Retrobiosynthesis of D-glucaric acid in a metabolically engineered strain of Escherichia coli

[摘要] (cont.) This colocalization led to enhancement of MIOX activity with concomitant productivity improvement, achieving 2.7 g/L of D-glucaric acid production from 10 g/L of D-glucose input. Secondly, retrobiosynthetic approach, a product-targeted design of biological pathways, has been proposed as an alternative strategy to exploit the diversity of enzymecatalyzed reactions. The first step in a glucaric acid pathway designed retrosynthetically involves oxidation of the C-6 hydroxyl group on glucose, but no glucose oxidase in nature has been found to act on this hydroxyl group on glucose. To create glucose 6- oxidase, a computational design and experimental selection was combined with the help of DNA synthesis technology. To this end, the sequence space of candidate mutations was computationally searched, the selected sequences were combinatorially assembled, and the created library was experimentally screened and characterized. Furthermore, the structure-activity relationship of the newly created glucose oxidases was elucidated, and the kinetic model mechanism for these mutants was proposed and analyzed. Collectively, parts, devices, and chassis engineering were applied to a synthetic pathway for the production of D-glucaric acid, and this synthetic biology approach was proven to be effective for new pathway design and improvement.

[发布机构] Massachusetts Institute of Technology