(484g) Biosynthesis of Fosfomycin

Phosphonic acids are a group of C-P bond containing natural products mainly produced by Streptomyces. Many of them show important biological activities such as antibacterial, antiviral, pesticidal, and anti-cancer properties. For example, fosfomycin is the only FDA approved drug for treatment of acute cystitis during pregnancy while fosmidomycin and FR900098 are potent antimalarial agents. However, very few biosynthetic pathways of phosphonic acids have been cloned, heterologously expressed, and biochemically characterized. Here we attempt to clone the fosfomycin gene clusters from Streptomyces wedmorensis and Streptomyces fradiae, and use Streptomyces lividans and E. coli as the heterologous producers. The fosfomycin gene cluster from Streptomyces wedmorensis has been studied over two decades, however, fosfomycin has never been produced in a heterologous host and its biosynthetic mechanism is not completely understood. We created fosmid libraries of genomic DNA from Streptomyces wedmorensis and Streptomyces fradiae, respectively, and screened them for the presence of the fosfomycin gene cluster using PCR with the primers specific to the known sequences in the gene cluster. We then transferred the positive fosmids containing the potential fosfomycin gene cluster into Streptomyces lividans and screened for fosfomycin production using a biological assay. One fosmid FM45 was found to enable the host to produce fosfomycin, which was further confirmed by enzyme inhibition assay and NMR study. Its insert was subsequently DNA sequenced. Gene knockout and deletion experiments showed that a previously unknown gene, orfC plays a crucial role for the formation of fosfomycin. Detailed characterization of the enzyme encoded by orfC showed that it catalyzes a NADPH-dependent dehydrogenation step, which occurs between the decarboxylation step and the methyl transfer step. In addition, phnDCE genes encoding an ABC type transporter were identified from the cluster, which might assist the transport of phosphonates between the organisms and the environment. Heterologous expression of the fosfomycin biosynthetic pathway in E. coli and engineering of Streptomyces lividans and E. coli for cost-effective production of fosfomycin are still in progress.