Deciphering the Late Biosynthetic Steps of Antimalarial Compound FR-900098

Abstract

FR-900098 is a potent chemotherapeutic agent for the treatment of malaria. Here we report the heterologous production of this compound in E. coli by re-constructing the entire biosynthetic pathway using a three plasmid system. Based on this system, whole cell feeding assays in combination with in vitro enzymatic activity assays reveal an unprecedented functional role of nucleotide conjugation and lead to the complete elucidation of the previously unassigned late biosynthetic steps. These studies also suggest a biosynthetic route to a second phosphonate antibiotic, FR-33289. A thorough understanding of the FR-900098 biosynthetic pathway now opens the possibilities for metabolic engineering in E. coli to increase production of the antimalarial antibiotic and combinatorial biosynthesis to generate novel derivatives of FR-900098.

Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

“…Conjugation of nucleotides to pathway intermediates is also found in the biosynthesis of FR-900098 and phosphinothricin. In both cases, a nucleotidyltransferase transfers a CMP moiety from CTP to one of the phosphonate oxygen atoms of a biosynthetic intermediate about halfway into the overall pathway (4,27). We heterologously expressed Psf7 in E. coli and purified the protein but to date have not been able to detect any activity using a series of nucleotides and potential phosphonate substrates (fosfomycin, PnPy, Pmm, 2-oxopropionylphosphonate).…”

New N-Acetyltransferase Fold in the Structure and Mechanism of the Phosphonate Biosynthetic Enzyme FrbF

“…Although significant research efforts have focused on identifying synthetic derivatives of these compounds with improved efficacy, such compounds are cost-prohibitive (11,33,34). We have recently characterized the biosynthetic pathway for FR-900098 and have reconstituted the pathway through heterologous expression of the biosynthetic genes in E. coli, opening new avenues for the production of derivative compounds by genetic manipulation (12,13). Such efforts will be aided by detailed biochemical and structural studies of the constituent enzymes in this pathway.…”

“…Protein Expression and Purification-The construction of an expression vector for the heterologous production of hexahistidine-tagged FrbF in E. coli has been described previously (13). E. coli expression strain BL21(DE3) was transformed with this expression vector, and single colonies of transformed E. coli were used to inoculate 5 ml of LB medium supplemented with kanamycin (50 g/ml).…”

“…More recently, the results of our whole cell feeding studies and cluster deletion analysis suggest that the enzyme FrbF catalyzes the acetyl transfer onto the hydroxylamine of the FR-900098 precursor N-hydroxy-3-aminopropylphosphonate (H3APn) 4 ( Fig. 1A, Compound 3) to generate the final FR-900098 product (13).…”

Expression and characterization of an <i>N</i>-oxygenase from <i>Rhodococcus jostii</i> RHAI

“…These enzymes were initially characterized from S. thioluteus, in the case of AurF, and Pseudomonas fluorescens, in the case of PrnD. More recently, other putative N-oxygenases have been characterized, FrbG (Johannes et al, 2010), CmlI (Lu et al, 2012;Makris et al, 2010) and PsAAO (Platter et al, 2011). The few enzymes characterized thus far have demonstrated limited substrate specificities with a preference for paminobenzoates and o-aminophenols (Platter et al, 2011;Winkler et al, 2006).…”