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The retentate is then sent to five CEX chromatography columns operating in parallel which was modeled based on unpublished data from Nomad Bioscience GmbH .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 , 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,vertical indoor farming 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. Transgenic production models were resized based on scenario design requirement for production levels ranging from 10–150 MT and expression levels ranging from 0.5–2.5 g/kg, while keeping the scheduling parameters the same from base case models. The significant impact of expression level on CAPEX and COGS is elucidated in Figure 4a–c.

Production level shows a very small decline in COGS for indoor upstream facility and a linear increase in CAPEX with increasing production level. On the other hand, the field upstream facility showed a significant increase in COGS at lower production levels due to the minimum ownership costs of field equipment regardless of the small acreage size. DSP followed the expected behavior that economy of scale dictates, with sharp decrease in COGS at lower production levels and diminishing returns at higher production levels. The deviation from linear trend at 150 MT/year in field upstream and DSP is likely due to the model’s specified equipment maximum rating, which allows for the inclusion of a new equipment in parallel beyond this rating.The impact of varying the highest cost drivers in each of the facility’s category by 25% on COGS is portrayed as a tornado diagram in Figure 5c. Field labor was the most sensitive cost variable, having the highest impact on the COGS, followed by the ultrafiltration membrane, which is replaced every 30 cycles. In this model, we assume a relatively high downstream recovery of the protein from harvest to formulation. The reason for this assumption is that spinach, being edible crop, allows for a lower target product purity and a consequently fewer DSP steps. It is particularly important to focus resources on maximizing downstream recovery during process development because it ultimately affects plant biomass and spray volume requirement upstream to appropriately compensate for these losses, which in turn affects equipment sizing in DSP based on the amount of plant material to be processed. The unit operations were resized according to the scenario design requirement for downstream recovery ranging from 50 to 95% while scheduling parameters were left unchanged. This effect of downstream recovery on the facility’s AOC and COGS is shown in Figure 5d and shows a 1.5× increase in AOC and COGS as downstream recovery decreases from 95% to 50%. Although our analysis indicates a relatively high COGS range for a sugar substitute, there are unrealized costs savings from thaumatin use due to its unique sweetness intensity. Thaumatin’s use in extremely small quantities is essentially why it is considered a noncaloric sweetener, as it provides only 4 calories per gram. Sensory evaluation studies have found that a sample with 5% sucrose +4.6 ppm thaumatin II had similar sweetness as a 10% sucrose control with minimal lingering aftertaste, suggesting that up to one-half of the sugar could be replaced by thaumatin II . SSBs including sodas, fruit drinks, and sport drinks account for 50% of the total added sugar in Western diets, and therefore provide an attractive avenue for thaumatin emergence as a sugar substitute.