A role for pabAB, a p-aminobenzoate synthase gene of Streptomyces venezuelae ISP5230, in chloramphenicol biosynthesis

Abstract

Mutagenesis of Streptomyces wenezuelae ISP5230 and selection for paminobenzoic acid-dependent growth in the presence of sulfanilamide yielded pab mutants 01551 9 and VS620) that continued to produce chloramphenicol (Cm), although with increased medium dependence. Transforming the mutants with pDQlO2 or pDQ103, which carried a pab-complementing fragment from 5. venezuelae ISP5230 in alternative orientations, restored uniformly high Cm production in VS620, but did not alter the medium dependence of Cm production in VS519. The cloned S. wenezuelae DNA fragment was subcloned and trimmed to the minimum size conferring pab complementation. The resulting 2.8 kb BamHI-Sac1 fragment was sequenced. Codon preference analysis showed one complete ORF encoding a polypeptide of 670 amino acids. Comparison of the deduced amino acid sequence with database proteins indicated that the N-and C-terminal regions resembled PabA and PabB, respectively, of numerous bacteria. The gene product showed overall sequence similarity to the product of a fused pabAB gene associated with secondary metabolism in Streptomyces griseus. Insertion of an apramycin resistance gene into pabAB cloned in a segregationally unstable vector and replacement of the S. wenezuelae chromosomal pabAB with the disrupted copy lowered sulfanilamide resistance from 25 to 5 pg ml-l and blocked Cm production.

Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4‐dimethylamino‐l‐phenylalanine precursor of pristinamycin I

“…The prototrophic phenotype of papA mutants for PABA indicates that PapA is only involved in secondary metabolism and that a different gene must be responsible for PABA synthesis for primary metabolism, even though a gene similar to papA could not be detected by Southern hybridization in the S. pristinaespiralis genome (data not shown). The same situation was observed for S. griseus (Gil et al, 1990) and S. venezuelae (Aidoo, 1989;Brown et al, 1996).…”

“…Both molecules are believed to have a biosynthetic relationship to the aromatic amino acids formed by the shikimate pathway, but neither is directly derived from phenylalanine or tyrosine. They appeared to come from chorismate by an additional branching reaction: PAPA, the precursor of chloramphenicol, is derived from chorismic acid through a set of reactions involving a PABA synthase (Vining and Westlake, 1984;Brown et al, 1996); PABA, the precursor of the p-aminoacetophenone moiety of candicidin, is also formed from chorismic acid by the enzyme PABA synthase (Gil et al, 1985;Criado et al, 1993). These examples prompted us to propose PAPA as a precursor of DMPAPA.…”

“…Database researches showed the ORF1 (designated papA ) product to be 52-53% identical with the p-aminobenzoic acid synthases (PABA synthases) from Streptomyces griseus, the producer of candicidin (Criado et al, 1993) and from Streptomyces venezuelae ISP5230, the producer of chloramphenicol (Brown et al, 1996) (Fig. 3).…”

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

“…Twelve of them are clustered on the approximately 18 kb of chromosomal DNA sequenced in this and previous studies (Mosher et al, 1990(Mosher et al, , 1995Brown et al, 1996;Chang et al, 2001;He et al, 2001). Functions for nine genes have been confirmed by insertional inactivation, and particular interest has focused on one (cmlP; see Fig.…”

Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine β-synthase and transsulfuration b bThe GenBank accession number for the sequence reported in this paper is AF319543.

“…Production of chloramphenicol was bioassayed as described by Doull et al (1985). For specific and more precise measurement of chloramphenicol produced in liquid cultures, the HPLC procedure of Brown et al (1996) was used.…”