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

这个小企业创新研究（SBIR）第一阶段项目是开发一个通用的过程，使微生物能够快速基因组工程。由于依赖于低效的DNA序列修饰，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）是一项颠覆性技术，它允许以大幅降低的成本快速工程微生物。MAGE通过同时在多个位置结合合成寡核苷酸实现大规模的高度特异性基因组修饰-类似于基因组的大规模并行重编程。然而，由于关键的遗传要求，MAGE的使用目前仅限于大肠杆菌。该项目的目标是鉴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，该项目将建立在制造一种具有mage能力的酵母菌株的进展上，这种酵母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酵母菌株进行MAGE的能力。该工艺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。这个项目的更广泛的影响/商业潜力，如果成功的话，将是快速和可逆地引入为特定功能（例如，生产特种化学品，酶等）重新编程多种微生物的能力。该工艺的成功应用将mage能力引入到广泛使用的出芽酵母（酿酒酵母）的菌株中，将带来立即的商业机会-使酵母基因组工程更快，成本显著降低。此外，该过程的演示为在其他高价值的商业酵母（如毕赤酵母和克卢维酵母菌）中部署mage能力铺平了道路。该项目将为将MAGE移植到其他酵母并最终移植到其他微生物的通用过程奠定基础。MAGE的引入带来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显著下降和信息学工具的日益丰富产生协同作用。每一个新的具有mage能力的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——为理解和工程生物学打开了新的机会之门。

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.