Characterizing a serendipitous pathway in E. coli that can overcome a block in pyridoxal-5'-phosphate synthesis

Haddox, Hugh

Undergraduate Honors Thesis

Characterizing a serendipitous pathway in E. coli that can overcome a block in pyridoxal-5'-phosphate synthesis Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/hq37vp03h

Abstract

Physiologically irrelevant metabolic pathways patched together from the reactions of multiple promiscuous enzymes can serve as a starting points for the evolution of novel pathways that degrade or generate new or pre‐existing metabolites. It has been shown that at least three such ‘serendipitous’ pathways in E. coli can be evolved to over‐come a block in the synthesis of pyridoxal‐5’‐phosphate (PLP). These pathways restore PLP synthesis by converting metabolites from core metabolism to intermediates in PLP synthesis that are downstream of the block, and can be elevated to a physiologically relevant level by the over‐production of certain enzymes of the pathway. Over‐production of HisB, Php, or YjbQ complements a strain of E. coli with a deletion in pdxB, which encodes an enzyme that is essential in PLP biosynthesis, by elevating flux through one or more serendipitous pathways that restore PLP synthesis by producing a novel source of 2‐oxo‐3‐ hydroxy‐4‐phosphobutanoate—an intermediate in PLP biosynthesis downstream of the step catalyzed by pdxB. The aim of this honors thesis was to further characterize the serendipitous pathway(s) involved in this process. First, this thesis indicated that HisB, Php, and YjbQ operate in the same serendipitous pathway. Although HisB, Php, and YjbQ are not epistatic, since over‐production of each enzyme still complements a deletion of pdxB when genes encoding the other two enzymes are deleted, complementation by each enzyme is inhibited by a single metabolite, indicating that HisB, Php, and YjbQ participate in a single serendipitous pathway that is down‐regulated by this metabolite. Second, I determined which catalytic domain of the bi‐functional HisB enzyme catalyzes the promiscuous activity in the serendipitous pathway that restores PLP synthesis upon HisB overproduction. When the two catalytic domains of HisB were individually over‐produced, only the imidazoleglycerol‐phosphate dehydratase domain complemented a deletion in pdxB. Moreover, mutating active site residues from this domain either partially or completely interfered with complementation by over‐production of the full length HisB protein. Lastly, I described a project that I am currently completing, which uses genome‐wide transposon mutagenesis to search for enzymes that participate in the same serendipitous pathway as HisB in rescuing PLP synthesis. I screened an insertion library for cells that could no longer synthesize PLP using this serendipitous pathway and am in the process of locating transposons in the genomic DNA of select insertion mutants.

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