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，使微生物快速基因组工程。由于依赖于低效的DNA连续修饰，目前的细胞工程技术既昂贵又耗时。多重自动基因组工程（法师）是一种破坏性技术，其允许以显著降低的成本快速工程化微生物。法师能够通过在多个位置同时掺入合成寡核苷酸来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，法师的使用目前仅限于E.大肠杆菌，由于关键的遗传要求。本项目的目标是一个通用的过程，以确定和优化所需的遗传特征，法师在新的微生物。作为第一步，该项目将建立在制造MAGE活性酵母菌株的进展基础上，该菌株尚未有效用于商业应用。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，因此有必要快速且可逆地赋予现有的酵母菌株经历法师的能力。该方法的成功应用将导致在现有的商业酵母菌株中快速和可逆地部署MAGE-感受态的能力。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重新编程许多微生物物种以实现特定功能的能力（例如，特种化学品、酶等的生产）。 该方法成功地将MAGE感受态导入到广泛应用的芽殖酵母S。酿酒酵母，将导致直接的商业机会-使酵母基因组工程更快，显着降低成本。此外，该过程的演示为在其他高价值的商业酵母（如巴斯德毕赤酵母和乳酸克鲁维酵母）中部署MAGE能力铺平了道路。该项目将为将法师移植到其他酵母菌并最终移植到其他微生物的通用过程奠定基础。 法师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信息学工具的日益丰富协同。 每一个新的有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.