Directed Evolution of Phosphite Dehydrogenase and Engineered Biosynthesis of Fr-900098
Johannes, Tyler William
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https://hdl.handle.net/2142/82408
Description
Title
Directed Evolution of Phosphite Dehydrogenase and Engineered Biosynthesis of Fr-900098
Author(s)
Johannes, Tyler William
Issue Date
2008
Doctoral Committee Chair(s)
Zhao, Huimin
Department of Study
Chemical Engineering
Discipline
Chemical Engineering
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Engineering, Chemical
Language
eng
Abstract
Another area in which biocatalysts are increasingly being used is in the production of small secondary metabolites, particularly antibiotics. Phosphonates are a small but growing class of compounds with many useful therapeutic properties. In particular, the phosphonates fosmidomycin and FR-900098 represent a new class of antimalarial compounds that can be used to inhibit the nonmevalonate pathway for isoprenoid biosynthesis in the malaria-causing parasite Plasmodium falciparum. Although no biosynthetic pathway for fosmidomycin has been elucidated as of yet, the biosynthetic pathway for FR-900098 has been cloned from Streptomyces rubellomurinus and heterologously expressed in Streptomyces lividans. In this work, we have created an E. coli strain capable of producing FR-900098 and have worked to decipher the late steps of FR-900098 biosynthesis. These studies revealed that the late steps of FR-900098 biosynthesis involve a unique bifunctional nucleotide transferase-decarboxylase (FrbH), an N-acetyltransferase (FrbF), a novel amide hydroxylase (FrbG), and a promiscuous nucleotide hydrolase (FrbI). The presence of cytidine 5'-monophosphate (CMP) conjugated phosphonate intermediates in the pathway also gives fascinating new insights into how phosphonate antibiotic pathways evolve to protect the host organism from the effects of toxic intermediates and promiscuous enzymes.
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