Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2‐2: designing new cytotoxic compounds

Abstract

SummarySafracin 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.

Characterization of the Saframycin A Gene Cluster fromStreptomyces lavendulaeNRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

“…1. The partial biosynthetic gene cluster of SFM-Mx1 (with a hydroquinone form of the E ring, a hydroxy group at the C-21 position, and a reserved ␣-amino group of Ala in comparison to SFM-A) and the entire biosynthetic gene cluster of SAC-B (one of the structurally simplest members in the SFM family) were cloned from Myxococcus xanthus in 1995 (32,33) and Pseudomonas fluorescens in 2005 (46), respectively, indeed revealing a nonribosomal peptide synthetase (NRPS) system for the formation of an identical tetrapeptide intermediate. In both cases, sequential incorporation of Ala, Gly, and Tyr derivatives into the backbone was speculated to be catalyzed by NRPSs in a colinear way according to the substrate specificity of the NRPS modules.…”

Biosynthetic Progress of the Tetrahydroisoquinoline Antitumor Antibiotics

“…[11] . 其中, SacF 和 SacG 是与 S-腺苷 甲硫氨酸依赖的甲基转移酶高度同源, 推测可能是负责 酪氨酸衍生物中两个甲基的引入; SacE 与 MbtH 家族蛋 白同源, 在这里的功能未知; SacD 仅与一些未知功能的 蛋白有很低的同源性, 根据异源表达的结果推测其可能 负责酪氨酸衍生物前体中 3-位羟基的引入.…”

Exploring the Biosynthesis of Natural Products and Their Inherent Suitability for the Rational Design of Desirable Compounds through Genetic Engineering

“…However, because its structure is extremely complex, complete synthesis of the compound did not afford the yield requisite for further studies and commercial use. Fortuitously, two varieties of NRPSs encoding biosynthetic gene clusters were identified in soil-borne bacteria which were identical to the genes responsible for synthesizing saframycin 69) and safracin, 70) both of which are structurally similar to ET 743. When expressed in a compatible heterologous expression system, these NRPSs might provide analogs of ET 743, along with effective intermediates.…”