Cloning, Expression, and Biochemical Characterization of Streptomyces rubellomurinus Genes Required for Biosynthesis of Antimalarial Compound FR900098

Abstract

The antibiotics fosmidomycin and FR900098 are members of a unique class of phosphonic acid natural products that inhibit the nonmevalonate pathway for isoprenoid biosynthesis. Both are potent anti-bacterial and anti-malarial compounds, but despite their efficacy, little is known regarding their biosynthesis. Here we report the identification of the Streptomyces rubellomurinus genes required for the biosynthesis of FR900098. Expression of these genes in Streptomyces lividans results in production of FR900098, demonstrating their role in synthesis of the antibiotic. Analysis of the putative gene products suggests that FR900098 is synthesized by metabolic reactions analogous to portions of the tricarboxylic acid cycle. These data greatly expand our knowledge of phosphonate biosynthesis and enable efforts to overproduce this highly useful therapeutic agent.

Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

“…The medium was supplemented with 10 mM fumarate as an electron donor and carbon source and 10 mM sulfate as an electron acceptor. The genomic DNA library from D. phosphitoxidans chromosomal DNA was constructed in a dual-cos fosmid, pJK050 (3,6), and transformed into a derivate strain of E. coli DH10B-WM3118. The pAE5 plasmid carries a mini-Mu transposon (mini-MuAE5) that was used for DNA sequencing.…”

“…The screening reaction mix consisted of 1 l LB-grown cell culture, 500 nM each primer, and Taq polymerase in Failsafe buffer G (Epicentre, Madison, WI) at an annealing temperature of 60°C. Two positive clones, pJK1043 and pJK1044, were chosen for DNA sequencing using pAE5 (mini-MuAE5) transposon insertions as mobile priming sites (6). Transposition reactions of BglII-digested pAE5 and either pJK1043 or pJK1044 were conducted in vitro using MuA transposase (MJ Research, Waltham, MA) per the manufacturer's instructions.…”

Discovery of phosphonic acid natural products by mining the genomes of 10,000 actinomycetes

“…NRRL S-474, and Streptomyces sp. NRRL S-481, were of particular interest because they elicited positive responses in a phosphonate-specific bioassay (18) and because the genomic information illustrated that their biosynthetic gene clusters were novel. Argolaphos A and B, Broad-Spectrum Antibacterial Phosphonopeptides.…”

“…acute cystitis; FR-900098 and fosmidomycin, antimicrobials undergoing clinical trials for malaria; and phosphinothricin, the active component in several commercial herbicides (Liberty, Basta, and Rely). Second, the methodology needed for gene-based discovery of phosphonate biosynthetic loci has been rigorously established (17)(18)(19)(20)(21)(22)(23). This method relies on the fact that all but two characterized phosphonate biosynthetic pathways begin with the enzyme phosphoenolpyruvate (PEP) mutase (encoded by pepM) (14).…”

Purification and Characterization of Phosphonoglycans from Glycomyces sp. Strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338

“…Plasmids and fosmids were isolated using Qiagen (Valencia, CA) miniprep or maxiprep kits. The genomic DNA from Glycomyces and Stackebrandtia strains, extracted using UltraClean microbial DNA isolation kit (MO BIO Laboratories, Carlsbad, CA), was used as the template for PCR amplification of a 406-bp pepM fragment with degenerate primers as described previously (21). PCR amplifications were performed with GoTaq Green master mix (Promega).…”