Source: EXOPOLYMER, INC. submitted to
NEXT GENERATION BIOPOLYMERS FOR HIGH VALUE MARKETS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1027313
Grant No.
2021-33610-35656
Cumulative Award Amt.
$648,366.00
Proposal No.
2021-06457
Multistate No.
(N/A)
Project Start Date
Sep 1, 2021
Project End Date
Aug 31, 2023
Grant Year
2021
Program Code
[8.8]- Biofuels and Biobased Products
Project Director
Wells, D.
Recipient Organization
EXOPOLYMER, INC.
737 INDUSTRIAL ROAD
SAN CARLOS,CA 940703310
Performing Department
(N/A)
Non Technical Summary
The development of fermentation-derived biopolymer (hydrocolloid) ingredients began in the U.S. with the discovery of xanthan gum in the 1960s by researchers at the USDA. In recent decades, however, production of these important ingredients has largely gone offshore, and the development of new and differentiated biopolymers has been stagnant. ExoPolymer intends to produce an entirely new portfolio of high-performance biopolymers that meet current and growing needs in several markets. Our technology and use of corn-derived feedstock will allow competitive manufacturing of these valuable ingredients to return to the U.S. and open new opportunities for U.S. agricultural products and production in rural communities.Biopolymers are widely used ingredients found in a diverse range of products and processes. Despite their important role, there has been little development of new products over the last fifty years. Existing products, such as alginate, xanthan gum, and guar gum, have limited capabilities and cannot provide needed functionality to a growing number of established and emerging markets. Another example is hyaluronic acid (HA), an animal-derived biopolymer that is used across multiple personal care and healthcare markets. HA is unique in its ability to bind water and provide lubrication - it is the predominant functional ingredient in topical anti-wrinkle and injectable joint health applications. HA is not broadly available, however, due to production complexities and sales costs that preclude its use in consumer products that are more financially accessible. Our main goal during this Phase II project is to finalize the commercial development of one or more biopolymer products that outperform HA, providing a needed upgrade to this valuable ingredient, and more importantly, making high performing products for anti-aging available to a substantially wider consumer group.During this project, we will identify high-yielding strains for commercialization. This step is necessary in order to reduce production costs by maximizing conversion of agriculture feedstock to end product. We will also finalize process conditions for production of biopolymers by performing multiple fermentation runs up to the 100-liter scale in a collaboration with the National Corn-to-Ethanol Research Center in Edwardsville, IL. Most importantly, we will conduct quantitative testing of biopolymers and their derivatives for moisture binding and skin penetration performance. Derivative molecules will be generated through a CRADA with the USDA ARS facility in Peoria, IL. The experiments carried out in this project will complete an initial data package for first target products and allow ExoPolymer to begin large scale production and sales to customers in the cosmetics market.Our technology will have broad impact in several areas. Economically, new biopolymers that are produced domestically will add considerable value to the U.S. agriculture industry, since new, high performance products will command a high price and generate a significant financial return. The use of domestic corn as the feedstock of choice for biopolymer production will decrease existing import and geopolitical complexities since industrial grade hydrocolloid products are made mostly in China and India. ExoPolymer's technology will also positively affect personal care market growth, which is being driven by consumer trends such as clean label, environmental awareness, non-GMO products, and alternative protein meat and dairy products. Final consumer products will benefit from the unique and differential properties of ExoPolymer's natural ingredients. By providing an entirely new portfolio of ingredients with novel and needed performances, ExoPolymer's biopolymers will enable success and expansion in several additional large and important markets while benefiting U.S. agricultural and rural communities.
Animal Health Component
33%
Research Effort Categories
Basic
33%
Applied
33%
Developmental
34%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51140101100100%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
ExoPolymer's goal for this Phase II project is the commercialization of new and differentiated biopolymers to compete with and replace hyaluronic acid (HA) in the personal care industry. Many high-end personal care creams and lotions with anti-wrinkle performance contain HA as the active ingredient. HA is a natural biopolymer that is derived either from animals, as a co-product from meat processing facilities, or the fermentation of a pathogenic Streptococcus species. When applied topically, low molecular weight HA can penetrate into the epidermis, bind water, and reduce the appearance of wrinkles. HA is the only commercially available biopolymer with these properties, and to date no alternative products are widely available. Both animal and pathogen production processes are costly, leading to a high price point for the raw ingredient ($1,000-$2,000/kg). Despite the high price, HA is a valued and sought-after ingredient due to its unique performance. The introduction of alternative biopolymers with improved performance relative to HA represents a major first commercialization opportunity for ExoPolymer in the personal care market. Because our products can be manufactured from agricultural feedstocks, our non-pathogenic strains are highly efficient at production, and our overall process is straightforward and scalable, we will be able to compete with HA on both performance and price.The technical objectives of this Phase II proposal are 1. to further develop production strains for maximum yields and titers, 2. to establish robust guidelines for strain propagation in bioreactors and purification of final products, and 3. to collect high resolution performance data to support commercialization. In Specific Aim 1, we will develop strains capable of high yields and desirable molecular weight distribution. We will use targeted mutations predicted to enhance production and will carry out directed genetic screens. Optimized strains will be used for subsequent scale up experiments. In Specific Aim 2 we will further optimize growth conditions that permit fermentation scale up to prepare for commercialization. We will focus on production strain performance at the bench scale, 5 liter, and 100 liter scale to determine rates, titers, and yields as affected by process variables, and to inform our technoeconomic model. We will generate sample quantities of product from these experiments, and further refine the process of generating functionalized derivatives in collaboration with the USDA ARS facility in Peoria, IL. In Specific Aim 3 we will perform detailed characterization of biopolymer products by examining traits important for their performance in personal care. We will examine their ability to dynamically bind water by quantifying absorption and desorption properties using a thermogravimetric instrument. Rheological properties of products will be measured to examine behavior of biopolymers on their own and in commercial lotion bases. We will further assay performance using an in vitro assay for transdermal penetration.
Project Methods
Efforts. During Phase II, ExoPolymer will conduct experiments in the three major focus areas that represent the major technical goals of the project. First, for strain engineering and improvement in titer and productivity, we will utilize several standard methods for genetic modification of production hosts. Molecular biology methods include PCR, cloning, homologous recombination, positive and negative selection, gene excision and promoter mutation. Other methods include directed and random mutagenesis and HTP screening for increased productivity. Second, we will develop the process to manufacture biopolymers at the bench scale up to 100 liters. The methods involved in process optimization will include in-depth surveys of carbon source, feeding strategies, nitrogen, and phosphate concentrations, as well as timing associated with inoculation and harvest. For fermentation in bioreactors, we will measure common parameters that are monitored during production - agitation, sparge rate, feed, pH, OUR, CER, DO, OD, and dissolved oxygen. Initially we will gather data from 5-liter volumes, and then directly scale to one or more 100-liter bioreactors for first target molecules. As part of the second objective, we will also optimize the formation of derivatives of biopolymer products in collaboration with the USDA ARS facility in Peoria, IL. Third, we will conduct performance assays of biopolymer products. These efforts include dynamic vapor sorption measurements and rheological assays of pure biopolymer as well as biopolymer in formulations. We will also measure performance in in vitro skin penetration assays in collaboration with a fee-for-service organization.Evaluation. Targeted genetic modifications in production strains will be confirmed by sequencing of local genomic regions. Genomes of strains that are derived from mutagenesis may be sequenced to rapidly identify alleles of interest. Alternatively, favorable alleles may be identified using traditional transposon-based genetic mapping. Increases in productivity will be quantified by directly measuring biopolymer output of improved strains. In some cases, a fluorescent dye may be used, and in other cases purification and mass of product will determine titer relative to control strains. Major milestones will include the generation of one or more strains that display improved titer over current production hosts. For process development, evaluation will include determining optimal values for the variables described above. Most importantly, productivity of strains on commercial feedstock under various scenarios will generate a robust protocol for larger scale manufacturing. A high yielding process at the 100-liter scale will be representative of behavior at larger volumes, and thus help to build a final, accurate cost model for production - this would be a major milestone in process development of our first target molecules. Thermogravimetric analysis and dynamic vapor sorption measurements will be generated using a Q5000 SA from TA instruments. Water absorption and desorption can be accurately measured across a range of temperatures using this instrument. Various rheological parameters will be measured using a TA Instruments DHR3 Rheometer. Different geometries may be utilized to measure rheological performance of purified biopolymer as well as fully formulated material in lotion base. For skin penetration assays, Franz cells will be used, and flow through will be measured for biopolymer content by one of several quantitative, optical assays. Milestones for these tasks include establishment of vapor sorption and rheological data sets, as well as generation of quantitative data for skin penetration.

Progress 09/01/21 to 08/31/23

Outputs
Target Audience:Target audiences for this effort include manufacturers of personal care, healthcare, and food products, where biopolymers are key ingredients that provide stability, viscosity, and other performance properties. Audiences can also include end consumers of products in these market spaces. Other interested communities are corporate partners or government laboratories. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The Phase II SBIR funding allowed ExoPolymer to hire a full time fermentation engineer. This individual is responsible for bench scale process development and fermentation and has had the opportunity to learn how to operate our bioreactor, as well as several basic principles of molecular biology and microbiology that are fundamental to our work. 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? IMPACT. During the final year of the Phase II project, we accomplished multiple goals across all objectives, with a focus on process development, elaborated testing of our first product, and partnering our technology. Biopolymers (aka hydrocolloids, polysaccharides, or more generally, carbohydrates) are essential ingredients used across a broad array of markets. They are used in personal care, pharmaceutical, and food applications, and even for lubrication in devices, machines, and for drilling. ExoPolymer has developed a state-of-the-art, agriculture-based approach to produce novel biopolymers using natural microbes, allowing innovation in this commercial and scientific space. The biopolymer industry has lagged behind other industrial biotechnology areas that have benefited from recent advances in synthetic biology. The current array of available products cannot meet new performance needs in the personal care, food, healthcare, and energy industries, and our approach is to apply modern genetic and molecular tools to the biopolymer industry. The Phase II funding from NIFA helped us develop a novel biopolymer for use in the personal care market segment as our first commercial product. During the second year of the Phase II project, we entered into a collaboration with CP Kelco for the manufacturing and sales of this valuable molecule. With early revenue from entry into the personal care market, we intend to aggressively pursue additional applications for our technology and products and to become the world leader in biopolymer design, biosynthesis, and manufacturing. Objective 1 - Strain Development. (1) Major activities completed - Initial and advanced strains were constructed for increased production of product. Small scale assays were carried out and top performing strains have been identified to bring forward for fermentation. (2) Data collected - critical strain information was acquired by sequencing and analysis of product titer at small scale. (3) Discussion of results - Candidate strains were identified and will be carried forward. These strains harbor stable expression elements that result in increased product rates and titers at the bench scale. (4) Key outcomes - We validated initial methodologies for strain construction by showing that targeted modifications can impact productivity of base strains, and high performing strains will be used in the scaled process. Objective 2 - Fermentation Optimization. (1) Major activities completed - We have developed a robust fermentation process and expanded our capabilities in-house to eight independent bioreactors, as well as scaled up to the 4000-liter scale with a manufacturing partner. As scale has increased, so has productivity and titer, indicating a clear path to commercially favorable production costs. (2) Data collected - For all fermentation runs, key data including dissolved oxygen, pH, temperature, glucose concentration, agitation, pitch rate, back pressure, foam control, and aeration levels were recorded. This information has guided progress towards full commercial scale. (3) Discussion of results - Our strains are capable of high levels of production on cost-competitive feedstocks and our process is well on the way to full commercial scale with our partner. (4) Key outcomes - Successful development of commercially relevant growth medium, successful growth, feedstock utilization, and production at larger fermentation scales, successful downstream processing of product, successful technology transfer to manufacturing partner. Objective 3 - Functional Testing. (1) Major activities completed - We have shown that our product can be incorporated into standard water-in-oil personal care formulations. Further, we have shown that formulated product provides a quantifiable increase in moisture levels in skin. We have begun to develop in-house formulation capabilities to identify key commercial attributes of our product. (2) Data collected - We have generated detailed data on formulations including information on concentrations, stability, and compatible ingredients. (3) Discussion of Results - Results from the Phase II work have proven that our high moisture-binding polymers can be successfully incorporated into standard personal care formulas. Our product can be utilized in formulations that normally include hyaluronic acid. While it is still necessary to generate additional data for more detailed, clinical skin hydration efficacy, initial testing indicates that our product may have a significant impact on skin moisture content. (4) Key Outcomes - We have gained a detailed understanding of formulation methods and ingredient compatibility and have successfully conducted early skin hydration studies.

Publications


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

    Outputs
    Target Audience:Target audiences for this effort include manufacturers of personal care, healthcare, and food products, where biopolymers are key ingredients that provide stability, viscosity, and other performance properties. Audiences can also include end consumers of products in these market spaces. Other interested communities are corporate partners or government laboratories. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The Phase II SBIR funding has allowed ExoPolymer to hire a full time fermentation engineer. This individual is responsible for bench scale process development and fermentation and has had the opportunity to learn how to operate our bioreactor, as well as several basic principles of molecular biology and microbiology that are fundamental to our work. 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?During the next reporting period, ExoPolymer will work less frequently with the USDA ARS facility in Peoria, and more with the commercialization partner.

    Impacts
    What was accomplished under these goals? IMPACT. Biopolymers (aka hydrocolloids, polysaccharides, or more generally, carbohydrates) are essential ingredients used across a broad array of markets. Uses range from personal care, pharmaceutical compounding, and food stabilization, to lubrication in devices, machines, and drilling. ExoPolymer has developed a state-of-the-art, agriculture-based approach, using natural microbes, that allows innovation in this commercial and scientific space. The biopolymer industry has lagged behind other industrial biotechnology areas that have benefitted from recent advances in synthetic biology. Additionally, the array of products that are currently available cannot always meet new performance needs in the personal care, food, healthcare, and energy industries. The time is right to apply modern genetic and molecular tools to the biopolymer industry, and ExoPolymer intends to be the first mover in this space. With early revenue from entry into the personal care market, we intend to aggressively pursue additional applications for our technology and products and to become the world leader in biopolymer design, biosynthesis, and manufacturing. During the initial year of the Phase II project, we accomplished multiple goals across all Objectives (Specific Aims), with a major focus on process development and formulation and testing of product in personal care formulations. Objective 1 - Strain Development. (1) Major activities completed - Initial strains were constructed for increased production of product. Small scale assays were carried out and top performing strains have been identified to bring forward for fermentation. (2) Data collected - critical strain information was acquired by sequencing and analysis of product titer at small scale. (3) Discussion of results - Candidate strains have been identified and will be carried forward. These strains harbor stable expression elements that result in increased product rates and titers at the bench scale. Further development of these strains may be required during the Phase II project, and top performers will be assessed at larger fermentation scales. (4) Key outcomes - We have validated initial methodologies for strain construction by showing that targeted modifications can impact productivity of base strains. Objective 2 - Fermentation Optimization. (1) Major activities completed - We have developed an initial fermentation process and expanded our capabilities in-house, as well as scaled up to the 14-liter scale with a manufacturing partner. As scale has increased, so has productivity and titer, indicating a clear path to commercially favorable production costs. (2) Data collected - For all fermentation runs, key data including dissolved oxygen, pH, temperature, glucose concentration, agitation, and aeration levels were recorded. Information from the most successful runs will guide future development of the process optimized for scale. (3) Discussion of results - Production data from larger scale fermentation has provided important foundational information for the next phases of scale up activity. We have shown that our strains are capable of high levels of production on cost-competitive feedstocks, and that purification at scale is feasible. (4) Key outcomes - Successful development of commercially relevant growth medium, successful growth, feedstock utilization, and production at larger fermentation scales, successful downstream processing of product, successful technology transfer to manufacturing partner. Objective 3 - Functional Testing. (1) Major activities completed - We have shown that our product can be incorporated into standard water-in-oil personal care formulations. Further, we have shown that formulated product provides a quantifiable increase in moisture levels in skin. (2) Data collected - We have generated detailed data on formulations that include our products, which include information on concentrations, stability, and compatible ingredients. For skin hydration testing, changes were measured using a corneometer, a device that is specifically designed to measure moisture content of the skin. (3) Discussion of Results - Results from the Phase II work thus far have proven that our high moisture-binding polymers can be successfully incorporated into standard personal care formulas. Our product can therefore be utilized in formulations that normally include hyaluronic acid. Further, the methods for use are identical to that of HA, substantially reducing the need for development of new procedures. While it is still necessary to generate additional data for skin hydration efficacy, initial testing indicates that our product may have a significant impact on skin moisture content. (4) Key Outcomes - Initial understanding of formulation methods and ingredient compatibility, successful incorporation into functional water-in-oil lotion base, successful testing and positive results for skin hydration.

    Publications