A phosphonate-induced gene which promotes Penicillium-mediate bioconversion of cis-propenylphosphonic acid to fosfomycin

Penicillium decumbens is able to epoxidize cis-propenylphosphonic acid (cPA ) to produce the antibiotic fosfomycin [FOM; also referred to as phosphonom ycin and (-)-cis-1,2-epoxypropylphosphonic acid], a bioconversion of consid erable commercial significance. We sought to improve the efficiency of the process by overexpression of the genes involved. A conventional approach of isolating the presumed epoxidase and its corresponding gene was not possib le since cPA epoxidation could not be achieved with protein extracts. As an alternative approach, proteins induced by cPA were detected by two-dimensi onal gel electrophoresis. The observation that a 31-kDa protein (EpoA) was both cPA induced and overaccumulated in a strain which more efficiently con verted cPA suggested that it might fake part in the bioconversion, EpoA was purified, its amino acid sequence was Partially determined, and the corres ponding gene was isolated from cosmid and cDNA libraries with oligonucleoti de probes. The DNA sequence for this gene (epoA) contained two introns and an open, reading frame encoding a peptide of 277 amino acids having some si milarity to oxygenases, When the gene was subcloned into P. decumbens, a fo urfold increase in epoxidation activity was achieved, epoA-disruption mutan ts which were obtained by homologous recombination could not convert cPA to FOM. To investigate the regulation of the epoA promoter, the bialaphos res istance gene (bar, encoding phosphinothricin acetyltransferase) was used to replace the epoA-coding region. In P. decumbens, expression of the bar rep orter gene was induced by cPA, FOM, and phosphorous acid but not by phospho ric acid.