The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus

Abstract

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Sfreptomyces uiridochromogenes genome. The S. uiridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. Zittidans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in frans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. uiridochromogenes bup genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. uiridochrornogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736

“…hygroscopicus cluster further substantiate previous mapping efforts that predicted the architecture of the two gene clusters were identical (13). The amino acid identity of S. viridochromogenes genes compared to published S. hygroscopicus homologs range from very highly conserved at 95.5% for the bcpA/phpI homolog pair from the core region of the PTT cluster to 63.9% for the phpR/brpA homologs located at the far downstream flank of the gene cluster.…”

“…The S. viridochromogenes genes and enzymes responsible for the initiation of phosphinothricin biosynthesis and production of phosphonoacetaldehyde (steps I and II), involving genes ppm and ppd, have been described previously (17,32,42) and were readily identified in our sequence; however, relatively little is known about the next two steps, the conversion of phosphonoacetaldehyde to hydroxymethylphosphonate (step III) and the subsequent oxidation of hydroxymethylphosphonate to phosphonoformate (step IV). S. hygroscopicus mutants blocked at both steps in the pathway could be complemented in trans by an unsequenced region of DNA closely linked to the ppm and ppd genes from either S. hygroscopicus (21,31) or S. viridochromogenes (13). The arrangement of the complementing genes within the unsequenced region was refined by restriction to at least two separate ORFs upstream of ppm, with step III complementing DNA localizing directly upstream from the step IV complementing sequence (31).…”

Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide

“…Previous analyses indicated that location of the group of genes comprised of phpC, phpD, and phpE in the PTT gene cluster suggested that they could have roles in the conversion of phosphonoacetaldehyde to phosphonoformate (Fig 1b, steps III and IV) 7,15 . The role of phpC was unknown and its homology to type III (iron-dependent) alcohol dehydrogenases did not suggest an obvious function for the gene in PTT biosynthesis.…”

“…To test if PhpC acts as a phosphonoacetaldehyde reductase, we purified a histidine-tagged PhpC fusion after overexpression in Streptomyces lividans. His-PhpC catalyzed the in vitro reduction of phosphonoacetaldehyde to hydroxyethylphosphonate (HEP, 14) with either NADH (15) or NADPH (16) as a cofactor using 31 P NMR spectroscopy (Fig. 2) and LC/MS (Table 1, Supplementary Fig.…”

Isolation and Characterization of the Carbon–Phosphorus Bond-forming Enzyme Phosphoenolpyruvate Mutase from the MolluskMytilus edulis

“…The pathways leading to the commercially important antibiotic fosfomycin (16) and herbicide bialaphos (17) have been elucidated (14,15,30), as has the pathway leading to AEP (18) (Fig. 1).…”