Aromatic Amino Acid Biosynthesis in Archaeoglobus fulgidus
H.G. Monbouquette
University of California Los Angeles

The aromatic amino acid synthesis pathway has been engineered successfully for the synthesis of natural and unnatural chiral amino acids, which are important drug intermediates, as well as other industrially important aromatics, such as indigo.  Production of aromatics via engineered microbes offers both environmental and economic advantages including exclusive use of aqueous solvent and non-toxic intermediates, and lower raw material cost.  Intense interest therefore has developed in the enzymes of these metabolic pathways.  A. fulgidus is representative of the third, most primitive domain of life, and the aromatic amino acid synthesis pathways have not been explored in these microorganisms despite the fact that they may offer a far more robust set of biosynthetic enzymes well suited both for in vivo and in vitro synthesis applications.  Recently, the entire genome of A. fulgidus was sequenced and a thorough study of open reading frames for sequences homologous to known enzymes was conducted.  It is noteworthy that a number of enzymes involved in common aromatic amino acid synthesis routes were not identified on the genome.  Our goal is to identify these new enzymes/pathways by a functional proteomics approach made possible by our demonstrated ability to culture A. fulgidus to the 100-liter scale, and to identify, isolate, sequence, clone and express (in E. coli) new enzymes from this microbe.  This project will establish a functional proteomics approach involving coordinated use of high-throughput LC/MS-based enzyme assays, DNA microarrays, and gene cloning and expression for fast screening of enzyme activities and for identification of genes in hypothesized metabolic pathways.

The following was accomplished in the first year of this project: (1) the 15 A. fulgidus open reading frames (ORFs) homologous to known genes in the aromatic amino acid synthesis pathways were cloned in E. coli and were sequenced, (2) a putative gene for a novel bifunctional phosphoribosyl (PRA) anthranilate transferase/indoleglycerol phosphate (IGP) synthase was found to be two separate genes, (3) prephenate dehdrogenase activity was confirmed for the over-expressed product of a putative trifunctional chorismate mutase/prephenate dehydratase/prephenate dehydrogenase gene, (4) over-expressed shikimate dehydrogenase was purified and partially characterized, and (5) a method for determining 95% confidence intervals for DNA microarray data was developed.  Of the 15 cloned ORFs, nine were over-expressed as soluble products.  An effort to obtain soluble products of the remaining genes and to characterize the recombinant enzymes is continuing.  A preliminary characterization of the recombinant shikimate dehydrogenase was conducted.  The enzyme exhibits similar kinetics to the E. coli enzyme, albeit at a temperature optimum of ~90 °C.  The prephenate dehydrogenase activity of the putative trifunctional enzyme suggests that this may indeed be a novel fusion of catalytic functions, although chorismate mutase and prephenate dehydratase activity has not been confirmed.  Work is ongoing to develop LC/MS as a tool for high throughput enzyme assays and to refine the DNA microarray technique such that LC/MS and DNA microarrays may be used in complementary fashion to identify new enzymes and metabolic pathways.  This approach will be used in the second year of the grant to identify the novel enzyme(s) catalyzing the first two steps in the shikimate pathway as well as the phosphorylation of shikimate.

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