Aromatic Biosynthesis in Archaeoglobus fulgidus

H.G. Monbouquette and J.C. Liao, Chemical Engineering, I. Schröder, Microbiology & Molecular Genetics, UCLA, Los Angeles, CA 90095

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 focused on the coordinated use of LC/MS-based enzyme assays, DNA microarrays, and gene cloning and expression for screening of enzyme activities and for identification of genes in hypothesized metabolic pathways.

The following has been accomplished in this project to date: (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, were sequenced and soluble products were expressed for most, (2) a putative gene for a novel bifunctional phosphoribosyl (PRA) anthranilate transferase/indoleglycerol phosphate (IGP) synthase was found to be two separate genes, (3) a putative trifunctional chorismate mutase/prephenate dehydratase/prephenate dehydrogenase gene was confirmed using LC/MS-based assays to be the first triple activity fusion of its kind in a single polypeptide, (4) over-expressed shikimate dehydrogenase was purified and fully characterized, (5) a method for determining 95% confidence intervals for DNA microarray data was developed, (6) a full-genome DNA microarray for A. fulgidus was created (the first for an archaeon), (7) the presence of a trp operon was confirmed using the DNA microarray.

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