The Neocarzinostatin Biosynthetic Gene Cluster from Streptomyces carzinostaticus ATCC 15944 Involving Two Iterative Type I Polyketide Synthases

Abstract

The biosynthetic gene cluster for the enediyne antitumor antibiotic neocarzinostatin (NCS) was localized to 130 kb continuous DNA from Streptomyces carzinostaticus ATCC15944 and confirmed by gene inactivation. DNA sequence analysis of 92 kb of the cloned region revealed 68 open reading frames (ORFs), 47 of which were determined to constitute the NCS cluster. Sequence analysis of the genes within the NCS cluster suggested dNDP-D-mannose as a precursor for the deoxy aminosugar, revealed two distinct type I polyketide synthases (PKSs), and supported a convergent model for NCS chromophore biosynthesis from the deoxy aminosugar, naphthoic acid, and enediyne core building blocks. These findings shed light into deoxysugar biosynthesis, further support the iterative type I PKS paradigm for enediyne core biosynthesis, and unveil a mechanism for microbial polycyclic aromatic polyketide biosynthesis by an iterative type I PKS.

Cloning of the Pactamycin Biosynthetic Gene Cluster and Characterization of a Crucial Glycosyltransferase Prior to a Unique Cyclopentane Ring Formation

“…In actinomycetes, this type of simple aromatic polyketides is generally biosynthesized by an iterative type I polyketide synthase (PKS) [15], which has been quite recently established and are encoded in the avilamycin [16], calicheamicin [17], neocarzinostatin [18], chlorothricin [19], biosynthetic gene clusters. Therefore, an iterative type I PKS was also anticipated to be involved in the biosynthesis of pactamycin.…”

A phosphopantetheinylating polyketide synthase producing a linear polyene to initiate enediyne antitumor antibiotic biosynthesis

“…We finally wished to extend the findings for SgcE and NcsE to demonstrate that all PKSE are likely functionally equivalent, thereby solidifying a common mechanism for initiation of enediyne biosynthesis. This was accomplished by plasmid-based expression of heterologous PKSE gene cassettes within two Streptomyces hosts: SB1005 and SB5002, ⌬sgcE and ⌬ncsE mutant strains devoid of C-1027 and NCS production, respectively (13,14). Plasmids pBS1049, pBS5020 (14), and pBS10005 (15), which contain sgcE, ncsE, and mdpE (the PKSE gene from the MDP cluster), respectively, were prepared and introduced into SB1005 by conjugation.…”

“…This was accomplished by plasmid-based expression of heterologous PKSE gene cassettes within two Streptomyces hosts: SB1005 and SB5002, ⌬sgcE and ⌬ncsE mutant strains devoid of C-1027 and NCS production, respectively (13,14). Plasmids pBS1049, pBS5020 (14), and pBS10005 (15), which contain sgcE, ncsE, and mdpE (the PKSE gene from the MDP cluster), respectively, were prepared and introduced into SB1005 by conjugation. Using HPLC and matrix-assisted laser-desorption ionization-MS to monitor production, all of the recombinant strains gave [MϩH] ϩ ions at m/z ϭ 844.259 and 846.272, consistent with the molecular formula for C-1027 and its aromatized product and identical to those isolated from the wild-type strain (SI Table 3) (13).…”

“…Inspired by the ability to map the ACP active site of recombinant SgcE, we prepared an E. coli expression plasmid for NcsE, the PKSE involved in NCS biosynthesis in Streptomyces carzinostaticus (14), and the protein was purified to near homogeneity (SI Fig. 6).…”

“…The biosynthetic gene clusters for C-1027 (13), NCS (14), MDP (15), and CAL (16) have been cloned and sequenced, and the first biosynthetic step leading to the enediyne core was speculated to be catalyzed by a new type of iterative polyketide synthase (PKS) that is shared among the entire enediyne family (12)(13)(14)(15)(16). PKSs catalyze decarboxylative condensation similar to fatty acid synthases (FASs) and likewise share homologous ketosynthase (KS) domains, responsible for decarboxylation and condensation, and acyltransferase (AT) domains, responsible for selection and loading of an acyl CoA (CoA)-substrate to a free thiol of an acyl carrier protein (ACP).…”