food and pharmaceutical industries.

Protein oxidation‐induced changes in the physicochemical properties and quality of plant food

SummaryProtein oxidation is widely present in food systems and is susceptible to internal factors involving lipids and enzymes as well as external factors such as temperature and oxygen promoters. Protein oxidation leads to modification of the peptide skeletons and amino acid side chains, which in turn alters the structural characteristics of proteins, including protein cleavage, aggregation and derivative formation, and severely affects their physicochemical properties and bioavailability, thereby reducing the edible and storage qualities of foods. This review summarises the main factors that cause and regulate oxidation of plant protein, the effects of protein oxidation on food quality and strategies to ameliorate protein over‐oxidation, aiming to provide meaningful information for the development of high‐quality plant protein products.

Implications and impacts of aligning regional agriculture with a healthy diet

One of the most intractable challenges currently facing agricultural systems is the need to produce sufficient food for all to enjoy a healthy balanced diet while minimising impacts to the environment. Balancing these competing goals is especially intractable because most food systems are not locally bounded. This study aims to investigate the likely impacts on production, profit and the environment that result from aligning food systems to a healthy diet, as defined by EAT-Lancet. For this, we consider two distinct areas of the UK, one in East Anglia and the other in South Wales. These two regions reflect different ecosystems and therefore differing specialisations in UK agriculture. We used the Rothamsted Landscape Model (a detailed agroecosystems process-based model) to predict soil carbon dynamics, nutrient flows and crop production for the dominant crops grown in these regions, and the IPCC inventory models to estimate emissions from six livestock systems. Two scenarios were considered, one in which the study regions had to meet healthy diet requirements independently of each other and another in which they could do so collectively. To map their production to healthy diets, both study areas require increases in the production of plant proteins and reductions in the production of red meat. While changes in production can feed more people a healthy diet compared to the business-as-usual state, the overall calories produced reduces dramatically. Emissions and leaching decrease under the healthy diet scenarios and pesticide impacts remain largely unchanged. We show that local infrastructure and environment have a bearing on how “localised” food systems can be without running into substantial constraints. Whilst isolation of the farming system to a regional level, as explored here, is unlikely to be practical, we nevertheless demonstrate that aligning agricultural production towards healthier diets can generate food systems with many associated benefits in terms of agroecosystems' health and resilience to shocks in the food supply chain.

Oil binding capacity and related physicochemical properties of commercial plant protein products

Providing sufficient food to a growing population while addressing the rise of food-related diseases requires innovative solutions. The use of plant proteins as structuring agents for oils has shown potential because of their healthier fatty acid profiles compared to solid fats. Thus, this study aims to investigate the potential correlations between the properties of proteins and their oil binding capacity (OBC) to provide insights into their applicability in structuring oil. Two samples each of sunflower, rapeseed, and soy protein products obtained from different manufacturers were denatured by heat and dispersed in oil after freeze-drying. Whey protein isolate (WPI) was used as a reference. The results indicate that, with some modifications, rapeseed and sunflower proteins can bind amounts of oil (3.7 oil [g]/protein [g]) comparable to that of WPI (4.04 oil [g]/protein [g]). However, the extraction process affects the final results and the texture of the protein-oil dispersion. Moreover, the protein solubility in NaCl, carbohydrate content, particle size, and the quantity of α-helical structures are positively correlated with the OBC, whereas an increase in the firmness of the dispersion is negatively associated with it. There was no correlation found between the changed β-sheet structure resulting from the heat treatment and the OBC. This study emphasizes the ability of plant proteins to structure oil and explains how to evaluate the physical and structural properties of plant proteins. Technical insights that may lead to the production of more sustainable and healthier food products are provided.

Production of Plant Proteins and Peptides with Pharmacological Potential.

The use of plant proteins or peptides in biotechnology is based on their identification as possessing bioactive potential in plants. This is usually the case for antimicrobial, fungicidal, or insecticidal components of the plant's defense system. They function in addition to a large number of specialized metabolites. Such proteins can be classified according to their sequence, length, and structure, and this has been tried to describe for a few examples here. Even though such proteins or peptides can be induced during plant-pathogen interaction, they are still present in rather small amounts that make the system not suitable for the production in large-scale systems. Therefore, a suitable type of host needs to be identified, such as cell cultures or adult plants. Bioinformatic predictions can also be used to add to the number of bioactive sequences. Some problems that can occur in production by the plant system itself will be discussed, such as choice of promoter for gene expression, posttranslational protein modifications, protein stability, secretion of proteins, or induction by elicitors. Finally, the plant needs to be set up by biotechnological or molecular methods for production, and the product needs to be enriched or purified. In some cases of small peptides, a direct chemical synthesis might be feasible. Altogether, the process needs to be considered marketable.

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality.

Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality. Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1. Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

Citizen science boosts fundamental and applied research for sustainable soybean cultivation in North‐Western Europe

Societal Impact StatementTo help save our planet, we need to shift to plant‐based protein food and enhance sustainable agricultural practices. Cultivation of legumes, including soybean, will be key because they produce protein‐rich beans without high applied fertilizer input. This complex challenge involves many stakeholders beyond the agricultural sector. In the ‘Soy in 1000 Gardens’ project, we engaged more than thousand citizens in a 6‐month gardening project aiming at facilitating sustainable soybean cultivation in Belgium. Our work shows that with the right approach, citizen science can provide insights to develop more sustainable agri‐food systems when integrated with fundamental and applied science.Summary The global food system faces numerous challenges in its pursuit of sustainability. Shifting to more plant‐based protein sources as well as transitioning to self‐reliant agri‐food systems is one way to meet these challenges. This transition requires the involvement of multiple stakeholders beyond the agricultural sector such as the citizens themselves. In this study, we employed a citizen science approach through the ‘Soy in 1000 Gardens’ project, which engaged more than 1000 citizen scientists in a 6‐month gardening project during which citizens not only observed plant growth but also executed plant growth measurements that meet scientific standards. We aimed at increasing the awareness about the power of soybean and its symbionts for sustainable plant protein production and at isolating efficient nitrogen‐fixing rhizobia to be used by local farmers to produce protein‐rich soybeans. The results suggest that the success of citizen science projects depends on the level of engagement and the provision of adequate support, among other factors. This study thus highlights the potential of citizen science to address complex challenges and contribute to more sustainable agri‐food systems when properly integrated. Unique in its scope, the project provided important insights into the drivers of participation, attrition and data quality.

Removal of plant endogenous proteins from tobacco leaf extract by freeze–thaw treatment for purification of recombinant proteins

Plants are useful as a low-cost source for producing biopharmaceutical proteins. A significant hurdle in the production of recombinant proteins in plants, however, is the complicated process of removing plant-derived components. Removing endogenous plant proteins, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major photosynthetic plant enzyme that catalyzes photosynthesis through carboxylation and oxygenation, is important for the purification of recombinant plant proteins. In particular, RuBisCO accounts for 50% of the soluble leaf protein; thus, the removal of RuBisCO is critical for the purification of recombinant proteins from plant materials. An effective conventional method, known as freeze–thaw treatment, was developed for the removal of RuBisCO from Nicotiana benthamiana, which expresses recombinant green fluorescent protein (GFP). Crude extracts or supernatants were frozen at − 30 °C. Upon thawing, most of the RuBisCO was precipitated by centrifugation without significant inactivation and/or yield reduction of GFP. Based on the proteomics analysis, using this method, RuBisCO large and small subunits were reduced to approximately 10% and 20% of those of the unfrozen supernatant solutions, respectively, without the need for specific reagents or equipment. The proteomic analysis also revealed that many ribosomal proteins were removed from the extracts. This method improves the purification process of recombinant proteins from plant materials. Prolonged freezing damaged recombinant β-glucuronidase (GUS), suggesting that the applicability of this treatment should be carefully considered for each recombinant protein.

Effect of synergism of sucrose ester and xanthan gum on the stability of walnut milk.

Single emulsifiers have an effect on the stability of plant protein drinks, giving some improvement. Emulsifiers are more effective in maintaining emulsion stability when combined with polysaccharides such as xanthan gum. In this paper, we studied the food-grade emulsifier sucrose ester and measured the average particle size, polydispersity value, zeta potential, microrheological properties, microstructure and creaming index related to walnut protein emulsion by constructing a walnut protein emulsion simulation system. SDS-PAGE and low-field NMR were used to analyze the relative molecular masses of emulsions and the water distribution of emulsions, respectively, to further investigate the synergistic effects of sucrose esters and xanthan gum on the ease of emulsification and intrinsic mechanisms of different molecular weight proteins of walnut protein emulsions. The results indicate that the synergistic effect of sucrose esters and xanthan gum was to stabilize emulsions better than single emulsifiers. Xanthan gum and protein may form protein-polysaccharide complexes, as well as the hydrophobic interaction between sucrose ester and xanthan gum. The properties of xanthan gum can improve the stability of the emulsion by affecting the mechanical properties of walnut protein emulsion, and the combination of sucrose ester and xanthan gum can better stabilize large protein molecules. The results not only provide a theoretical basis for the stability of plant protein emulsion systems, but also provide technical support for the production and processing of large-molecule plant proteins into emulsions in this field for improving their stability, and also provide more possibilities for other types of emulsions. © 2023 Society of Chemical Industry.

Efficient production of single cell protein from biogas slurry using screened alkali-salt-tolerant Debaryomyces hansenii

Production of single cell protein (SCP) by recovering ammonia nitrogen from biogas slurry shows great potential against protein scarcity and unsustainable production of plant and animal proteins. Herein, a high-alkali-salt-tolerant yeast strain, Debaryomyces hansenii JL8-0, was isolated and demonstrated for high-efficient SCP production. This strain grew optimally at pH 8.50 and 2500 mg/L NH4+-N, and it could efficiently utilize acetate as the additional carbon source. Under optimal conditions, SCP biomass of 32.21 g/L and productivity of 0.32 g/L·h−1 were obtained in fed-batch fermentation. Remarkably, nearly complete (97.40 %) ammonia nitrogen from biogas slurry was recovered, probably due to its high affinity for NH4+-N. Altogether, this strain showed advantages in terms of cell biomass titer, productivity, and yield. A cultivation strategy was proposed by co-culturing D. hansenii with other compatible yeast strains to achieve high-efficient SCP production from biogas slurry, which could be a promising alternative technology for biogas slurry treatment.

Evaluation of sono-physico-chemical and processing effects in the mixed sarcoplasmic protein/soy protein isolate system

Since it may be employed to guide the production of high-quality plant protein as a partial substitute for animal protein using sono-physico-chemical effects, it is important to investigate the mixing of animal and plant protein in ultrasound (UID)-assisted processing systems. A study group of sono-physico-chemical processing with five distinct soy protein isolate (SPI)/ sarcoplasmic protein (SPN) ratios was developed in this work. The results showed that adding additional SPN to the mixed protein can increase its sono-physico-chemical impact, and this effect is greatest when the ratio of SPI to SPN is 1:3. The high SPN group's grafting rate rose from 39.13% to 55.26% in comparison to the high SPI content group. Quercetin (Que) may more readily modify SPN than SPI in the “dual protein” system used in this work, highlighting the critical function of plant protein in controlling the effects of UID-assisted processing in the “dual protein” system. Changes in apparent viscosity and microstructure are the primary parameters that affect the severity of sono-physico-chemical effects in SPI/SPN mixed protein systems, in addition to structural variables.

Protein Hydrolysate from Underutilized Legumes: Unleashing the Potential for Future Functional Foods.

Proteins play a vital role in human development, growth, and overall health. Traditionally, animal-derived proteins were considered the primary source of dietary protein. However, in recent years, there has been a remarkable shift in dietary consumption patterns, with a growing preference for plant-based protein sources. This shift has resulted in a significant increase in the production of plant proteins in the food sector. Consequently, there has been a surge in research exploring various plant sources, particularly wild, and underutilized legumes such as Canavalia, Psophocarpus, Cajanus, Lablab, Phaseolus, and Vigna, due to their exceptional nutraceutical value. This review presents the latest insights into innovative approaches used to extract proteins from underutilized legumes. Furthermore, it highlights the purification of protein hydrolysate using Fast Protein Liquid Chromatography. This review also covers the characterization of purified peptides, including their molecular weight, amino acid composition, and the creation of three-dimensional models based on amino acid sequences. The potential of underutilized legume protein hydrolysates as functional ingredients in the food industry is a key focus of this review. By incorporating these protein sources into food production, we can foster sustainable and healthy practices while minimizing environmental impact. The investigation of underutilized legumes offers exciting possibilities for future research and development in this area, further enhancing the utilization of plant-based protein sources.

Improved protein extraction technology using deep eutectic solvent system for producing high purity fava bean protein isolates at mild conditions

This study aimed to extract fava bean proteins using an eco-friendly deep eutectic solvent (DES) containing choline chloride and a glycerol-based system. Protein extraction conditions were optimized using response surface methodology, and the extracted protein was characterized in comparison to the protein extracted using a conventional alkaline solution. The optimum conditions obtained using the RSM model were: 28:1 w/w liquid: solid ratio, 1:2 w/w choline chloride: glycerol molar ratio, 50 °C temperature, 1 h extraction time, and 40% w/w water content. Under these optimum conditions, protein content, protein yield, and protein recovery rates of 92.33 ± 2.28%, 65.42 ± 6.53%, and 23.15 ± 2.31% were achieved. Alkaline extraction showed similar protein content (92.50 ± 1.36%) but lower protein yield (60.76 ± 1.16%) and recovery rate (21.74 ± 0.19%) when compared to DES-extracted proteins (P < 0.05). The secondary structure analysis revealed an increased α-helix (21.37%) content in DES-extracted proteins compared to alkaline-extracted proteins (10.68%). Moreover, the alkaline-extracted proteins showed intermolecular β sheets as protein aggregates (7.61%) and an increased percentage of β-turns (19.71%). The molecular weight distribution pattern of proteins extracted from both methods showed more similarities. Overall, DES protein extraction could be an alternative to the conventional alkaline extraction method, promoting sustainable plant protein production from fava beans for use in new food formulations.

Fusing the 3’UTR of seed storage protein genes leads to massive recombinant protein accumulation in seeds

The demand for recombinant proteins is rising dramatically, and effective production systems are currently being developed. The production of recombinant proteins in plants is a promising approach due to its low cost and low risk of contamination of the proteins with endotoxins or infectious agents from the culture serum. Plant seeds primarily accumulate seed storage proteins (SSPs), which are transcribed and translated from a few genes; therefore, the mechanism underlying SSP accumulation has been studied to help devise ways to increase recombinant protein production. We found that the 3’UTR of SSP genes are essential for SSP accumulation and can be used in the production of recombinant proteins in Arabidopsis. Fusion of the 3’UTR of SSP genes to the 3’ ends of DNA sequences encoding recombinant proteins enables massive accumulation of recombinant proteins with enzymatic activity in Arabidopsis seeds. This method is also applicable to the production of human Interferon Lambda-3 (IFN-lambda 3), a candidate biopharmaceutical compound against hepatitis C infection. Considering the low cost and ease of protein production in Arabidopsis, as well as the rapid growth of this plant, our method is useful for large-scale preparation of recombinant proteins for both academic research and biopharmaceutical production.

Developing and modelling of sustainable protein extraction using ammonium hydroxide – A recoverable and reusable solvent

Plant protein demand is growing as more people adopt a healthier and sustainable lifestyle. Since plant proteins are cell wall protected, extraction process that breaks cell-wall barrier is needed to obtain protein. However, the conventional extraction methods use non-reusable and non-recoverable solvents. Therefore, the goal of this research is to develop and model sustainable protein extraction using NH4OH—a recoverable and reusable solvent. Soybean protein was extracted at a temperature of 55 °C, varying particle sizes (>1, 0.425 – 1, 0.25–0.425, and<0.25 mm), times (0, 3, 6, 12, and 48 h), NH4OH concentrations (1%, 2.5%, 5%, 10% and 15%) and solid loading (1:10) using a full factorial experimental design. The empirical kinetic models for soybean protein extraction were analyzed with parabolic diffusion model, So & MacDonald’s model, hyperbolic, and first order model. The highest protein yield of 59% was obtained at 0.425 – 1 mm particle size, 12 h, and 1% NH4OH. Amine concentrations of extracted proteins were poorly correlated with protein yield evincing that extraction conditions to obtain the high yield did not cause protein hydrolysis. Three out of the four models gave a good fit. The best fit was So & MacDonald’s model, with a minimum root mean square error (RMSE) of 0.14 and maximum coefficient of linear correlation (R) of 0.89. In conclusion, NH4OH is a viable and sustainable solvent to extract plant protein and can be utilized for industrial plant protein production.

Formulation and Physical Stability of High Total Solids Lentil Protein-Stabilised Emulsions for Use in Plant Protein-Based Young Child Formulae.

The demand for high-quality plant protein products is increasing and the aim of this work was to evaluate the impact of increasing the total solids content on the formation and stability of lentil protein stabilised oil-in-water emulsions. A series of emulsions were formulated using different proportions of total solids: 23, 26, 29, 32, and 35% (w/v). The emulsions were formulated using three ingredients-lentil protein, sunflower oil, and maltodextrin-which made up 15.85, 27.43, and 56.72% (w/w) of the total solids, respectively. The changes in apparent viscosity, particle size distribution, and colour during thermal processing were evaluated, with the physical stability investigated using an analytical centrifuge. The apparent viscosity of the solutions increased with total solids content (25.6 to 130 mPa.s-1), as did redness colour intensity (a* value increased from 5.82 ± 0.12 to 7.70 ± 0.09). Thermal processing resulted in greater destabilisation for higher total solids samples, as evidenced by greater changes in particle size, along with decreased redness colour. These results bring a better understanding of high total solids plant protein emulsions and factors affecting their stability, which could be used for the development of cost-effective and sustainable processing solutions in the production of plant protein young child formulae.

Depletion of the NbCORE receptor drastically improves agroinfiltration productivity in older Nicotiana benthamiana plants.

Nicotiana benthamiana is increasingly used for transient gene expression to produce antibodies, vaccines, and other pharmaceutical proteins but transient gene expression is low in fully developed, 6-8-week old plants. This low gene expression is thought to be caused by the perception of the cold shock protein (CSP) of Agrobacterium tumefaciens. The CSP receptor is contested because both NbCSPR and NbCORE have been claimed to perceive CSP. Here, we demonstrate that CSP perception is abolished in 6-week-old plants silenced for NbCORE but not NbCSPR. Importantly, older NbCORE-silenced plants support a highly increased level of GFP fluorescence and protein upon agroinfiltration. The drastic increase in transient protein production in NbCORE-depleted plants offers new opportunities for molecular farming, where older plants with larger biomass can now be used for efficient protein expression.

The giant diploid faba genome unlocks variation in a global protein crop

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

A new journal dedicated to the science of sustainable food proteins

Harjinder Singh, Editor-in-Chief In the next decade, we will see a dramatic dietary shift toward more plant-based and alternative proteins. Global population growth and the dietary changes associated with rising standards of living are placing tremendous pressure on the systems that provide our proteins. How will we reconcile the creation of new protein foods that are not only sustainable but also affordable and highly nutritious? Sustainable Food Proteins (SFP) is a new international, open access, peer-reviewed journal focused on the research attempting to answer this question. The journal publishes high-quality original research articles, reviews, and opinion pieces on all aspects of the science dedicated to the advancement of sustainably derived protein foods. It covers the entire spectrum from protein sourcing to food formulations, to analytical characterization, to digestion and nutrition, and the nuanced research in between. The food industry is clamoring for knowledge on novel protein sources, structures, and functionality to inform formulations, as well as sensory information that may drive consumer perceptions. We are interested in manuscripts on these topics, as well as on novel techniques used to characterize and enhance these functionalities. For example, we are accepting papers on processing and extraction technologies, especially for edible proteins that are not sourced from traditionally farmed livestock. We welcome submissions on recombinant technologies for protein production (in bacteria, yeast, or plants) and cell culture-based production of animal protein. We also seek papers on addressing scientific challenges in food product formulations and product development using alternative protein ingredients. We welcome research on the digestibility, nutritional quality, and health impacts of alternative proteins and protein-based foods. There is currently a significant focus on protein science and innovation to transform our food system to become more environmentally sustainable. The academic community and scientists in the food industry now have a dedicated forum to showcase their important work. The editorial board of SFP is eager to share those findings in our journal and our goal is for SFP to be the leading journal where anyone interested in this burgeoning field can find cutting-edge protein research.

Development of a Molasses-Based Medium for Agrobacterium tumefaciens Fermentation for Application in Plant-Based Recombinant Protein Production

The Agrobacterium-mediated transient gene expression system is a rapid and efficient method for heterologous recombinant protein expression in plants. The fermentation of genetically modified Agrobacterium tumefaciens is an important step in increasing the efficiency of recombinant protein production in plants. However, the limitation of this system that makes it economically non-competitive for industrial-scale applications is the Agrobacterium suspension production cost. In this study, the utilization of sugarcane molasses as an alternative low-cost source of carbon at a concentration of 8.7 g/L and nitrogen at a concentration of 2.4 g/L for Agrobacterium cultivation was investigated. Molasses pretreatment using sulfuric acid (SA) was applied before fermentation, and it resulted in a maximum specific growth rate of 0.232 ± 0.0063 h−1 in the A. tumefaciens EHA105 culture. The supplementation of antibiotics in the molasses-based medium was shown to be unnecessary for plasmid maintenance during fermentation in both Agrobacterium strains, which helped to reduce the production cost. We evaluated recombinant protein production using an Agrobacterium culture without antibiotic supplementation in the growth media by demonstrating green fluorescent protein expression in wild-type Nicotiana benthamiana leaves. In the evaluation of the culture medium cost, the molasses-based medium cost was 6.1 times lower than that of LB. Finally, this study demonstrated that the newly developed molasses-based medium for Agrobacterium fermentation is a feasible and effective medium for transient recombinant protein production in plant tissues.