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.
Current technologies to engineer cells for specific functions (e.g., chemical or fuel production) are inefficient, expensive and extremely time consuming due to reliance on decades-old recombinant DNA technologies. Multiplex Automated Genome Engineering (MAGE) is a disruptive technology that provides a powerful platform to engineer microorganisms at tremendously reduced cost. MAGE enables large-scale genome modifications at multiple genomic locations simultaneously – akin to massive parallel reprogramming of the genome. Until recently, MAGE was limited to a laboratory strain of E. coli, possessing key genetic features that enable MAGE. Recent advances have allowed us to port MAGE to yeast, an important commercial organism for the production of biofuels, enzymes, and a variety of specialty chemicals. However there is significant room for increases in efficiency. Furthermore, since potential industrial partners use their own strains for production, it is necessary to endow existing strains of yeast with the capacity to undergo MAGE. This Small Business Innovation Research project aims to develop a process to identify and optimize the requisite genetic features for MAGE in yeast, a process we call xMAGE. To establish feasibility of our system design in Phase I, we constructed and tested an initial xMAGE system, which allows us to vary protocol parameters and alter genetic components in our yeast strains. We created and tested an initial library of yeast strains for introduction into the xMAGE system, each with varying components thought to have an impact on the efficiency of MAGE in yeast. We found marked differences in the efficiency with which these strains were able to perform MAGE, with some showing promise for use in commercial projects. This demonstrated the great potential in screening a larger library with this system in Phase II. We also identified potential areas in which our yeast protocol can be improved to make it more robust and scalable. When Phase II of this Small Business Innovation Research project is complete, enEvolv will have the ability to efficiently introduce the capacity to reprogram or optimize industrial strains of yeast for the production of fuels, chemicals, and other valuable commercial molecules. The successful application of the xMAGE system offers immediate commercial, government, and academic applications – making whole genome engineering of virtually any yeast strain orders of magnitude faster and less expensive. Additionally, demonstration of this process paves the way for rapid introduction of MAGE-competence in other high-valued commercial yeasts. This project will establish the basis for a generalized process to port MAGE to other species of industrial yeast, and 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 and utilize new sequence information at an extraordinary pace. Thus, each new MAGE-competent species opens a door to understanding and engineering biology in a way that was not previously possible.
