Consequently, there is currently an increase in the production of natural sugar alternatives based on the shift in consumer preferences toward more natural products to meet their dietary need and restrictions. Stevia, the common name for glycoside extracts from the leaves of Stevia rebaudiana, is a natural, sweet-tasting calorie-free botanical that is currently gaining popularity as a sugar substitute or as an alternative to artificial sweeteners. Recent reports project the annual growth rate of stevia compounds to be 6.1% and 8.2%, during 2015–2024 and 2017–2024, respectively. Stevia has gained industry acceptance in recent years due to its ease of cultivation in several countries across the globe and its high sweetness index . This shows that the growth of stevia’s use as a sugar substitute, despite taste limitations of the marketed glycosides, was contingent on the feasibility of its large-scale manufacturing. Thaumatin, monellin, manbinlin, pentadin, brazzein, curculin, and miraculin are sweet tasting proteins that are naturally expressed in tropical plants. Studies have found that human T1R2-T1R3 receptors expressed in taste buds in the mouth and recognize natural and synthetic sweetness while T1R1-T1R3 recognize the umami taste. These receptors, which have several binding sites, are activated when the compounds that elicit sweet taste bind to them. However, these proteins have unique binding properties and do not all bind at the same sites, which leads to varying perception of sweetness. This work focuses on thaumatins, a class of intensely sweet proteins isolated from the arils of the fruits of the West-African plant Thaumatococcus daniellii. The distinctiveness of thaumatin lies in its sweetness index being up to 3500 times sweeter than sugar. According to the 2008 Guinness World Records, it is the sweetest natural substance known to mankind.

Thaumatin I and II, the two main variants of the protein,benefits of vertical farming are comparable in their biological properties, structure, and amino acid composition. The structure consists of a single polypeptide chain of 207 amino acids that are linked together by 8 disulfide bonds. The two variants differ by only five amino acid residues. Through chemical modifications and site-directed mutagenesis, it has been determined that the residues on the cleft-containing side of the protein have the strongest effect in eliciting sweetness to taste receptors on the tongue. The specificity of these residues demonstrates the importance of the protein structure in inducing thaumatin’s sweetness. In the USA, extracted thaumatin and thaumatin B-recombinant were initially affirmed Generally Recognized as Safe flavor enhancers/modifiers, but not as sweeteners. In the USA, plant-made thaumatin I and/or thaumatin II were granted GRAS status by the FDA in 2018 for use as a sweetener . In 2020, the FDA granted GRAS status to recombinant thaumatin II produced in Nicotiana plants for use as a sweetener and as a flavor enhancer/modifier . In the EU, thaumatins are allowed as both sweeteners and flavor enhancers. Thaumatin’s safety has been extensively documented. The Joint FAO/WHO Expert Committee on Food Additives report claims that the protein is free from any toxic, genotoxic, or teratogenic effects. Thaumatin is currently used as a flavor modifier in food applications such as ice creams, chewing gum, dairy, pet foods, soft drinks, and to mask undesirable flavor notes in food and pharmaceuticals. The current top global thaumatin manufacturers are Naturex , France; Beneo Palatinit, Germany; Natex, UK and KF Specialty Ingredients, Australia. The global production of thaumatin increased to 169.07 metric tons in 2016 from 138.47 MT in 2012. However, the current production method through aqueous extraction from the fruits of the tropical plant T. daniellii limits its availability while the demand is increasing. T. daniellii is not cultivated and harvesting of the arils takes place in plants growing wild in rainforests of West Africa ranging from Sierra Leone to the Democratic Republic of Congo.

The current production process is substantially dependent on the availability and quality of the native plant from year to year, which limits thaumatin’s use as a commodity product. The emergence of recombinant DNA technology and the use of cultured cells have allowed the production of proteins in large quantities. Enzymes and structural proteins are used in many industrial applications including the production of food and beverages, bio-diesel, cosmetics, bio-polymers, cleaning materials, and waste management. Most importantly, recombinant production allows for the expression of a protein outside its native source. Therefore, there exists a viable alternative to secure the desired quantities of thaumatin reliably and sustainably, without impacting rainforest ecosystems. Notably, there have been many attempts to produce thaumatin by means of genetically engineered microorganisms and plants. Despite successfully expressing thaumatin in yeast , bacteria , fungi , and transgenic and transfected plants , biotechnological large-scale production facilities have yet to be established. Molecular farming, the production of recombinant proteins in plants, offers several advantages over bioreactor-based systems. In this application, plants are thought of as nature’s single use bioreactors, offering many benefits such as reduced upstream production complexity and costs , linear scalability, and their inability to replicate human viruses. Specifically, open-field growth of plants has the potential to meet the market’s need for a large-scale, continuous demand of a commodity product at a competitive upstream cost. It has been marked suitable for this operation as plants can be easily adapted on an agricultural scale to yield several metric tons of the purified protein per year. Here, we present a feasibility study for a protein production level of tens of metric tons per year.

The success of a new product in the biotechnology process industry depends on well-integrated planning that involves market analysis, product development, process development, and addressing regulatory issues simultaneously, which requires some decisions to be made with limited information. This generates demand for a platform to help fill in those gaps and facilitate making more informed process and technology decisions. Process simulation models can be used in several stages of the product life cycle including idea generation, process development, facility design, and manufacturing. For instance, based on preliminary economic evaluations of new projects, they are used to eliminate unfeasible ideas early on. During the development phase of the product, as the process undergoes frequent changes, such models can easily evaluate the impact of these changes and identify cost-sensitive areas. PSMs are also useful for directing lab and pilot-scale studies into areas that require further optimization. Additionally, PSMs are widely used in designing new manufacturing facilities mainly as a tool for sizing process equipment and supporting utilities, as well as for estimating the required capital investment and cost of goods. This ultimately helps companies decide on building a new facility versus outsourcing to contact manufacturers. There are currently few published data-driven simulations of techno-economic models for plant-based manufacturing of proteins for pharmaceutical, bio-fuel, commercial enzyme, and food safety applications. However, to the best of our knowledge, no studies have proposed or assessed the feasibility of plant-based protein bio-production platforms on the commodity scale in tens of metric tons per year. The feasibility of production at this scale is critical for the emergence of thaumatin as a sugar substitute. Here, we present a preliminary process design, process simulation, and economic analysis for the large-scale manufacturing of thaumatin II variant by several different molecular farming production platforms. The base case scenario assumes an annual production capacity of 50 MT thaumatin. To achieve this level of production in a consistent manner, manufacturing is divided into 157 annual batches. Upstream production is attainable through open-field,urban vertical farming staggered plantation of Nicotiana tabacum plants. Each batch has a duration of 45 days and a recipe cycle time of 2 days. A full list of process assumptions can be found in Table S1. The proposed design achieves the expression of thaumatin in N. tabacum leaves using magnICON® v.3. This technology developed by Icon Genetics GmbH allows for the separation of the “growth” and the “expression” phases in a manufacturing process. Moreover, this process obviates the need to use agroinfiltration, which requires more capital and operational costs for inoculum preparation and implementation of expensive units for the infiltration process, containment of the genetically engineered agrobacteria, and elimination of bacteria-derived endotoxins.

In this design, transgenic N. tabacum or N. benthamiana plants carry a double-inducible viral vector that has been deconstructed into its two components, the replicon and the cell-to-cell movement protein. Background expression of recombinant proteins prior to induction remains minimal; however, inducible release of viral RNA replicons—from stably integrated DNA proreplicons—is triggered upon spraying the leaves and/or drenching the roots with a 4% ethanol solution resulting in expression levels as high as 4.3 g/kg fresh weight in Nicotiana benthamiana. Nonetheless, Nicotiana tabacumhas several advantages that make it more suitable for large-scale open field production such as field hardiness, high biomass yields, well-established infrastructure for large-scale processing, plentiful seed production, while attaining expression levels up to 2 g/kg FW. Furthermore, it is unlikely that transgenic tobacco material would mix with material destined for the human food or animal feed chain, unless it is grown in rotation with a food crop, but further development of strict Good Agricultural Practice for transgenic plants should overcome these issues.An alternative upstream facility design scenario was developed to evaluate the process economics of a more controlled supply of thaumatin by growing the plant host in a 10-layer vertical farming indoor environment. Nicotiana benthamiana is chosen as a host because it is known to be a model for protein expression for both Agrobacterium and virus-based systems, but its low biomass yield and difficulties regarding adaptation in the field hinder its application for open outdoor growth. However, this species grows very well in indoor, controlled environments and has high recombinant protein production. This upstream production facility uses the same method of expression and follows the same schedule as the base case upstream facility. Transient expression in plants is a method of recombinantly producing proteins without stable integration of genes in the nuclear or chloroplast genome. The main advantages of using this method are reducing the extensive amount of time needed to develop a stable transgenic line and overcoming bio-safety concerns with growing transgenic food crops in the field expressing heterologous proteins. Transient expression is attainable through several systems including biolistic delivery of naked DNA, agrobacteria, and infection with viral vectors. Notably, the use of viral vectors has been marked suitable for application on a field-scale due to the flexibility of production, and the quick accumulation of target proteins while achieving high yields. A new report has shown efficacy in delivering RNA viral particles using a 1–3 bar pressure, 1–4 mm atomizer nozzles spray devices in the presence of an abrasive to cause mechanical wounding of plant cell wall. GRAS notices GRN 738 and GRN 910 describe production of thaumatin in edible plant species and N. benthamiana, respectively. The expression of thaumatin in leaf tissue of the food crops Beta vulgaris , Spinacia oleracea , or Lactuca sativais generally lower than in N. benthamiana. However, despite having lower expression levels, the absence of pyridine alkaloids that are present in Nicotiana species is a major advantage for production in food crops because of the significant downstream resources needed to remove alkaloids in Nicotiana-based products. The ultimate solution may be a high-expressing engineered Nicotiana host devoid of alkaloid biosynthesis, but that option was not modeled in this study. The transient production facility is designed to produce 50 MT of purified thaumatin in spinach, annually, over 153 batches due to longer turnaround time required for S. oleracea compared to N. tabacum crops. Each batch has a duration of 67.8 days and a recipe cycle time of 1.94 days. The proposed base case upstream field production facility, displayed in Figure 1, consists of a 540 acre block of land divided into 22 plots, each of which is suitable for growing 318,000 kg FW of N. tabacum, carrying 477 kg of thaumatin, accounting for downstream recovery of 66.8%. It is assumed that the facility is located in a suitable climate where the growth of N. tabacum is attainable throughout the year, ignoring variations in production between batches . Each batch starts with direct seeding of transgenic N. tabacum plants in the field . The seeds are left to germinate for two weeks followed by vegetative growth for 3 more weeks post germination .