SBIR Phase I: Boosting Industrial Bio-Fermentation with Microbial Stem Cells

这个小企业创新研究（SBIR）第一阶段项目的更广泛影响是用一种新的平台技术重新构想生物制造，这种技术可以提高许多产品的产量，包括食品添加剂，生物材料前体，生物燃料和药品。技术进步解决了限制传统生物发酵的一个根本问题，即生产细胞遭受有限的健康和活力以换取更高的产量。在这项提议中，遗传工具将被用来将细胞繁殖和产品合成的劳动分为两种不同的细胞类型，称为干细胞和工厂细胞。当旧的工厂细胞耗尽时，生产力由新的工厂细胞维持，这些细胞从干细胞群中产生。这种方法可能特别适用于生物燃料和其他难以通过传统生物发酵大量生产的分子，因为产品对生产它的细胞有毒。它可以用于增加现有生物过程的盈利能力，也可以用于将新产品推向市场，目前很难生产。在这个项目中，该团队试图证明生产燃料（柠檬烯）和乳制品酶（凝乳酶）的好处，以证明其在生物燃料和农业部门的应用。广泛的工业实施将推动生物制造走向“绿色”革命，有助于清洁工业的发展，减少美国和全球对化石燃料的依赖。这几乎是该行业普遍存在的问题。由于缺乏代谢资源、产物的细胞毒性作用和破坏生物合成途径的突变，高产细胞可能变得无活性。在这个项目中，微生物干细胞技术（MiST）通过建立一个多细胞系统来分离生长和生产。一种细胞类型专门用于产品合成（工厂细胞），而另一种（干细胞）负责细胞分裂和新工厂细胞的产生。随着较老的工厂细胞失去生产力，生物反应器不断补充来自干细胞群的新工厂细胞。通过保持活跃的工厂细胞群，MIST支持的培养物预计将表现出比常规生物发酵更长的生产寿命和更高的总产率。本项目旨在验证该技术在E.大肠杆菌被改造以生产柠檬烯，柠檬烯是生物柴油和其他有用化学品的前体。在工厂细胞中，T7RNAP将驱动一系列生物合成酶的高水平表达。由于柠檬烯对生产细胞具有细胞毒性作用，因此与传统的柠檬烯生产菌株相比，MIST支持的工厂细胞补充有望提高生产率2倍以上。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.