Retro-Biosynthesis for the Microbial Production of Organic Compounds.

摘要

This project was concerned with the development of a methodology for the specification of novel biosynthetic pathways towards organic compounds. Our overall objective is to expand the potential for biological production of small molecules, especially for compounds that have either unknown or intractable natural routes. As a model compound, we chose to design and assemble pathways for the production of glucaric acid, a 'top value-added compound from biomass' that has a fully elucidated but very lengthy biological route. As an alternative to the natural pathway, we designed 5 potential routes to glucaric acid and chose two to implement in a recombinant Escherichia coli host. During the granting period, we successfully assembled one pathway, the so-called 'benchmark pathway,' that resulted in the production of glucaric acid at over 1 g/L. Pathway assembly required the isolation of a gene encoding uronate dehydrogenase from the bacterium Pseudomonas putida. In order to improve flux through the pathway, we initiated a collaboration with investigators at the University of California, Berkeley, to employ novel enzyme co-localization techniques. A second collaboration was initiated to perform computation-based enzyme engineering for the second pathway. Finally, a publicly-available database of enzymatic transformations (www.retro-biosynthesis.com) was created to aid future pathway designs.