SBIR Phase I: Novel Proteolysis-based Tools for Metabolic Engineering of Amino Acid Producing Strains

这个小企业创新研究（SBIR）第一阶段项目旨在设计微生物，以经济有效地生产氨基酸l -苏氨酸。目前，工程微生物承受突变，通过抑制细胞来增加感兴趣的苏氨酸的产生。S产生其他氨基酸的能力。这些突变是至关重要的，因为它们有效地引导细胞？苏氨酸的代谢通量，从而提高生产效率和简化下游净化。不幸的是，这些突变也会降低细胞适应性，因此，生长培养基必须补充昂贵的营养物质。本文的技术研究将评估在没有昂贵培养基补充的情况下，应用新型调节蛋白水解技术直接代谢通量生产苏氨酸的可行性。该项目有3个主要目标：1)产生含有非通路代谢酶的大肠杆菌菌株，通过生长阶段依赖的蛋白水解系统标记降解；2)测试这些菌株在无添加剂培养基上生长的能力；3)检测这些工程菌株生产苏氨酸的能力。我们预计我们的工程菌株将在最小的，无补充的培养基上健壮地生长。在诱导我们的蛋白水解系统后，我们希望我们的菌株能够特异性地消除脱靶代谢途径，从而导致我们的目标产品苏氨酸的产量大幅增加。该项目更广泛的影响/商业潜力是产生更具成本效益的l -苏氨酸生产微生物菌株。据估计，到2013年，纯化氨基酸将构成13亿美元的美国市场。这些化学物质被用作动物饲料补充剂、人工甜味剂阿斯巴甜生产的前体，并有潜力作为生物燃料的前体。目前，利用高度工程化的微生物将低成本的糖源（如葡萄糖）转化为最终的氨基酸产品，商业化地生产关键氨基酸。为了提高转化效率和便于下游净化，微生物？S合成其他非通路氨基酸的能力经常被消除。然而，由于这些其他氨基酸对细菌生长至关重要，它们必须作为补充添加到生长培养基中，这大大增加了生产成本。这里提出的技术将允许高效、稳健地生产易于纯化的氨基酸，而不需要培养基补充，大大降低了生产成本。此外，本文开发的调节降解技术将为其他工业代谢工程应用提供下一代调节工具。

This Small Business Innovation Research (SBIR) Phase I project aims to engineer microbes for the cost-effective production of the amino acid, L-Threonine. Currently, engineered microbes bear mutations that increase the production of Threonine of interest by inhibiting the cell?s ability to produce other amino acids. These mutations are critical as they effectively channel the cell?s metabolic flux toward Threonine, thereby boosting production efficiency and easing downstream purification. Unfortunately, these mutations also decrease cellular fitness and, thus, the growth media must be supplemented with costly nutrients. Technical research herein will assess the feasibility of applying novel regulated proteolysis technology to direct metabolic flux toward Threonine production in the absence of costly media supplementation. The project has 3 key objectives: 1) generate E. coli strains containing off-pathway metabolic enzymes tagged for degradation by a growth-phase dependent proteolysis system, 2) test the ability of these strains to grow on supplement-free media, and 3) assay for production of Threonine by these engineered strains. We anticipate that our engineered strains will grow robustly on minimal, un-supplemented media. Upon induction of our proteolysis system, we expect our strains to specifically eliminate off-target metabolic pathways, leading to a substantial increase in production of our target product, Threonine.The broader impact/commercial potential of this project is the generation of more cost-efficient L-Threonine producing microbial strains. Purified amino acids are estimated to constitute a U.S. market of $1.30 billion by 2013. These chemicals are used as animal feedstock supplements, precursors in production of the artificial sweetener aspartame, and have potential as biofuel precursors. Currently, key amino acids are produced commercially using highly engineered microbes that convert low-cost sugar sources (e.g. glucose) to the final amino acid product. To improve the conversion efficiency and ease downstream purification, the microbe?s ability to synthesize other, off-pathway amino acids is often eliminated. However, because these other amino acids are critical for bacterial growth, they must be added as a supplement to the growth medium, substantially increasing production costs. The technology proposed here would allow for efficient, robust production of easily purified amino acids without the need for media supplementation, dramatically reducing production costs. Moreover, the regulated degradation technology developed herein will provide next-generation regulatory tools for other industrial metabolic engineering applications.