Progress 09/01/21 to 03/20/24

Outputs
Target Audience:Chi Botanic was demonstrating the functional application of plant cell agriculture to multiple audience types throughout the project. First, the ability to transform the vanilla pod into a fast growing cell suspension is a real application of an abstract commercial possibility for financial investors looking to capitalize from this new form of agriculture. Representing this audience was a reporter from the weekly newletter of the venture capital firm AgFunder, who wrote an article detailing our USDA-funded vanilla project. The second target audience are those commercial sectors with applications of this new way to produce vanilla flavor and fragrance. The value that it could provide to their customer base goes beyond the organoleptic qualities of the vanillin molecule. We are developing an alternative to the vanilla plant that is more ethical, sustainable, and a potential domestic source beyond the Hawaiian islands. Perhaps most importantly, we target a far cheaper source for our audience of customers. Finally, we are also targeting the young scientists that are drawn to this new technology. Many have found the application to make vanilla very interesting because it is something of an exotic plant. It has been refreshing to watch students and young scientists gravitate towards learning in vitro methods and sterile technique in order to be responsible for transforming a multi-cellular plant into a shaken, microbe-like cell suspension. Changes/Problems:No work or budgetary expenditures occurred during the 1 year no cost extension. What opportunities for training and professional development has the project provided?Training and professional development has been facilitated by this project under multiple settings. 1) Chi Botanic Laboratories:Independently, two young scientists received training that helped developed their botanical research skills to include cell suspension culture. This includes differences in how plant cells in this format are maintained and improved including how they are kept sterile, maintained, and tested accurately. 2) The Jinkerson Laboratory:Students in the Jinkerson laboratory in the University of California, Riverside Botany and Chemical Engineering Departments used this opportunity in two ways. The first was to independently develop ways to analyze aloe using Nuclear Magnetic Resonance microscopy to reveal that our cell were unique among any aloe ever tested in the type of polysacharride they produce. In addition, a Jinkerson lab student assisted Chi Botanic in writing provisional patent applications to protect the technology we are developing while expanding their ability to assimilate, process, and create new information. 3) The UC Riverside Plant Transformation Research Center (PTRC):The PTRC was able to enlist young students in developing methods to genetically transform our aloe vera plants. They expanded the plantlets we have in sterile culture, tested their range of sensitivity to antibiotics, and attempted the first DNA transfers by agrobacteria for Chi Botanic. How have the results been disseminated to communities of interest?Chi Botanic has disseminated information about this project via attendance of public synthetic biology and food technology conferences, a journal article, non-provisional patent, and articles about Chi Botanic in online media like magazines and technical press. We also have attempted to make a presence on Chi Botanic's pages in social media platforms like Meta and X. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1) A cell-culturedaloe ingredient:Because aloe vera cell culture does not require the removal of toxin like aloe inner leaf, at least 19 valuable bioactives are retained that include moisturizing, exfoliating and texture improvement, humectant and moisture retentation via improved barrier, antioxidants, and skin-conditioning effects. Through secondary testing we proved that our aloe cell culture has more niacin (Vit B3) than steak, more folate (Vit B9) than spinach or eggs, and more Vitamin C than oranges. Aloe cell culture also contains: Vitamins A, B6, B12, K, Minerals, Salicylic acid, Chromones, and Ceramides not measurable in processed aloe inner leaf.This extremely healthy composition is guarenteed without heavy metals, herbicides, or pesticides due to the way it is grown indoors. We targeted the development of ascalable approach to the conversion of aloe plant cell cultures into ingredients, multiple common processes for growing, filtration, milling, drying, and packaging were analyzed and tested. Key Metric 1.1: Using microscopy and dry weight analysis, successfully showed that no more than 5% of starting biomass is lost via filtration. Key Metric 1.2:Developed a liquid product with much lower energy requirements than spray drying the elmination of significant loss made possible by the application of natural preservative formula providing long-term antibacterial sterility at room temp and above. Key Metric 1.3: Successfully proven incorporated into a lotion and facial mask shown with 2X higher max incorporation than aloe inner leaf juice and long-term stability by Above Renaldi formulator. Also tested as meeting criteria for use by cosmetic ingredient supplier Unigen. Objectie 2) Comparable gel content:Our main objective is to physically transformed our cell culture before it can be used by aloe product manufacturers as a drop-in replacement for traditionally-grown aloe inner leaf gel. Key Metric 2.1: We aimed to reach10% polyacetylated mannan (acemannan) gel by dry weight during the course of the project. While progress was made in the techniques that will make this possible, we did not reach this goal. However, we did develop gel detection techniques that can be applied in high-throughput, allowing us to differentiate from huge populations or environmental triggers for higher gel content that we will apply in the future to extend these lines from safer, fast-growing aloe to an aloe that can replace what is today grown in the field! Objective 3)The firstnon-toxic Aloe vera: As of a April 8th, 2021 the legal limit for the anthraquinonesaloins, aloe-emodins, and danthrones is set below 1 ppm in the EU & Korea and 10 ppm in the US. Our first aloe vera cell cultures had alreay been in liquid cell suspension for nearly two years! Key Metric 3.1: We aimed to obtain a minimum of 5-10 individual gene-edited lines. Instead, we found that our liquid aloe cell suspensions produce less than 100X the maximum aloin by even the strictest regulation that is nearly beyond the limit of detection, even by gas chromatography. Key Metric 3.2: We obtained aloe cell suspensions from two independent aloe varieties and aloe cell cultures from a total of 7 different varieties. Key Metric 3.3: Show that at least 1 aloin-free line is stable after 6 months of growth. Our first aloin-free Aloe cell culture has been grown in stable, sterile culture for over 5 years!

Publications

• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Hann EC, Harland-Dunaway M, Garcia AJ, Meuser JE, Jinkerson RE. Alternative carbon sources for the production of plant cellular agriculture: a case study on acetate. Front Plant Sci. 2023 Oct 26;14:1104751. doi: 10.3389/fpls.2023.1104751. PMID: 37954996; PMCID: PMC10639172.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Over 100 investors and venture capital firms were introduced to this technology during the reporting period. In addition, many multi-national personal carecompanies were introduced to the primary product, an aloe stem cell ingredient for lotions, face masks, body sprays, and other moisturizing and nurturning products for the skin. Students and research staff at University of California, Riverside were also introduced to this new way to produce aloe during the 2022-2023 year. Perhaps most importantly, Chi Botanic uses articles in the media and social media to reach thousands of people interested in plant technologies making a meaningful difference of all ages. Changes/Problems:One of the biggest challenges is increasing levels of acemannan. However, we've made major technical strides in how we select cells with the trait of interest in ultra-high-throughput from literally millions of cells. Using this Plant Cell Foundry approach, we expect to isolate high acemannan cell lines in the coming months. What opportunities for training and professional development has the project provided?Two young scientists were trained through funding for this project. Each made significant strides in their technical, presentation, and organizational skills during the course of this work. How have the results been disseminated to communities of interest?Typically results have been disseminated via investorpresentations and public talks. Recently, an article titled, "Plant cell culture deep dive: As cracks emerge in botanical supply chains, 'It's a huge space to watch'" was published online by "Ag Funder News". In addition. progress has been posted across Chi Botanic'ssocial media platforms like Twitter, Instagram, Facebook, and LinkedIn for thousands of followers to witness. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we expect to publish a journal article in "Frontiers in Plant Science" that explores the future of plant cell culture technology. In addition, we are looking to sell our Aloe cell culture lysate and introduce it into a retail cosmetic product via B2B (business to business) sales.

Impacts
What was accomplished under these goals? 1) A downstream bioseparations process was developed to produce a "aloe vera cell culture lysate" that is a direct replacement of existing aloe products on the market in standard formulations. This includes two optionalnatural preservative formulas that can be selected by the customer. 2) We created ultra-fast growing strains and fully characterize the polysaccharide content. The increase in acemannan (polyacetylated mannan) content is ongoing, though it is clear this is not important for all applications. 3) We have shown that aloe cell cultures produce 100X less aloin that is required by current regulations. Work to "knock-out" the genes for aloin production is ongoing.

Publications

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

Outputs
Target Audience:Aloe product manufacturers and their customers in the personal care and cosmetics industry are the primary target audience, including Unigen, Estee Lauder, Unilever, and indie brand Freestyle Beauty. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training Activities This project supported the master's and PhD training of one graduate student and the research training of one undergraduate student. Professional development This project supported the attendence of the SynBioBeta Built with Biology Global Conference, April 12-14, 2022, by the project director and two Chi Botanic scientists. How have the results been disseminated to communities of interest?The results of this project have been disseminated to potential investors and customers, including Estee Lauder, Freestyle Beauty, andUnigen. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, the focus will be on: 1. Optimization of thedrying and milling procedures forharvested aloe vera cells. 2. Optimization of polyacetylated production by the CB3 cell line using various elicitors, including abscisic acid, mannose, and sorbitol. 3. Targeted disruption of the aloin biosynthesis pathway currently in progress at UC Riverside.

Impacts
What was accomplished under these goals? Objective 1. Establish downstream bioseparations processes to convert aloe cell cultures into high-quality aloe products by establishing and optimizing filtration, drying, and milling procedures. Downstream bioseparation processes were focused on the creation of a product stable, high value, aloin-free lysate created from the previously optimized CB3 aloe vera cell line grown in dark conditions. This work was done to address the needs and specifications of our firstcommercial customer, Freestyle Beauty, which included optimizing a desalting procedure for washing the cells to allow for the lysate to be usable in cosmetics and adjusting the pH of the lysate for optimal addition to cosmetics. The bench scale (1-5L) conversion of the aloe cell culture to this market-ready ingredient was used to generate material for formulation and testing of this new product. The next steps will be focused on taking the process to pilot scale (50-100 L) for the delivery of 250L of SpectraBotanic Aloe vera Full-spectrum Lysate for the launch of Freestyle Beauty's "Tigerlove" cosmetics brand early next year. Performed by: Chi Botanic Current Status: In progress Objective 2. Increase polyacetylated mannan production by aloe cell cultures by using elicitors and selection strategies. Over the past year, production research and development focused on increasing biomass growth potential both in flasks and bioreactors. Initial challenges included browning of the aloe vera cells, contamination of the bioreactors, low yields, and time to inoculation. Optimization of the bioreactor set-up, autoclave sterilization, and growth conditions (i.e. type of bubbling, airflow rate, stirring speed, additives, and time to harvest) have significantly improved the color and quality of the aloe vera cells from last year. We are currently able to consistently grow >100 g/L (wet weight) of aloe vera cells of a light color in our 3, 6, and 15 L bioreactors, with a maximum harvest of 240 g/L (wet weight), by doubling the starting cell density and filling up the reactor with media as the cells grow. Initial testing for polyacetylated mannan production suggests the acemannan content of our aloe vera cells is about 0.1% (weight percentage of dried solids; data processed by UC Riverside) and about 5 mg/L in our processed lysate solution (data processed by Process NMR). This is about 70 x less than typical aloe verainner leaf juice. Further development in the processing of aloe vera cells, elicitors, and selection strategies are underway toimprovethe polyacetylated mannan content. Aloe vera cells are currently harvested at Chi Botanic for lysate production using a series of washes to remove salts responsible for breaking desired emulsions in cosmetics; however, the media from growing the aloe vera cells cultures has been shown to contain about 1% acemannan (weight percentage of dried solids; data processed by UC Riverside), a ten fold increase from the cells themselves. This suggests that acemannan could be harvested directly from the cell culture media. Additional testing performed by Unigen on our freeze-dried aloe vera cells for polysaccharide content using HPLC - size exclusion chromatography found the total polysaccharide content to be greater than 20%. These results suggest that while the acemannan content may be low for the suspension cell culture cells, they may be producing other polysaccharides of interest, primarily in the 50K-5K molecular weight range from which >95% of the polysaccharides were found. Preliminary exploration into the response of the aloe vera CB3 suspension cell cultures to elicitors known to increase acemannan production have been performed, including with abscisic acid. An initial 6-well plate screening for cell viability at 0, 5, 10, and 20 uM abscisic acid (ABA) concentrations suggests that at 0 - 10 uM concentrations of abscisic acid the aloe vera cells have less browning after 11 days, while after 17 days cells at all concentrations of ABA had significant browning. Performed by: Chi Botanic and UC Riverside Current Status: In progress Objective 3. Develop aloe plant cell lines that do not produce the carcinogen aloin by disrupting genes involved in anthraquinone biosynthesis. 1. We have established that our CB3 aloe vera cell line does not produce aloin (< 1 ppm aloin) without the use of gene disruption. Additionally, HPLC analysis from Unigen did not detect anthraquinones in freeze-dried samples from the CB3 aloe vera cell line. Performed by: Chi Botanic Current Status: Complete 2. Transformation to eliminate aloin production in aloe vera is currently underway in the Plant Transformation Research Center at UC Riverside. Over 300 aloe vera shoots are currently being grown at this facility from which stem discs were cultivated and are being used for Agrobacterium-mediated plant transformation with the Aglo1 strain. Various hygromycin concentrations were tested for optimal selection concentrations. Hygromycin is currently being used at a concentration of 25 mg/L along with 250 mg/L Timentin for selection of the transformed tissue cultures. Performed by: UC Riverside Current Status: In progress

Publications