Progress 09/01/20 to 12/31/22

Outputs
Target Audience:American Tobacco Producer: The Phase II effort supports two of the five theme areas outlined in the USDA Science Blueprint (A Roadmap for USDA Science from 2020 to 2025): Sustainable Ag Intensification and Value- Added Innovations. The Phase II goal supports the Sustainable Ag Intensification theme because increasing the terpene yield per harvest will directly reduce the environmental impacts associated with plant production and processing. SynShark's molecular genetics and purification technology aligns well with many of the theme strategies which include (i) using genomic technology to accelerate breeding progress, (ii) using precision agriculture technologies, innovative input technologies and stand improvement to optimize resource use and reduce the gap between actual yield and yield potential, and (iii) enhancing the utility and value of metabolites, and other constituents of plant products. As for the Value-Added Innovations theme, SynShark's technology to generate an alternative source of squalene from tobacco plants aligns well with this theme objective, which is fostering sustainable non-food, feed and other high-value chemical and biophysical applications of plants (pharmaceutical, nutraceutical, industrial products, and feedstock ingredients). The proposed technology development effort addresses two of USDA Strategic Plan Goals. Creating a new alternative source for high value terpene products from tobacco would greatly benefit the American tobacco producer. The outcome of SynShark's new technology would provide an effective financial safety net for tobacco farmers to sustain economically viable agricultural production and support rural jobs and economic growth, which is an objective of USDA's Strategic Goal #2. Increasing the yield of targeted value-added products through molecular genetics and purification would facilitate economic development and stewardship of private lands. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Extraction was conducted at Wagon Trail Hemp, using cryo EtOH as solvent on September 22nd. This is not the preferred solvent, however SynShark has tried other various methods. This option is easier to find service contracts for and reduces extraction costs. Dried and shredded tobacco material was extracted at a 10:1 solvent to biomass ratio to increase possible extracted titers of larger non-polar molecules such as larger terpenes. The dynamic step was also ran through twice to ensure the best possible results while using a less optimal solvent. Although filters were kept, removing coagulated fats and waxes to do analytical to determine if squalene became bound to FAAs or other lipid molecules. The 1,187lbs of dried and shredded tobacco yielded 5.82 kg of crude, degummed/dewaxed tobacco oil. Samples of the crude extract and solid phase (spent tobacco material) were analyzed by Hong Ma at NCSUTable 5. Pictures of Extraction found inFigure 6below. Figure 6. Primary Extraction and Solvent Recovery at Wagon Trail Hemp ? Initial purification steps, fractioning the oil, were completed using a Short Path Distillation assembly from Summit Research made available to SynShark through Synergy Labs. Molecular distillation parameters of squalene are published, and served as benchmarks in this purification step as well as distillation models by mass and vacuum. Similar to the extraction step, SynShark found difficulty in purification partnership opportunities. Synergy Research Laboratories in Columbia, TN allowed the team to utilize their short path distillation unit but not any other equipment for biphasic or counter current separation of polar species prior to distillation. Molecular Distillation was separated into 2 stripping runs to remove light weight molecules before a separate 3rd target range for squalene fraction(s). A hotter run was also done on the 4th day to remove squalene that was not pulled at the target temp due to poor vacuum. These fractions from each run were separated on whether they collected at the condenser (lighter molecules not condensing in the short path arm) and those that were collected in the collection flask (molecules much closer to the boiling flask's actual temperature). Furthermore, these fractions were separated by density and polarity when collected creating sub-fractions, shown in the molecular distillation qualitative figure below in Results. Collected fractions were labeled by succession and alphabetically labeled top to bottom in the sub-fractions developed. Analytical was performed by Dr Hong Ma at NCSU, and data is inTable 6. Pictures and descriptions of fractions are inFigure 7below. Figure 7. Short Path Distillation at Synergy Labs(A)Benchtop Short Path Molecular Distillation unit - vacuum not shown.(B)1. Collections from a main body from the collection flask settling in the separation flask. 2. Lower fraction (denser or water-soluble layer) removed for analytical testing. 3. Multiple layers separated - showing oil bodies in the emulsion layer between the hydrophilic bottom and hydrophobic top.(C)Collections flask and cold trap flask collections of fractions before being separated into sub-fractions for analytical.(D)1. TOP and 2. SIDE view of sub-fractions before being sent out for analytical testing.(E)1. Boiling flask after reaching over 200 C, and 2. The thermal probe (2.1 close up) - showing the heavy weight material left creating a heavy tarlike substance before cooling to a biopolymer hard plastic like material. The Research Findings or Results (1) Given that the overall terpenoid metabolic pathway appeared to be upregulated inyabby5mutant lines, it was reasonable to hypothesize that squalene accumulation would also be increased in these tobaccos. To test this, construct FST (containing FPS + SQS + T-oleosin cassettes engineered to transport their respective proteins into the chloroplast) was introduced into the background TI1068, and theyabby5knockout line referred to as TI1068 P53a (shown in Fig. 1). Although the transformation efficiency was relatively low, we were able to recover 5 T0plants for each line. These plants were grown in a greenhouse and assayed for squalene just prior to flowering. The results are shown below inTable 1. Although the sample size was small, the results did not support the notion that one can realize higher squalene yields within theyabby5mutant background. Figure 2. Field-grown tobacco plants of lines pT10, pT8 and pT5. As can be seen fromFig. 2, pT5 plants were generally shorter than those of lines pT10 and pT8, and more of these plants died after being transplanted from float trays in a greenhouse to the field (see the gaps in the pT5 row). Because pT5 was segregating for the transgene containing the DXPS, MS, GO, and CAT constructs, each plant from that row was genotyped to determine whether it contained [FPS + SQS]and[DXPS, MS, GO, and CAT], or just [FPS + SQS] alone. Squalene analysis was conducted for all surviving plants of line pT5, and four representative plants from lines pT8 and pT10. Furthermore, two wild type TI1068 plants were included as negative controls. The result of this analysis are presented inTable 3. Field analysis of the three pT lines revealed several interesting trends. Comparisons the squalene data from line pT5 plants that possessed the additional [DXPS, MS, GO, and CAT] transgene cassettes with those that lacked it, suggested that these extra transgenes did not serve to enhance squalene accumulation above and beyond that which could be attributed to FPS + SQS alone. Furthermore, none of the pT8 or pT10 individuals showed squalene levels that were higher than pT5 plants expressing only the FPS + SQS transgenes. These results differed from what was observed at Texas A&M in their growth chamber/greenhouse evaluations. Given that there was repeated used of regulatory elements (promoters and transcription terminators) in the multiple cassettes that were included in these transgenes, a likely explanation is that these transgenes had become silenced. (2) Squalene was detectable in all plants taken at harvest, however in the lab the highest observed content had always resulted from a full day of light exposure. This was not observed in the field grown plants. A few had no detectable squalene, a possible sign of segregating or silencing and thus no longer contain or are expressing the transgenic cassettes for squalene production. No detectable squalene was lost through either waste fractions (aqueous or surfactant) from the on-site mulching or screwpress. The dried material mass concentration calculations place our expected yield from wet leaf below 2mg/g but we observed about that in the squalene concentration of milled and dried material. This 2000ug/g material is stable, and is the input biomass for extraction. Data collected at harvest give us on average average (arithmetic mean) 165ug/g fresh weight biomass, after drying roughly 0.2% of processed biomass. The low yield we believe is now largely due to segregation in the pT10 line. This line had been chosen for increased yields previously shown in both greenhouse and field study, but did not perform well in this pilot scale operation. The data below is for the pilot plot at Pat Short's Farm (Table 4). Table 4. Analytical Squalene Data for Harvest and Dried Material How have the results been disseminated to communities of interest? As shown below inTable 5, the cryo EtOH extracted only about 30% of the available squalene. Improved efficacy can be obtained by using a non-polar solvent, however traces of ethanol are more desired than other trace solvents. SynShark has performed super-critical CO2and hexane in previous work, where scCO2had low efficacy and hexane proved to have many hazards and the concern of residual solvent. Ideally an organic solvent followed by a counter current system would allow for only non-polar molecules of a specified mass to be isolated before moving on to purification steps. Table 5. Extraction Efficiency from Pre- and Post- Yields 2021 SynShark Extraction Samples Squalene content (ug/g DW) Mean Post-Extraction Tobacco (Rep1) 1589.95 2243.32 (70.14%) Post-Extraction Tobacco (Rep2) 2896.69 Pre-Extraction Tobacco (Rep1) 3070.79 3198.20 (100%) Pre-Extraction Tobacco (Rep2) 3325.61 Extracted Oil Sample 30667ppm (3%)w/v Extraction Efficacy Pre%-Post% 29.86% ?Purification of the crude tobacco oil for squalene would have been higher throughput by using a washing step to strip water-soluble molecules from hydrophobic oil target fraction. The facilities we were able to partner with had the same concerns as others with GE tobacco oil going into a reactor also used for consumer products, but did allow our team use of one of their short path vacuum distillation units to attempt to take fractions of our known target window of approximately 165-185C under deep vacuum. InTable 6below, the starting crude oil tested at about 7%. All of the Run 3 at temps from 150C-200C collected squalene, and can be combined to reconstitute the majority fraction. Oddly, overshooting the material on Run 4 up to 222C yielded squalene. This overshot temp fraction was at 30% squalene but was of much smaller volume. Table 6. Short Path Distillation Fractions Squalene Analytical Potential Applications (Commercial or Other) of the Research The utilization of the current technology is being evaluated at SynShark currently. The team is now working on evaluation of expression in new background lines, this will allow the team to breed stable lines of transgenic tobacco for squalene production. Additional efforts are being placed on the ability to express transgenes transiently or through inducible promoters. The project also has a new design focusing on expression in the trichome allowing for isolation of trichomes prior to primary extraction while alleviating the stress and regulation put on constitutive production of squalene on the plant. From a production and processing position, SynShark wishes to refocus on vaccine adjuvant production, requiring an indoor cultivation and short plant cycling time. Inducible promoters and transient expression systems favor this style of production but also require more capable extraction and isolation partners. With the correct partner in pharmaceutical research the team sees potential for rapid vaccine adjuvant production. Part II. Comprehensive Description of Results The team is still at work evaluating our current partners in post-harvest processing, however we have a few conclusions from this work. On genetic improvement of squalene producing tobacco lines, it was concluded that theyabby5mutant background line did not appear to show any additive impact on squalene yield. Both theyabby5mutant line and Ti1068 line as the background had comparable yield and yield ranges. Within the field trial conducted at NCSU of various improved squalene lines originating from Texas A&M, it was concluded that the additional enzymes utilized for improved yield showed no impact on yield. It appears that lines are segregating as observed in pT5 line 250 previously. The field trial also has shown yields are lower than expected and variable, making the constitutive over expression of chloroplast targeted enzymes a less viable approach for production. This has led to a refocus of Dr Dewey's efforts toward novel approaches to produce squalene in tobacco in a non-constitutive system. SynShark, through its partnerships, successfully produced, harvested and dried stable, extractable tobacco biomass. The yield was lower than expected, but consistent with observations made by Dr Dewey on the pT lines. It was shown that we had no losses through onsite processing, delivery, and drying compared to traditional flue-curing. This creates a cost saving avenue in the future. Although traditional ethanol extraction systems are now more frequent than before the 2016 farm bill, the extraction efficacy is only around 30%. Despite not having the capacity to separate polar species from the ampiphillic extracted crude tobacco oil, fractioning the oil was able to produce a set of fractions with 2x and 4x concentration of squalene (15% and 30%). These fractions will be reconstituted and further processed by more capable partners using counter current separation and centrifugal partitioning chromatography. Part III. Commercialization Success In this research, SynShark and its partners proposed to accomplish two main objectives (1) The genetic improvement of squalene producing GE tobacco lines, and (2) The production of field grown GE tobacco leaf into extracted and purified high squalene oil. In our first objective, the sub-awardee lab at NCSU were unable to increase squalene content. This failure has exposed an underlying issue with the genetic technology supplied by Texas A&M, and has allowed the team to redirect efforts to more impactful approaches. While the team does not see this as a pivot, production approaches will be altered to cater to the rapid production of vaccine adjuvant. Due to the nature of the material being of a GMO and it being tobacco, many potential partners were uninterested in allowing SynShark to utilize their equipment for extraction. Super critical CO2and hexane have been used in the past, being ineffective and too hazardous/toxic for our application, respectively. Low yield plants and low efficiency solvent produced enough oil for attempting downstream purification. Inability to have access to certain equipment constrained the efforts to produce higher purity squalene. Currently, SynShark has been able to produce a fraction from GE tobacco at 30% squalene. For these reasons, SynShark is currently seeking partnerships with pharmaceutical labs capable of taking a primary extract or low purity fraction and produce an adjuvant level purity squalene isolated oil. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A Brief Description of the Research Carried Out (1) The task of genetic improvement of squalene producing GE tobacco lines was led by Dr Ralph Dewey at NCSU through the sub-award. This was investigated in two fronts. First, one of the novel avenues that the team pursued during this past year was to determine whether expressing squalene-enhancing transgene constructs in a genetic background containing mutations in the gene called YABBY5 could lead to higher levels of squalene accumulation in tobacco. Our rationale was based on a publication in 2016 that showed that the transcription factor YABBY5 served as a negative regulator of terpene biosynthesis, as well as unpublished results from NCSU where tobacco plants with YABBY5 knockout mutations were shown to accumulate nearly double the amount of diterpenes in comparison to its parental control line (TI1068). The NCSU data supporting this is shown below inFigure 1. Figure 1. Double homozygous knockout mutations in YABBY5 enhance diterpene production in tobacco accession TI1068.Lines P53a, P22a and P36a possess CRISPR-Cas9-mediated indels in both alleles of the two YABBY5 homologs found within the tobacco genome. Leaf surface content of the three predominant diterpenes (A-DVT, B-DVT and cis-abienol) in TI1068 are shown in separate graphs. N=9 for each genotype tested (data courtesy of Ramsey Lewis, NCSU). Given that the overall terpenoid metabolic pathway appeared to be upregulated inyabby5mutant lines, it was reasonable to hypothesize that squalene accumulation would also be increased in these tobaccos. To test this, construct FST (containing FPS + SQS + T-oleosin cassettes engineered to transport their respective proteins into the chloroplast) was introduced into the background TI1068, and theyabby5knockout line referred to as TI1068 P53a (shown inFig. 1). Although the transformation efficiency was relatively low, we were able to recover 5 T0plants for each line. These plants were grown in a greenhouse and assayed for squalene just prior to flowering. The results are shown below inTable 1. The other task Dr Dewey and his team evaluated was the field performance of multiple squalene enhancing genetic technologies. At the beginning of this research project, materials were received from Texas A&M University (TAMU), in which several genes constructs had introduced in addition to the chloroplast localized FPS and SQS constructs that originally were shown to be effective in producing measurable amounts of squalene in the tobacco plant. Although each of these technologies appeared to increase squalene accumulation in comparison to the FPS + SQS only control in experiments conducted at TAMU, these were conducted separately in growth chamber and/or greenhouse conditions. In order to directly compare the different gene combinations in a field environment, several plants of the following lines were grown within the same field: pT5, pT8 and pT10. The different gene cassettes contained in each of these lines is shown inTable 2. These three lines were generated by transforming a line called G1, which contained the FPS and SQS constructs on a single transgene construct, with a separate transgene construct containing the all of the additional cassettes listed above. Although each of the lines provided to NCSU were fixed for the FPS and SQS cassettes, there was a potential that the transgene containing the other constructs was segregating. Therefore, prior to growing these lines in the field, we grew 24 plants from each line in the lab and genotyped them to determine whether or not the line was fixed for all of the transgenes shown inTable 2. From this analysis we concluded that lines pT8 and pT10 were fixed, and that line pT5 was still segregating for the transgene possessing the [DXPS, MS, GO, and CAT] cassettes. We took advantage of the fact that this line was segregating to simultaneously generate an internal control that could determine the degree to which the additional transgenes were able to enhance squalene production above and beyond the FPS + SQS cassettes alone. A picture of this experiment in the field is shown below inFigure 2. (2) Production of GE tobacco with engineered traits for increased squalene production, originating from Texas A&M University through Phase I, were grown in a traditional tobacco cultivation scheme of row cropping transplants. Biocontainment was achieved by removal of "buttons" - or floral meristems - upon development and treatment with a suckercide. This keeps plants from going into reproduction and seed production, and instead keeps sugars in the primary leaves. This is advantageous to SynShark's GE tobacco production as it keep primary metabolism feeding the tertiary metabolism which squalene production is a part of. Furthermore it ensures there is not pollen flow (thus gene flow) to other tobaccos, and keeps from setting seed to overwinter and produce volunteers. This material produced is the input biomass for our production and processing plan shown below inFigure 3below. Map and pictures from the pilot scale field are below inFigure 4. Figure 3. Production and Processing Plan for SynShark, llc Figure 4. Pilot Scale Plot Day of Harvest Pictures Half of the harvested bulk material, as leaves, were hand-picked in the morning of Aug 27thand fed into a mulcher. The output of the mulcher fed the screw press. This method produced milled and pressed biomass at 75% moisture to be loaded into a closed box truck. This material was driven to Wilson, NC to a stem line drier (barrel dryer) and was dried below 10% (at approx. 5-7%) and was transported to a walk in refrigerated room prior to extraction. The other half was loaded into a traditional bulk barn (flue cure dryer) and later transported to TRP with the milled and pressed material. Material is stored in c30 boxes with oxylo to reduce oxidation or degradation before extraction. Materials were determined to have similar quantities and no determinable loss through traditional flue curing the tobacco leaf prior to extraction. More detail can be found through the APHIS-BRS filing 21-071-101n. Analytical of fresh harvested leaves, milled material, and dried material from the barn dried and barrel dried material were conducted by Hong Ma at NCSU and shown in results (Table 4). Pictures of the drying process can be seen inFigure 5below. Figure 5. Drying of Milled and Shredded GE Tobacco at Tobacco Rag Processors

Publications

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:American Tobacco Producer: The Phase II effort supports two of the five theme areas outlined in the USDA Science Blueprint (A Roadmap for USDA Science from 2020 to 2025): Sustainable Ag Intensification and Value- Added Innovations. The Phase II goal supports the Sustainable Ag Intensification theme because increasing the terpene yield per harvest will directly reduce the environmental impacts associated with plant production and processing. SynShark's molecular genetics and purification technology aligns well with many of the theme strategies which include (i) using genomic technology to accelerate breeding progress, (ii) using precision agriculture technologies, innovative input technologies and stand improvement to optimize resource use and reduce the gap between actual yield and yield potential, and (iii) enhancing the utility and value of metabolites, and other constituents of plant products. As for the Value-Added Innovations theme, SynShark's technology to generate an alternative source of squalene from tobacco plants aligns well with this theme objective, which is fostering sustainable non-food, feed and other high-value chemical and biophysical applications of plants (pharmaceutical, nutraceutical, industrial products, and feedstock ingredients). The proposed technology development effort addresses two of USDA Strategic Plan Goals. Creating a new alternative source for high value terpene products from tobacco would greatly benefit the American tobacco producer. The outcome of SynShark's new technology would provide an effective financial safety net for tobacco farmers to sustain economically viable agricultural production and support rural jobs and economic growth, which is an objective of USDA's Strategic Goal #2. Increasing the yield of targeted value-added products through molecular genetics and purification would facilitate economic development and stewardship of private lands. Changes/Problems: Nothing Reported 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? Part I. Executive Summary Purpose of the Research SynShark's goal has been to produce a viable solution for an environmentally unsustainable demand for squalene through implementation of an economically favorable option for US tobacco farmers. This objective was set by introducing disruptive technology, in the form of GE tobacco, to farm squalene rich tobacco oil. During Phase II of the USDA-SBIR, the team were tasked with two areas of work, (1) The genetic improvement of squalene producing GE tobacco lines, and (2) The production of field grown GE tobacco leaf into extracted and purified high squalene oil. A Brief Description of the Research Carried Out (1) The task of genetic improvement of squalene producing GE tobacco lines was led by Dr Ralph Dewey at NCSU through the sub-award. This was investigated in two fronts. First, one of the novel avenues that the team pursued during this past year was to determine whether expressing squalene-enhancing transgene constructs in a genetic background containing mutations in the gene called YABBY5 could lead to higher levels of squalene accumulation in tobacco. Our rationale was based on a publication in 2016 that showed that the transcription factor YABBY5 served as a negative regulator of terpene biosynthesis, as well as unpublished results from NCSU where tobacco plants with YABBY5 knockout mutations were shown to accumulate nearly double the amount of diterpenes in comparison to its parental control line (TI1068). The NCSU data supporting this is shown below in Figure 1. Figure 1. Double homozygous knockout mutations in YABBY5 enhance diterpene production in tobacco accession TI1068. Lines P53a, P22a and P36a possess CRISPR-Cas9-mediated indels in both alleles of the two YABBY5 homologs found within the tobacco genome. Leaf surface content of the three predominant diterpenes (A-DVT, B-DVT and cis-abienol) in TI1068 are shown in separate graphs. N=9 for each genotype tested (data courtesy of Ramsey Lewis, NCSU). Given that the overall terpenoid metabolic pathway appeared to be upregulated in yabby5 mutant lines, it was reasonable to hypothesize that squalene accumulation would also be increased in these tobaccos. To test this, construct FST (containing FPS + SQS + T-oleosin cassettes engineered to transport their respective proteins into the chloroplast) was introduced into the background TI1068, and the yabby5 knockout line referred to as TI1068 P53a (shown in Fig. 1). Although the transformation efficiency was relatively low, we were able to recover 5 T0 plants for each line. These plants were grown in a greenhouse and assayed for squalene just prior to flowering. The results are shown below in Table 1. The other task Dr Dewey and his team evaluated was the field performance of multiple squalene enhancing genetic technologies. At the beginning of this research project, materials were received from Texas A&M University (TAMU), in which several genes constructs had introduced in addition to the chloroplast localized FPS and SQS constructs that originally were shown to be effective in producing measurable amounts of squalene in the tobacco plant. Although each of these technologies appeared to increase squalene accumulation in comparison to the FPS + SQS only control in experiments conducted at TAMU, these were conducted separately in growth chamber and/or greenhouse conditions. In order to directly compare the different gene combinations in a field environment, several plants of the following lines were grown within the same field: pT5, pT8 and pT10. The different gene cassettes contained in each of these lines is shown in Table 2. These three lines were generated by transforming a line called G1, which contained the FPS and SQS constructs on a single transgene construct, with a separate transgene construct containing the all of the additional cassettes listed above. Although each of the lines provided to NCSU were fixed for the FPS and SQS cassettes, there was a potential that the transgene containing the other constructs was segregating. Therefore, prior to growing these lines in the field, we grew 24 plants from each line in the lab and genotyped them to determine whether or not the line was fixed for all of the transgenes shown in Table 2. From this analysis we concluded that lines pT8 and pT10 were fixed, and that line pT5 was still segregating for the transgene possessing the [DXPS, MS, GO, and CAT] cassettes. We took advantage of the fact that this line was segregating to simultaneously generate an internal control that could determine the degree to which the additional transgenes were able to enhance squalene production above and beyond the FPS + SQS cassettes alone. A picture of this experiment in the field is shown below in Figure 2. (2) Production of GE tobacco with engineered traits for increased squalene production, originating from Texas A&M University through Phase I, were grown in a traditional tobacco cultivation scheme of row cropping transplants. Biocontainment was achieved by removal of "buttons" - or floral meristems - upon development and treatment with a suckercide. This keeps plants from going into reproduction and seed production, and instead keeps sugars in the primary leaves. This is advantageous to SynShark's GE tobacco production as it keep primary metabolism feeding the tertiary metabolism which squalene production is a part of. Furthermore it ensures there is not pollen flow (thus gene flow) to other tobaccos, and keeps from setting seed to overwinter and produce volunteers. This material produced is the input biomass for our production and processing plan shown below in Figure 3 below. Map and pictures from the pilot scale field are below in Figure 4. Figure 3. Production and Processing Plan for SynShark, llc Figure 4. Pilot Scale Plot Day of Harvest Pictures ?

Publications

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:American Tobacco Producer: The Phase II effort supports two of the five theme areas outlined in the USDA Science Blueprint (A Roadmap for USDA Science from 2020 to 2025): Sustainable Ag Intensification and Value- Added Innovations. The Phase II goal supports the Sustainable Ag Intensification theme because increasing the terpene yield per harvest will directly reduce the environmental impacts associated with plant production and processing. SynShark's molecular genetics and purification technology aligns well with many of the theme strategies which include (i) using genomic technology to accelerate breeding progress, (ii) using precision agriculture technologies, innovative input technologies and stand improvement to optimize resource use and reduce the gap between actual yield and yield potential, and (iii) enhancing the utility and value of metabolites, and other constituents of plant products. As for the Value-Added Innovations theme, SynShark's technology to generate an alternative source of squalene from tobacco plants aligns well with this theme objective, which is fostering sustainable non-food, feed and other high-value chemical and biophysical applications of plants (pharmaceutical, nutraceutical, industrial products, and feedstock ingredients). The proposed technology development effort addresses two of USDA Strategic Plan Goals. Creating a new alternative source for high value terpene products from tobacco would greatly benefit the American tobacco producer. The outcome of SynShark's new technology would provide an effective financial safety net for tobacco farmers to sustain economically viable agricultural production and support rural jobs and economic growth, which is an objective of USDA's Strategic Goal #2. Increasing the yield of targeted value-added products through molecular genetics and purification would facilitate economic development and stewardship of private lands. Changes/Problems: Nothing Reported 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?Plans for next reporting period (Technical): In addition to following through with objectives as outlined in the original proposal, we are pursuing additional strategies designed to either enhance the overall squalene yield, or increase our ability to extract the squalene in a manner more amenable to downstream extraction in a highly purified form. Briefly, we are taking replacing the constitutive promoters that are currently driving transcription of the individual gene cassettes and replacing them with trichome-specific promoters. Producing squalene exclusively within trichomes will enable extraction of the triterpenoid using simple organic washed of the leaf surface, and thus avoid the problems associated with extracting the compound of interest from macerated whole leaf extracts. Plans for next reporting period (Commercialization): During the course of the first year of the SBIR, the SARS-CoV-2 pandemic created a major new market opportunity for SynShark. At a price point of $19 to $32 per kilogram and a lack of a US adjuvant market, SynShark's internal cost structures made it difficult to compete with Shark or Olive Oil sources of cosmetic grade squalene. But its use as a vaccine adjuvant is causing a major change in the squalene market as it is made in human livers and transferred to the skin through the circulatory system. Adjuvatis, Croda International, GlaxoSmithKline (GSK), Merck and Novavax are the largest makers of vaccine adjuvant. GSK is supplying squalene adjuvants for 1 billion SARS-CoV-2 vaccine doses through partnerships with the vaccine developers Sanofi, Clover Biopharmaceuticals, Medicago, and Innovax, which are admittingly all shark derived.[i] Another manufacture of squalene shark adjuvant for SARSCoV- 2 vaccines is Seqirus. Shark liver sourced squalene carries the risk of associating persistent organic pollutants like PCB, PBDE, organochlorine pesticides, polycyclic aromatic hydrocarbons, dioxin, and heavy metals;[ii],[iii] and the possible presence of various pathogens with which sharks could be infected.[iv] Due to risks and public outcry over sharking, an alternative source of squalene is being sought out. SynShark is working with Fujifilm Diosynth, a major manufacture of SARS-CoV-2 vaccines currently without squalene, and Dr. Barry Holtz (former president of iBio), to evaluate the market opportunity. It is clear that associated pesticides in Olive Oil Production inhibit its use, and Amryisis synthetic product lacks the bioactivity of natural squalene (industrial disinterest was also confirmed to SynShark by Dr. Rumyantsev of Sanofi). This summer, SynShark harvested, dried, and extracted one acre of its squalene tobacco into a 5.8kg of crude oil. After the purification step is validated in this activity, SynShark will directly provide samples of its tobacco squalene to the chemical manufactures, like Clariant and Sanofi, (both of whom have requested SynShark samples) and adjuvant suppliers. In addition, corporate development will be started with downstream end users to stimulate demand. [i] https://cen.acs.org/pharmaceuticals/vaccines/hunt-alternatives-shark-squalene-vaccines/98/i47 [ii] Misbah, S.A., Peiris, J.B. and Atukorala, T.M. "Ingestion of shark liver associated with pseudotumor cerebri due to acute hypervitaminosis A." J. Neurol. Neurosurg. Psych. 47: 216 (1984). [iii] Chen, S. and Li, K.W. "Mass spectrometric identification of molecular species of phosphatidylcholine and lysophosphatidylcholine extracted from shark liver." J. Agri. Food Chem. 55: 9670-9677 (2007). [iv] Seo, J.B., et al. "Shark liver oil-induced lipoid pneumonia in pigs: correlation of thin-section CT and histopathologic findings." Radiology 212: 88-96 (1999).

Impacts
What was accomplished under these goals? Impact Statement SynShark LLC aims to create an economically viable source of squalene from American tobacco. Squalene is a triterpene important to the cosmetic and pharmaceutical industry. Unfortunately, squalene is mainly sourced through the "livering" of millions of deep-sea sharks, threatening their populations. Through the Company's exclusively licensed technology for triterpene squalene, which is extracted from metabolically engineered tobacco, SynShark demonstrated that it can express high squalene levels through a novel engineering method. Specifically, the Company showed a capacity to shift carbon flux into the MEP-pathway, allowing for greater accumulation of squalene in engineered tobacco. During this Phase II reporting period, the Company has started optimizing the previously studied method for increased terpene production and storage within SynShark tobacco. This includes combinations of genetic technologies and superior background genomes. Because the cropping methods and postharvest infrastructure are already in place due to tobacco's historical importance in agriculture and the American economy, tobacco can be easily commercialized. Tobacco offers an ideal synthesis platform for modification for commercial applications due to high transformation efficiency. SynShark has previously produced stable squalene producing tobacco varieties at pilot scale and processed fractioned bioproduct for the market. Phase II research has been focusing on optimizing this method and creating a quantification of the portfolio of high quality ingredients being expressed in SynShark's tobacco, beyond squalene. The know-how developed to bring squalene to market will support a rapid go-to-market scenario beyond Phase II. Through the optimizing of squalene extraction from SynShark tobacco, we plan to expand our platform of natural products to satisfy the highly demanded market. Task #1 Outcome Combining Independent Squalene Enhancement Technologies: Produces a higher yield of total squalene in the farmed product and at scale. Task #1 Technical Progress: In the original proposal, multiple potential squalene enhancing technologies were to be combined in ways that had not been previously attempted. This was to be accomplished by crossing the plant materials shown in the table below (which corresponds to Table 4 of the proposal): Table 1. Transgenic Lines, Constructs and Modes of Action Line ID Individual transgene cassettes Mechanism T10-15 FPS*, SQS*, GO, MS, CAT, DXPS, TOleosin C2 redirection + oil droplet FSRT-8 FPS, SQS, RibB(G108S), TOleosin C5 redirection + oil droplet Anti-SQE-14 FPS, SQS, amiSQE NtSQE inhibition *The FPS and SQS transgenes in this line originated from parental line G1; all other cassettes were introduced by transforming G1 with vector pT10. The lines shown above originated from the lab of Dr. Joshua Yuan at Texas A&M University (TAMU) and were provided to NCSU, along with the constructs that they used to generate the transgenic lines. Because these lines weren't developed "in house", we thought it would be important to validate each line. This was to be accomplished by designing PCR primers that would be specific for the individual transgene cassettes and assaying several progenies from each line. Unexpectedly, only line T10-15 tested positive; lines FSRT-8 and Anti-SQE-14 appeared to simply be wild type plants, as no amplification was observed using any primer pairs targeting the various transgene cassettes. The conclusion that T10-15 was the only legitimate transgenic line was further validated by squalene analysis on a subset of the individuals. Squalene was readily detected in the T10-15 plants and nondetectable in those coming from seed lots marked FSRT-8 or Anti-SQE-14. Because of this discrepancy, it was not possible to combine the different squalene enhancing technologies by crossing the lines provided to us by TAMU. As a result, these activities were transferred to Task #2 using materials generated by NCSU as described below. Task #2 Outcome Introducing Squalene Enhancing Constructs into a High Yielding Commercial Grade Tobacco Background: Increases yield of the target bioproduct and is aligned with current North Carolina tobacco cultivation processes Task #2 Technical Progress: The plant expression vectors used to generate the transgenic lines shown in Table 1 are designated as follows: pT10 [containing GO + MS + CAT + DXPS + TOleosin]; pFSTR [containing FPS + SQS + RibB + TOleosin] and pAnti-SQE (containing FPS + SQS + amiSQE]. As mentioned above, these vectors were provided to NCSU by TAMU researchers. Prior to using these vectors for transformation, we conducted a series of restriction digests and PCR amplications to confirm their integrity. These tests validated plant expression vectors pFSTR and pAnti-SQE, but not pT10. Our attempts to obtain alternative sources of pT10 (as well as additional transgenic FSTR and Anti-SQE seed stocks) from TAMU were unsuccessful. Therefore, in order to meet those goals of this proposal, it was decided that only the validated pFSTR and pAnti-SQE vectors would be used to transform the high yielding tobacco variety K326. Crosses between FSTR and Anti-SQE transgenic plants in K326 could be conducted to test the effects of combining the T-oleosin and C5 redirection cassettes with the amiSQE technology, while crosses of these same materials to the validated line T10-15 obtained from TAMU could be used to combine the C2 redirection technology (with TOleosin) with the C5 redirection and amiSQE cassettes. Standard Agrobacterium-based transformation protocols were used to introduce the pFSTR and pAnti-SQE vectors into K326. A total of 9 T0 plants transformed with pFSTR and 10 T0 individuals transformed with pAnti-SQE were recovered. Young T0 plant from both experiments were assayed for SQS transgene expression levels using quantitative real-time PCR (qRT-PCR). The pRT-PCR results are shown below in Figure 1. Figure not allowed Figure 1. qRT-PCR analysis of T0 plants transformed with either pFSTR or pAnti-SQE. Plant FSTR#53 was arbitrarily select as the individual against which the expression levels were compared (defined as "1"). Once the T0 plants were sufficiently large, they were transplanted to larger pots and grown to maturity in a greenhouse. Just prior to flowering, an upper leaf sample was harvested from each plant, frozen in liquid nitrogen and analyzed for squalene content. The results of the squalene analysis are shown below in Table 2. Table 2. Squalene analysis of T0 generation K326 plants Figure not allowed In general, there was a very good correlation between the levels of SQS transgene expression and squalene content. It is also worth noting that the values presented in Table 2 represent the quantity of squalene on a fresh weight basis. Given that approximately 90% of tobacco leaf weight is water, the squalene concentrations are predicted to be approximately 10 times higher on a dry weight basis. In order to test the effects of combining the C5 redirection and TOleosin constructs with amiSQE transgene, the three highest producing MiSQE T0 plants (MiSQE#5, MiSQE#27 and MiSQE#46) were cross with the highest producing FSTR individual (FSTR#86). The progeny of these crosses will be evaluated for squalene accumulation over the next several months. Task #3 Outcome Quantitation of Additional High Value Compounds from the Tobacco Oil Fraction: Selection of target products from tobacco agriculture in the area, create scale and expression (and Intellectual Property), fraction it, and deliver a unique product Task #3 Technical Progress: A GCMS method was validated for the quantitation of Squalene and other compounds of interest in engineered tobacco. Squalene was easily detected and well-separated from all other compounds in the sample preparation. Figure not allowed

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