Progress 07/01/19 to 02/29/20

Outputs
Target Audience: American Tabocco Farmer, e.g., Vick Family Farms: As a major tobacco farmer in the largest tobacco farming state, SynShark's crop is critical to the Vick Family Farms. The concept of having valuable terpenes extracted from one of Vick Family Farm's important crops is an exciting opportunity. Vick Family Farms have been working with SynShark on this pursuit including the Phase I period. Vick Family Farms has provided a strong letter of commitment to invest in the growth of SynShark - details included in SynShark's Phase II application. OEMs, e.g., Deutsche Process: Deutsche Process is a sanitary processing equipment manufacturer for a broad range of multiple industries specializing in liquid processing and down stream refinement. Deutsche Process is a sister company of the Deutsche Beverage Technology and Ink Keg brands, and are an industry leader in turnkey beverage solutions for the craft beer sector. During the Phase I period, Deutsche Process and Synshark have collaborated on the extraction of high-yield terpene production from tobacco. This year, working with SynShark, Deutsche Process was able to demonstrate an effective extraction of SynShark's squalene rich oil by using Ethanol and a lab scale version of our Counter-Current Rotax Extraction System, which can now be scaled to industrial capacity. Deutsche Process' expertise in extraction can be leveraged to address downstream challenges to provide seamless integration of Synshark's novel approach. Deutsche Process provided a stong letter of support for our Phase II application. US Tobacco Manufacturer, e.g., Tobacco Rag Processors, Inc. (TRP): TRP's interest in SynShark is based on its strategic intent to process local agriculture for future markets. TRP belives that the combination of SynShark's technology combined with TRP's farming reach and manufactirng expertise is a compelling partnership for both entities. TRP has provided a letter of intent to invest in capiral shares of SynShark - details included in our Phase II application. Changes/Problems:The deliverables from Phase I toward the Carbon Repartition task (Task 1) were altered due to a sub-awardee change. Partnership with a tobacco breeding research group at NCSU has led to the incorporation of an organized breeding scheme using pairwise crosses for combining squalene accumulation technologies. The genetically uniform F1 progeny resulting from these crosses allowed comparison of C2 and C5 strategies when coupled with other successful storage and downstream inhibition technologies. This new shift in strategy resulted in increases in squalene titer and more stability in yield prediction than has been observed in previous iterations of the various technologies. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The Phase I project work addresses the use of non-food biobased products whereby SynShark's new technology unlocks the competitiveness of value added terpenes from tobacco. Through optimization of squalene extraction from SynShark tobacco as a competitive substitute to the status quo, the Company can enhance agriculture's role as a supplier of a raw materials market reliant on animal mass from the seas. This approach to delivering squalene addresses two sustainability issues, 1) the immediate need for switching in a declining American tobacco farming sector, and 2) ocean ecology issues resulting from the needless 'livering' of deep water sharks for this product. The Phase I work supports tobacco farmers looking to replace low value smoking leaf with a specialty tobacco that can bring extra income. Much can be said about the continued loss of tobacco growing contracts in the American South, but more needs to be understood about the high cost of switching to different crops. Through our current scale-up of squalene accumulating tobacco lines, the Company addressed simultaneously the market need for squalene rich oils as well as the agricultural community's need for new high value crops that can be cultivated with existing infrastructure and knowledge. During the Phase I period, through terpenoid metabolic engineering, the Company has successfully shifted the fundamental carbon allocation pathways within tobacco allowing for increased accumulation of the triterpene squalene. Squalene as an adjuvant is one of the critically studied aspects of delivering a vaccine for Coronavirus 2019 (COVID-19), and shark derived squalene is unthinkable for manufactures.[i] The Team has the specific expertise and experience to reach enough squalene yield in SynShark tobacco to achieve commercialization success. Phase I accomplishments focus on transforming SynShark's scientific discovery on enhanced terpene production from tobacco into products with commercial potential and societal benefit. SynShark has successfully proved the feasibility of this technology in the Phase I period. Over the course of the Phase I period, SynShark has proven that this early or "seed" stage research and development effort in molecular genetics and purification has commercial potential, most notably in creating an alternative source for squalene and help build a strong, biomanufacturing-based economy. Phase I results showed that the data from this redistribution channel revealed increased carbon allocation into the MEP for terpene synthesis when coupled with strong downstream squalene sink. SynShark was able to localize it away from the downstream pathway by synthesizing these molecules in the stroma. Further efforts to compartmentalize the compound was successful by the addition of modified oleosin proteins to sequester the squalene within protective oil bodies. SynShark hypothesizes that part of the success of squalene accumulation in Company plants is due to the fact that squalene production was engineered into the energetically favorable MEP pathway, whereas native squalene synthesis occurs in the cytosolic MVA pathway. This provides a natural sequestration from the enzymes involved in downstream squalene metabolism. The Phase I research was focused on optimizing the squalene production in SynShark tobacco plants. Through the three objectives explained below, the Company was able to gain enough information to control the squalene yield in SynShark tobacco plants. This has been a crucial achievement as squalene yield is the most important step to long-term return on investment. This drives both the ability to purify an end product and a pathway to control other valuable terpenes. The team believes, however, that they have the specific know-how and experience to further improve the squalene yield in Phase II and bring tobacco-based terpene production closer to commercialization. Over the course of the Phase I period, SynShark made significant progress in addressing all three technical barriers. #1. Carbon repartition: The first objective of the Phase I effort was to address the technical barriers associated with carbon repartition, the key limiting factor for higher plant biomass and terpene yield. Even though multiple approaches can be exploited to increase photosynthesis limited progress has been made to increase terpene yield by improving carbon fixation alone. In the Phase I period, SynShark focused on re-channeling and re-distributing photorespiration products, enabling it to couple intermediate channeling with terpenoidbiosynthesis. In particular, considering that G3P exists abundantly in chloroplast, the available pyruvate has greatly promoted carbon flux channeling to MEP pathway for squalene biosynthesis. The increase of pyruvate has leveraged more G3P to be condensed by DXPS to DXP, the first committed step to downstream MEP pathway terpene production. Overall, the Phase I results showed that can increase terpene yield. This strategy was shown to increase carbon allocation to the MEP and will be optimized in the Phase II period to further increase squalene yield. The second objective of the Phase I effort was the storage of final products in planta. For yield purposes, the produced squalene has to be stored in the proper organelles or structures to reduce loss. As aforementioned, several structures including glandular trichomes are involved in terpenoid accumulation. Subcellular droplets have been indicated as a mechanism for squalene storage, an option that has been explored, including SynShark's recent work with squalene. To enhance the squalene yield by the storage organelle, SynShark LLC has licensed the synthetic droplet technology addressing the success criterion of this Phase I objective. Prevention of downstream consumption of squalene: The third objective of the Phase I effort was to prevent the downstream consumption of squalene. Even though chloroplast compartmentation of squalene biosynthesis has led to a significant increase in squalene accumulation, various lines of evidence suggested that squalene might leak out the plastid membrane to be consumed by squalene expoxidase (SQE), which prevented further increases of squalene. Thus,downstream degradation remained the major challenge for squalene production. The instability of squalene in cytosol has not been solved and still hampered squalene production. In order to address this barrier, SynShark has applied Overall, during the Phase I period, SynShark has made significant progresses with three sets of technologies to increase the squalene yield. However, maximizing commercial potential of the technology depends on further increasing the yield. The Company's current model indicates that a 4-6% or more of squalene yield is needed to generate a viable purified squalene product. The combination of these technologies provides realistic approaches to consistently reaching the 4-6% squalene yield target, which will be pursued in the Phase II period along with other novel yield improvement approaches. In addition to addressing the above mentioned squalene yield increasing terpene pathway modification objectives, during the Phase I period, SynShark has explored addressing the commercial bottlenecks in feasibility, mainly harvesting to post-harvesting processing, followed by further processing steps such as molecular distillation and remediation. Overall, this effort was composed of harvest and drying (Task 2), and post-harvest processing; extraction, molecular distillation, and chromatography (Task 3).

Publications