Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2-2: designing new cytotoxic compounds.
Safracin is an antibiotic with anti-tumour activity produced by Pseudomonas fluorescens A2-2. The entire safracin synthetic gene cluster spanning 17.5 kb has been identified, cloned and sequenced. The safracin cluster comprises 10 open reading frames (ORFs) encoding proteins for three non-ribosomal peptide synthetases (NRPS), three safracin precursor biosynthetic enzymes, two safracin tailoring enzymes, a safracin resistance protein and a small hypothetical protein of unknown function. These genes are organized in two divergent operons of eight and two genes respectively. This pathway exhibits unusual features when compared with other NRPS systems. We have demonstrated by heterologous expression of the cluster that it is able to direct the synthesis of safracin in other strains. Cross-feeding experiments have confirmed that 3-hydroxy-5-methyl-O-methyltyrosine is the precursor of two amino acids of the molecule. Genetic analyses have allowed us to demonstrate that the bicistronic operon encodes the hydroxylation and N-methylation activities of the pathway. The cloning and expression of the safracin cluster has settled the basis for the in vivo and in vitro production of a wide variety of compounds, such as the promising ecteinascidins anti-cancer compounds.
Velasco
Ana
A
Drug Discovery Department, PharmaMar, S.A. E-28770-Colmenar Viejo, Madrid, Spain. avelaco@pharmamar.com
Acebo
Paloma
P
Gomez
Alicia
A
Schleissner
Carmen
C
Rodríguez
Pilar
P
Aparicio
Tomas
T
Conde
Susana
S
Muñoz
Rosario
R
de la Calle
Fernando
F
Garcia
Jose Luis
JL
Sánchez-Puelles
Jose M
JM
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial; D007546:Isoquinolines / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D007546:Isoquinolines; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase