Sunday, May 4, 2008
2-24

Chorismate superpathway mediated 3-hydroxypropinic acid growth inhibition of E. coli

Tanya Warnecke1, Michael D. Lynch2, Anis Karimpour-Fard1, and Ryan T. Gill1. (1) Department of Chemical and Biological Engineering, University of Colorado, 1111 Engineering Drive, UCB 424, Boulder, CO 80309, (2) OPX Biotechnologies, Inc., 5541 Central Ave., Suite 270, Boulder, CO 80301

We have previously developed Scalar Analysis of Library Enrichment (SCALEs), a new high-resolution, genome-wide approach that can be used to monitor enrichment and dilution of individual clones within a genomic-library population. We have employed this method in the selection of tolerance phenotypes specific to a commercially relevant chemical, 3-hydroxypropionate. Biosynthetic processes yielding 3-HP have previously been demonstrated from development of recombinant host.  However, severe growth inhibition has been observed for extracellular acid levels as low as 10 g/L in minimal media (pH 7.0), which limits the economic feasibility of 3-HP production and points to the importance of 3-HP tolerance in a successful biological strategy in 3-HP production. The genome-wide, high resolution data generated by the SCALEs method has allowed for the diagnosis of several key 3-HP toxicity mechanisms, one being the limitation of intermediates in the chorismate metabolic superpathway. Chorismate is the common precursor to a number of aromatic amino acids (tyrosine, phenylalanine, and tryptophan) and vitamins (folate, ubiquinone, and meniquinone) required for cell viability.  Addition of each downstream product from chorismate shows partial regeneration of specific growth and final cell density.  Similarly, addition of shikimate an intermediate in this pathway leads to a 20% regeneration of growth compared with wild-type growth, indicating that inhibition occurs prior to the formation of shikimate.  Finally, clones overexpressing a feedback resistant form of the aroH gene active in the first step of chorismate synthesis show a significant increase in growth rate and demonstrate a viable genetic route to address this 3-HP toxicity mechanism.

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