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.
Bio-based production of chemicals is critical for US economic growth and positively impacts the environment. It will also help to free the US from reliance on foreign oil which improves our energy security and national defense. Production of bio-based chemicals requires the conversion of sugar into downstream chemicals via the action of enzymes within bacterial cells. In order to overproduce a particular chemical it is essential to "turn off" side reactions that would divert the flow of carbon to the target molecule. Traditionally, such off-target enzyme pathways have been completely removed from cells, however in many cases these pathways are essential to cellular growth and thus cannot easily be removed. Ginkgo BioWorks has developed novel methodologies to conditionally regulate the off-pathway enzymes upon an external trigger. This allows for the overproduction of valuable chemicals such as amino acids, flavors, fragrances, pharmaceuticals, and many others. In this Phase 1 SBIR we have demonstrated that our regulated proteolysis technology can operate effectively in a microbe commonly used for industrial fermentation. Additionally, we have demonstrated the effectiveness of the technology for overproduction of an amino acid and a terpene molecule. These are two classes of chemicals with large, preexisting markets with substantial growth potential. Cost savings associated with using out technology for production of these chemicals will hasten the transition to a bio-based economy. Ginkgo BioWorks will continue to develop this technology and has already begun to apply it to a broad range of industrial chemicals through commercial partnerships.