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Title:Microbial synthesis of antimalarial compound FR-900098: pathway characterization and engineering
Author(s):Desieno, Matthew
Director of Research:Zhao, Huimin
Doctoral Committee Member(s):van der Donk, Wilfred A.; Leckband, Deborah E.; Schroeder, Charles M.
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
metabolic engineering
Abstract:Phosphonates represent a small, but potent group of compounds with many useful therapeutic properties. FR-900098 is a novel chemotherapeutic agent for the treatment of malaria and has been effective in humans and other animals. This compound inhibits 1-deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase, the second step in the nonmevalonate pathway for isoprenoid biosynthesis for many organisms including Plasmodium falciparum, the parasite responsible for the most virulent form of the disease. The biosynthetic cluster for FR-900098 has recently been cloned from Streptomyces rubellomurinus and heterologously expressed in Streptomyces lividans and Escherichia coli. In this work, we sought to obtain complete elucidation of the FR-900098 biosynthetic pathway. Detailed biochemical studies with purified enzymes were used to verify each of the reactions in the antimalarial biosynthesis. In doing so, we revealed an unprecedented functional role for nucleotide conjugation to intermediates within the pathway. We also describe the characterization of one of the enzymes involved in FR-900098 biosynthesis, the FAD and NADPH dependent N-hydroxylase FrbG. A series of biochemical analyses and the solved crystal structure provided insights into the catalytic mechanism, including nucleotide conjugation as a recognition mechanism for FrbG activity. A thorough understanding of the FR-900098 biosynthetic pathway also provided a platform for directed biosynthesis to generate novel derivatives of the compound and metabolic engineering in E. coli to increase production of the antimalarial. Although not required for FR-900098 biosynthesis, the Fe(II) and α-ketoglutarate dependent hydroxylase FrbJ was determined to be involved in the synthesis of another phosphonate antibiotic FR-33289. We demonstrated its ability to catalyze the hydroxylation of additional substrates as part of the creation of a new library of potential antimalarial compounds. Through a series of bioassays and in vitro experiments, we identified two new prospective antimalarials. Metabolic engineering to increase FR-900098 production in E. coli was approached in two ways, the first of which was the targeted increase of phosphoenolpyruvate (PEP) concentration within the cell. This metabolite is a common precursor of phosphonates and is also involved in several reactions of primary metabolism. The second approach was to balance flux through the FR-900098 biosynthetic pathway through ribosome binding site (rbs) engineering.
Issue Date:2012-02-06
Rights Information:Copyright 2011 Matthew Desieno
Date Available in IDEALS:2012-02-06
Date Deposited:2011-12

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