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 biosafety 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 sativa is 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, air racking 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 . After a total of 35 days post seeding, a tractor sprayer applies 4900 L of a 4% ethanol solution to the plot’s crop, triggering the synthesis and accumulation of thaumatin in plant biomass. The plants are incubated for 7 more days, during which time they continue to uptake nutrients and express thaumatin. After 42 days from seeding, the batch is harvested through two mechanical harvesters andfour hopper trucks at a rate of 17,000 kg/h and transported to downstream processing facility using a conveyer belt . The plot undergoes a turnaround period of three days for which the labor and equipment cost is included. No pesticides, fungicides, or herbicides costs are added due to the assumption that not enough growing degree days are accumulated during the batch cycle duration , for disease-causing organisms to be a concern.
The base case downstream processing facility is designed to purify and formulate 318.5 kg/batch of thaumatin with 98% purity. A DSP batch starts with shredding plant biomass using two industrial shredders , each processing 40,000 kg of plant biomass/h. This step is designed to homogenize the leaves and stems to facilitate the extraction process. Shredded plant material is then mixed with an acetate buffer in a 0.8 L of buffer to 1 kg of biomass ratio. This step leverages stability of thaumatin at low pH to precipitate host plant proteins that aren’t stable under acidic conditions. The extraction buffer consists of 50 mM acetic acid and 150 mM sodium chloride mixture at a pH of 4.0. The resulting plant slurry is then fed into a screw press to separate most of the dry plant material. A screw press is recommended for this step because it minimizes the amount of extraction buffer needed by forcing out more plant sap with the increasing pressure inside the chamber. The crude extract stream obtained from the screw press unit is sent to three parallel P&F filtration units for initial clarification, each having a membrane area of 190 m2 . Furthermore, the model assumes the use of food-grade filter membranes designed to include 10 filter sheets with decreasing particle retention size from 25 to 0.1 µm. The acetate buffer is applied once again as cake wash with a 0.2 L buffer to 1 L extract ratio. Diatomaceous earth is added to this step as a filter aid in a 6:100. The stability of thaumatin at low pH and high temperatures facilitates the precipitation of more host cell proteins as well as other undesired plant-derived compounds. Using seven heating tanks , the plant extract is then heated to 60 ◦C for 60 min. Following heat incubation, the stream is sent to a P&F filtration unit to capture the heat-precipitated proteins. It is assumed that a 90% reduction of N. tabacum total soluble proteins is attainable following the heat incubation and precipitation steps. Concentrating the thaumatin stream prior to the ultrafiltration/diafiltration step is necessary to avoid processing large liquid volumes ~573,000 L further downstream.
It has been reported that thaumatin experiences a loss in sweetness when heated above 70 ◦C at a pH of 7.0; therefore, the product stream undergoes concentration by evaporation prior to neutralizing the solution since the protein can sustain higher temperatures at a low pH.The triple effect evaporation unit is designed to evaporate 90% of the water content in the stream at 109 ◦C, 77 ◦C, and 40 ◦C in the first, second, and third effect, respectively, over 4 h.The exiting stream is then neutralized with 1:1 molar ratio and mixed in V-101 for 30 min and sent to the P&F filtration unit to remove any precipitated materials. An additional 1.5% loss of thaumatin during this step is assumed. Because soluble impurities such as nicotine and other pyridine alkaloids are abundant in N. tabacum plants, a UF/DF step is necessary to eliminate small molecules. The UF/DF unit consists of 4 stacked cassette holders, each containing twenty 3.5 m2 cassettes. Since thaumatin is a 22 kDa protein, a membrane with MWCO of 5 kDa is used per working process knowledge. Assuming a conservative flux of 30 L/, the inlet stream is concentrated using a concentration factor of 5, diafiltered 10 times against reverse osmosis water, then re-concentrated using a CF of 5 over 20.6 h, resulting in a 75% pure thaumatin and nicotine content of 1.08 mg/kg thaumatin. A retention coefficient of 0.9993 was assumed for thaumatin, resulting in 5.8% thaumatin loss in UF/DF . The retentate is then sent to five CEX chromatography columns operating in parallel which was modeled based on unpublished data from Nomad Bioscience GmbH . GE Healthcare Capto S resin with an assumed binding capacity of 150 g/L was used in this analysis. Table S2 shows the downstream losses breakdown per unit operation. Spray drying is used as a final formulation step over other means of industrial drying due to the heat sensitivity of thaumatin.The simulated facility consists of three sections—Virion production laboratory , curing cannabis spinach field growth, and DSP. A list of base case design parameters and assumptions is shown in Table S3. The VPL process is adopted from a recent article entailing the production of RNA viral particles from agrobacteria carrying a PVX construct. The laboratory is sized to produce 7900 L of spray solution per batch for application in the field. Nicotiana benthamiana plants are used as the host to produce the viral particles to inoculate spinach. N. benthamiana seeds are germinated in soilless plant substrate at a density of 94 plants per tray. Seedlings are grown hydroponically , under LEDs, until reaching manufacturing maturity at day 35. Agrobacterium tumefaciens is grown for 24 h, before being left in a 4 L flask overnight, and the A. tumefaciens suspension is added to MES buffer in V-101. N. benthamiana infiltration takes place in a vacuum agroinfiltration chamber for 24 h followed by incubation for 7 days in . N. benthamiana biomass production, agrobacterium growth, agroinfiltration, and incubation parameters are adapted from. After the incubation period, 41.5 kg of N. benthamiana fresh weight are ground and mixed with PBS buffer in a 5:1 buffer:biomass ratio.
The extract is then sent to a decanter centrifuge to separate plant dry matter from the liquid phase which is clarified by dead-end filtration , followed by mixing the permeate with 35.9 kg of diatomaceous earth and 7780 L of water to reach a final concentration of 1014 viral particles/L and 4.55 g diatomaceous earth/L. Diatomaceous earth is used as an abrasive to mechanically wound plant cell walls allowing the virions to enter the cytoplasm of the cell. The final spray is stored in for 13 h before field application. Field operation starts at the beginning of each batch with the direct seeding of 28.3 million Spinacia oleracea seeds over 22.6 acres. Spinach is planted over 80-inch beds with an assumed 3 ft spacing between beds, resulting in 14,520 linear bed feet per acre. Seeds are germinated and grown in the field for 44.5 days, during which time a drip irrigation system delivers irrigation water and soluble fertilizer to the soil. It is assumed that 200 acre-inches of irrigation water and 64 tons of fertilizer are needed per batch. A tractor on which multiple high-pressure spray devices are mounted is used to deliver the viral particle solution at a rate of 2 acres/h. This method of delivery has shown high effectiveness. Spinach plants are incubated in the field for 15 days post-infection. During that period, thaumatin starts to accumulate in the crop at an average expressionlevel of 1 g/kg FW after 15 days post-spraying. At day 60, two mechanical harvests collect a total of 344 MT spinach biomass, carrying 344 kg thaumatin, with the aid of four hopper trucks, which is transferred to a 500-m-long conveyor belt that extends from the field collection site to the DSP section of the facility. Harvesting occurs at an average rate of 17,000 kg FW/h, which is estimated based on a harvester speed of 5 km/h and 14,520 linear bed feet per acre. A more simplified downstream processing, enabled by the use of spinach as a host, starts with mixing plant material with 65 ◦C water before extracting the green juice through a screw press . The resulting GJ is heated for 1 h at 65 ◦C in ten jacketed tanks , then concentrated by evaporation to reduce product stream volume for further purification steps. Since thaumatin is not stable at temperatures above 70 ◦C at neutral pH, evaporation is performed at a low temperature of 40 ◦C and 0.074 bar vacuum pressure. Thermally degraded host cell proteins and impurities are eliminated in a P&F filtration unit designed to include 10 filter sheets with decreasing particle retention size from 25 to 0.1 µm. Smaller impurities are removed using a diafiltration unit with 5 kDa molecular weight cut off cassettes in a similar process as described in Section 3.3, the retentate is spray dried in to obtain a final product which has 5% water content, and 348 kg of solid material containing 94% pure thaumatin and 6% spinach impurities. These impurities are expected to be water soluble, heat stable molecules in the range of 5–100 kDa, according to the theoretical design of the filtration scheme.As shown in Figure 3a, field labor is the highest contributor to the upstream field facility followed by consumables. Detailed labor requirement and cost estimation calculations can be found in Tables S7 and S8. Consumables include mechanical harvester and tractor’s fuel, lubrication, and repair costs and other field equipment repair costs. Upstream indoor facility AOC breakdown elucidates a high cost of consumables due to the cost of soilless plant substrate, followed by high energy consumption from the LED lighting system used for plant growth.