275963 Bacteria Engineered to Produce the Chiral Building Block (S)-Styrene Oxide From Renewable Feedstocks

Monday, October 29, 2012: 9:24 AM
Cambria East (Westin )
Rebekah McKenna1, Shawn Pugh1, Matthew Sawtelle2 and David R. Nielsen1, (1)Chemical Engineering, Arizona State University, Tempe, AZ, (2)Chemical Engineering, Arizona State University

Aromatic compounds represent a diverse class of fine and commodity chemicals with important commercial applications ranging from their use as molecular building blocks, flavor agents, and monomers. Today, conventional chemocatalytic synthesis routes for all aromatic compounds rely upon petroleum-derived BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds as feedstock.  However, through de novo pathway engineering, our lab has been exploring the ‘bottom up’ development of microbial biocatalysts to produce a number of useful aromatics renewable feedstocks.  One such example is the chiral building block (S)-styrene oxide whose conventional applications include as a reactive plasticizer and as a chemical intermediate for cosmetics, surface coatings, and agricultural and biological chemicals.  Enantiopure (S)-Styrene oxide production from glucose was accomplished by the co-expression of styrene monooxygenase (SMO) in a previously engineered styrene producing strain of Escherichia coli.  SMO from 3 strains of styrene-degrading Pseudomonas was assayed for function and performance.  Ultimately, styAB from P. putida S12 was found to display the greatest inherent activity.  Expressing styAB with PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae in a phenylalanine over-producing strain of E. coli, initial titers of more than (S)-styrene oxide titers exceeded 1 g/L in shake flask cultures.  Metabolite flux has been improved and balanced through modulation of pathway enzyme expression, including PAL2 the initial pathway bottleneck.  This was achieved through the systematic tuning of each of gene copy number, promoter strength, and RBS strength. As a result, current titers now approach the toxic threshold of (S)-styrene oxide, determined as ~1.6 g/L.  The (S)-styrene oxide pathway has most recently been translated into the aromatic tolerant host P. putida S12 upon its engineering to: i) over-produce phenylalanine and ii) eliminate its natural ability to degrade (S)-styrene oxide.  The resultant strain has been characterized with regards to its ability to produce (S)-styrene oxide from different renewable substrates and survive in presence of elevated product titers.

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