| NSF Org: |
TI Translational Impacts |
| Recipient: |
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| Initial Amendment Date: | April 27, 2023 |
| Latest Amendment Date: | September 30, 2024 |
| Award Number: | 2222602 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Erik Pierstorff
epiersto@nsf.gov (703)292-0000 TI Translational Impacts TIP Directorate for Technology, Innovation, and Partnerships |
| Start Date: | May 1, 2023 |
| End Date: | December 31, 2024 (Estimated) |
| Total Intended Award Amount: | $274,100.00 |
| Total Awarded Amount to Date: | $274,100.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
1938 HARNEY ST STE 305 LARAMIE WY US 82072-3037 (307)761-2329 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
1938 E Harney St Laramie WY US 82072-3037 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | SBIR Phase I |
| Primary Program Source: |
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| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.084 |
ABSTRACT
The broader impact of this Small Business Innovation Research (SBIR) Phase I project is to reimagine bio-manufacturing with a novel platform technology that could boost the yields of many products, including food additives, biomaterials precursors, biofuels, and pharmaceuticals. The technological advancement addresses a fundamental issue that limits conventional bio-fermentation, which is that producing cells suffer limited health and viability in exchange for higher yields. In this proposal, genetic tools will be used to divide the labor of cell reproduction and product synthesis into two different cell types, called stem cells and factory cells. As older factory cells become exhausted, productivity is maintained by new factory cells, which are born from the stem cell population. The approach may be particularly well suited to biofuels and other molecules that are difficult to produce in large quantities by conventional bio-fermentation because the product is toxic to the cells that make it. It could be applied toward increasing the profitability of existing bio-processes and also for bringing new products to market, which are currently too difficult to produce. In this project, the team seeks to demonstrate the benefits of producing a fuel (limonene) and a dairy enzyme (chymosin), as proof of its application in biofuel and agricultural sectors. Broad industrial implementation will advance bio-manufacturing toward the ?green? revolution, contributing to the development of cleaner industries and decreasing US and global reliance on fossil fuels.
This project aims to solve two major limitations of microbial fermentation processes: metabolic exhaustion and genetic drift. These are nearly universal problems in the industry. Highly producing cells can become inactive due to the lack of metabolic resources, cytotoxic effects of products, and mutations that break the biosynthetic pathway. In this project, Microbial Stem Cell Technology (MiST) uncouples growth and production by establishing a multicellular system. One cell type is dedicated to product synthesis (factory cells), while another (stem cells) is responsible for cell division and the generation of new factory cells. As older factory cells lose productivity, the bioreactor is continuously replenished with new factory cells, derived from the stem cell population. By maintaining an active factory cell population, MiST-supported cultures are expected to exhibit increased production longevity and higher overall yield than conventional bio-fermentations. This project aims to validate the technology in E. coli engineered to produce limonene, a precursor for biodiesel and other useful chemicals. In the factory cells, T7RNAP will drive high-level expression of a suite of biosynthetic enzymes. Since limonene has a cytotoxic effect on producing cells, MiST-supported factory cell replenishment is expected to increase productivity by more than 2-fold compared to the conventional limonene-producing strains.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Industrial bioreactors utilize microbial organisms as living factories to produce a wide range of commercial products through fermentation. This technology spans various sectors: pharmaceutical industries use genetically modified bacteria to produce therapeutics, supplements, and bioactive molecules; the food industry uses microbes for enzymes and additives; and the cosmetic industry utilizes them for active ingredients. Furthermore, microbial production holds immense potential for biofuel production, offering a sustainable alternative to traditional chemical processes.
However, current bio-fermentation technologies face limitations in efficiency and scalability. This inefficiency primarily stems from the metabolic burden and toxicity of fermentation products on the producing cells. To achieve industrially relevant production, a mechanism for replenishing these cells is crucial.
AsimicA has developed the Microbial Stem Cell Technology (MiST) platform to address this challenge. MiST enhances bio-fermentation efficiency through asymmetric cell division. MiST cultures divide asymmetrically, generating a stem cell and a "factory cell" for product synthesis. The stem cell undergoes further asymmetric division, continuously replenishing the factory cell population. This cyclical process leads to continuous production, higher product yields, and increased bioreactor productivity.
This SBIR Phase-I project focused on: 1) developing the MiST platform for industrial-scale production, and 2) demonstrating increased limonene production using MiST. Limonene, used in dietary supplements, cosmetics, solvents, and jet fuel, is toxic to producing organisms, making it an ideal candidate for showcasing MiST's impact on bioreactor yields. Moreover, limonene belongs to the terpenoid family, a broad class of chemicals with diverse industrial applications. The similarity in biosynthesis between limonene and other terpenoids highlights the commercial potential of MiST for AsimicA.
The project involved modifying the MiST genetic circuit for high-level expression in factory cells. Asymmetric cell division and factory cell differentiation were quantified using fluorescence microscopy, confirming over 90% of stem cells undergo continuous asymmetric division and differentiation within 45 minutes. MiST cultures exhibited a significantly higher ratio of actively producing factory cells compared to conventional cultures.
The MiST genetic circuit was incorporated into E. coli, with limonene biosynthesis genes encoded on a plasmid. Produced limonene was recovered and quantified using GC-MS. Results indicated a MiST-mediated increase of over 4-fold in limonene titers compared to controls, and over 30% compared to the best-in-literature system.
These Phase-I results demonstrate MiST's superiority for terpenoid bioproduction. Before commercialization, the remaining tasks include: (a) validating MiST in bench-top bioreactors; (b) expanding the terpenoid portfolio; and (c) scaling up production in 300L bioreactors. Once commercialized, MiST will revolutionize productivity in industries reliant on bio-fermentation.
Last Modified: 02/17/2025
Modified by: Nikolai Mushnikov
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