SBIR Phase I: Reversible Introduction of Multiplex Automated Genome Engineering (MAGE) Competence in Yeast

这个小型企业创新研究(SBIR)第一阶段项目是开发一个通用过程，使微生物能够进行快速基因组工程。由于依赖于低效的、连续的DNA修饰，目前的细胞工程技术既昂贵又耗时。多重自动化基因组工程(MAGE)是一种颠覆性技术，允许以显著降低的成本快速设计微生物。MAGE通过在多个位置同时合并合成的寡核苷酸，实现了大规模的高度特异性的基因组修改-类似于大规模的基因组并行重新编程。然而，由于关键的遗传要求，目前MAGE的使用仅限于大肠杆菌。该项目的目标是确定和优化新微生物中MAGE所需的遗传特征的一般过程。作为第一步，该项目将在制造具有MAGE能力的酵母菌株方面取得进展，该菌株目前还不能有效地用于商业应用。此外，由于潜在的工业合作伙伴使用他们自己的菌株进行生产，因此有必要迅速和可逆地赋予现有的酵母菌株经受MAGE的能力。这一过程的成功应用将导致能够在现有的商业酵母菌株中快速和可逆地部署MAGE能力。如果成功，该项目的更广泛的影响/商业潜力将是快速和可逆地引入为特定功能(例如，生产特殊化学品、酶等)的大量微生物种类重新编程的能力。成功地应用这一方法将MAGE-能力引入广泛使用的芽生酵母菌株，将立即带来商业机会--使酵母基因组工程速度更快，成本显著降低。此外，这一过程的展示为在其他高价值的商业酵母中部署MAGE能力铺平了道路，如巴斯德毕赤酵母和克鲁维酵母。该项目将为将MAGE转移到其他酵母菌，并最终转移到其他微生物的通用过程奠定基础。MAGE的引入产生了重写或编辑新基因组的能力，使我们的过程与测序成本的非凡下降和日益丰富的信息学工具协同作用。每一个新的法师能力物种都赋予了以非凡的速度重写、理解和利用序列信息的能力-打开了理解和工程生物学的新机会的大门。

This Small Business Innovation Research (SBIR) Phase I project is to develop a general process to enable microorganisms for rapid genome engineering. Current technologies to engineer cells are expensive and time consuming due to reliance on inefficient, serial modifications of DNA. Multiplex Automated Genome Engineering (MAGE) is a disruptive technology that allows for rapid engineering of microorganisms at substantially reduced cost. MAGE enables large-scale highly specific genome modifications via incorporation of synthetic oligonucleotides at multiple locations simultaneously - akin to massive parallel reprogramming of the genome. However, use of MAGE is currently limited to E. coli due to key genetic requirements. The goal of this project is a general process to identify and optimize the requisite genetic features for MAGE in new microorganisms. As a first step, the project will build on progress in making a MAGE-competent yeast strain, which is not yet efficient for use in commercial applications. Moreover, since potential industrial partners use their own strains for production, it will be necessary to quickly and reversibly endow existing strains of yeast with the capacity to undergo MAGE. The successful application of this process will result in the ability to rapidly and reversibly deploy MAGE-competence in existing commercial yeast strains.The broader impact/commercial potential of this project, if successful, will be the rapid and reversible introduction of the capacity to reprogram numerous species of microorganisms for specific functions (e.g., production of specialty chemicals, enzymes, etc.). The successful application of this process to introduce MAGE-competence to strains of the widely utilized budding yeast, S. cerevisiae, will result in immediate commercial opportunities - making yeast genome engineering faster and significantly less expensive. Additionally, the demonstration of this process paves the way for deployment of MAGE-competence in other high-valued commercial yeasts, such as Pichia pastoris and Kluyveromyces lactis. This project will establish the basis for a generalized process to port MAGE to other yeasts, and ultimately other microorganisms. The introduction of MAGE engenders the ability to rewrite or edit novel genomes, making our process synergistic with the extraordinary decline in sequencing costs and increasing wealth of informatics tools. Each new MAGE-competent species confers the ability to rewrite, understand, and utilize sequence information at an extraordinary pace - opening the door to new opportunities for understanding and engineering biology.